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CLONING OF SNAP-23, ITS TISSUE DISTRIBUTION AND SUBCELLULAR LOCALIZATION IN NON-NEURAL CELLS
Peggy Pui Chi Wong
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Biochemistry University of Toronto
O Copyright by Peggy Pui Chi Wong, 1997
395 Wellington Street 395, rue Wellington Ottawa ON K I A ON4 Ottawa ON KIA ON4 Canada Canada
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CLONING OF SNAP-23, ITS TISSUE DISTRIBUTION AND
SUBCELLULAR LOCALIZATION IN NON-NEURAL CELLS
A M.Sc. thesis by Peggy Pui Chi Wong submitted in conformity with the
requirements for the degree of Master of Science, Graduate Department of
Biochemistry, University of Toronto, 1997.
ABSTRACT
Correct targeting of proteins to specific cellular compartments is thought to occur
via transport vesicles. A current hypothesis proposes that in the nervous system, targeting
specificity of synaptic vesicles is achieved by interaction of a vesicle protein (VAMP) and
presynaptic plasma membrane proteins (syntaxin and SNAP-25). VAMP and syntaxin
isoforrns have been identified outside the nervous system, but SNAP-25 expression is
restricted to neural and neuroendocrine cells. 1 hypothesized that functional equivalent(s)
of SNAP-25 must exist in non-neural cells to allow intracellular traffic. This Thesis
describes the search for a non-neural equivalent of SNAP-25, SNAP-23, by screening a
melanoma cDNA library. cDNA clones were isolated, sequenced, and fusion proteins
were expressed. With a specific antibody, SNAP-23 was detected in al1 non-neural tissues
and ce11 lines examined. Subcellular fractionation of 3T3-L1 adipocytes suggested that
SNAP-23 was predominantly found at the plasma membrane.
PREFACE
The work presented in this Thesis was performed from 1995-1997 in the
laboratory of Dr. Amira Klip, Division of Ce11 Biology, The Research Institute of the
Hospital for Sick Children, Toronto, Ontario, Canada with the financial support of the
University of Toronto (University of Toronto Open Fellowship) and a grant from the
Medical Research Council to Dr. Amira Klip.
The results of this Thesis have been presented in two publications:
1. Wong, P. P. C., N. Daneman, A. Volchuk, N. Lassam, M. C. Wilson, A. Klip, W. S.
Trimble. 1997.
Tissue Distribution of SNAP-23 and Its Subcellular Localization in 3T3-L1 Cells.
Biochernical and Biophysical Research Communications 230: 64-68.
2. Gaisano, H. Y., L. Sheu, P. P. C. Wong, A. Klip, W. S. Trimble. 1997.
SNAP-23 is Located in the Basolateral Plasma Membrane of Rat Pancreatic Acinar Cells.
Submitted.
A B S T R A C T ................................................................... i i ... P R E F A C E ..................................................................... 111
TABLE O F CONTENTS ..................................................... i v LIST O F T A B L E S ............................................................ vi
L I S T O F F I G U R E S .......................................................... vii
L IST O F ABBREVIATIONS ................................................ viii
B A C K G R O U N D .............................................................. The SNARE Hypothesis: a Conceptual Framework for Studying
I n t r a c e l l u l a r T r a f f i c ..................................................... ................. Part 1: Components of the SNARE hypothesis
Soluble Proteins ............................................................. NSF .................................................................... SNAPs .................................................................
Membrane-associated Proteins ............................................ t-SNARES .............................................................
Syntaxins ....................................................... ....................................................... SNAP.25
v.SNAREs ............................................................ ......................................................... VAMPS
Synap totagmins ................................................ Part II: Models for Vesicle Targeting, Docking and Fusion ..
Vesicle Targeting and Docking ............................................ The SNARE hypothesis: a general vesicle targeting and
docking mode1 ........................................................ Recent modifications to the SNARE hypothesis ..................
Membrane Fusion ........................................................... Part III: Proteins Modulating the Accessibility of the
S N A R E S ............................................................ (1) n-Sec 1 / Muncl8 / rbSec 1 ............................................. (2) Rab .......................................................................
Part IV: Role of SNAREs in Insulin-stimulated Glucose
T r a n s ~ o r t ...........................................................
VAMPS ................................................................ 53
Synaptotagmins ....................................................... 54
t.SNAREs ............. .. .................................................... 55
Syntaxins .............................................................. 55 SNAP.25 .............................................................. 56
RATIONALE AND HYPOTHESIS .......................................... 5 7
IDENTIFICATION OF A SNAP-25 LIKE PROTEIN IN NON-NEURAL
............................................... CELL LINES AND TISSUES 5 8 I n t r o d u c t i o n ............................................................. 58
............................................... Experimental Procedures 59 Materials ............................................................................ 59 Methods ............................................................................. 59
............................... RNA isolation and Northern blot analysis 59 ............................................ cDNA cloning and sequencing 60
............. Bacterial expression of SNAP-23 and SNAP-25 proteins 61 .......................................... Generation of SNAP-23 antisera 61
................................................................... Ce11 culture 61
......................................................... Membrane isolation 62
......................... Subcellular fractionation of 3T3-L1 adipocytes 63 Immunoblotting ............................................................. 63
R e s u l f s ................................................................. 65
Isolation of SNAP-23 from human rnelanoma cDNA library ................ 65
Generation of two SNAP-23 specific antibodies .............................. 67
Expression of SNAP-23 protein in different ce11 lines and tissues .......... 67
Subcellular localization of SNAP-23 in 3T3-LI cells ......................... 68
Subcellular distribution of SNAP-23 in non-neural secretory cells .......... 69
D i s c u s s i o n .............................................................. 8 2
SNAP-23 is a SNAP-25-related gene product ................................. 82 Expression of SNAP-23 in different tissues and cells ......................... 85
Subcellular localization of SNAP-23 in non-neural cells .................. .,., 86
................. OVERALL DISCUSSION AND FUTURE DIRECTIONS Y 1
REFERENCES ............................................................... 97
........................................................ APPENDED PAPERS 117
LIST OF TABLES
Table B l Similarity between the subcellular localization of proteins involved in the
................................. nerve-terminal and yeast transport machinery 25
Table B2 Similarity between the nerve-termina2 and yeast membrane traffic mac hinery .......................................................................... 26
Table B3 A summary of molecules that interact with different tagmin isoforms ...... 27
BACKGROUND
Fig . B1 Schematic representation of the predicted coiled-coi1 dornains of SNAREs and a-SNAP which mediate protein-protein interactions ..................................... 23
Fig . B2 Schematic representation of the positions of cleavage of SNAREs by
different Clostridial neurotoxins ................................................................. 24
Fig . B3 Proposed mode1 for the docking and fusion of synaptic vesicles to the
presynaptic plasma membrane at a nerve terminal ........................................ 31
Fig . B4 Two proposed modeIs for synaptic vesicle docking and fusion ................. 35
Fig . B5 Scaffold mode1 for membrane fusion during exocytosis ......................... 41
IDENTIFICATION OF A SNAP-25 LIKE PROTEIN IN NON-NEURAL CELL LINES AND TISSUES
Fig . 1 A SNAP-25 cDNA probe did not hybridize with RNA from 3T3-L1 cells ...... 72
Fig . 2 Immunodetection of a SNAP-25 like protein in membranes from 3T3-L1
adipocytes .......................................................................................... 73
Fig . 3 Nucleotide sequence of human melanoma SNAP-23 and its predicted
arnino acid sequence ............................................................................... 74
Fig . 4 Cornparison of amino acid sequences of human melanoma SNAP-23 and
SNAP.25B ......................................................................................... 75
Fig . 5 Characterization of SNAP-23 and SNAP-25 antibodies ............................ 76
Fig . 6 Expression of SNAP-23 in ce11 lines and tissues .................................... 77
Fig . 7 Localization of SNAP-23 in 3T3-LI cells .......................................... 78
Fig . 8 Expression of SNAP-23 in different types of white blood cells ................... 79
Fig . 9 Subcellular distribution of SNAP-23 in human neutrophils ........................ 80
Fig . 10 Distribution of SNAP-23 in pancreatic acinar cells ................................ 81
LIST OF ABBREVIATIONS
ADP
Ala
A% ATP
BoNT ~ a 2 + cDNA CNS
CYT
EDTA
ER
EST Gln GLUT
GST
Hepes DDM
Ile
IRS- 1
Kb
kDa LDM
LYS
Mg rnM
mRNA MW
NEM
NIDDM
NSF PC12
PCR PDGF
Adenosine diphosphate Alanine
Arginine Adenosine triphosphate Botulinurn neurotoxin Calcium Complementary deoxyribonucleic acid Central nervous system Cytosol Eth ylenediamine tetraacetic acid Endoplasmic reticulum Expressed sequence tag Glutamine Glucose transporter Glutathione-S- transferase N-[2-Hydroxyethyl] piperazine-N' [2-ethanesulfonic acid] Insulin-dependent diabetes mellitus Isoleucine Insulin receptor substrate- 1
Kilobase Kilodalton
Light density microsornes Lysine Magnesium
Millirnolar Messenger ribonucleic acid Molecular weight N-ethylmaleimide
Non-insulin-dependent diabetes mellitus NEM-sensi tive factor Rat pheochromocytoma ce11 line Polymerase chain reactions Platelet-derived growth factor
1 I
Pi
PI PKC PM
SDS-PAGE Ser SH2 SNAP SNAP-23 SNAP-25 SNARE TeTx val VAMP ZGM
L l l V O ~ l 1 V l 1 l V O I L u l
Inorganic orthophosphate Isoelectric point Protein kinase C Plasma membrane Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Senne Src-homology-2 Soluble NSF attachment protein SynaptosomaI-associated protein of 23 kDa
Synaptosomal-associated protein of 25 kDa SNAP Receptor Tetanus toxin Valine Vesicle-associated membrane protein Zymogen granule membrane
THE SNARE HYPOTHESIS: A CONCEPTUAL FRAMEWORK FOR STUDYING INTRACELLULAR TRAFFIC
A fundamental question in ce11 biology is how are proteins targeted to specific
cellular compartments by means of transport vesicles. The transport machinery is crucial
for generation of specific organelles such as the plasma membrane, endosomes and Golgi
stacks, as well as maintaining their cornpartmental integrity. This would require the
constitutive transport of vesicles to and from their destined compartments. Another aspect
of the life cycle of a ce11 is its ability to respond to extracellular stimuli which involvcs
regulated exocytic processes. Regulated exocytosis is responsible for cellular events such
as neurotransrnitter release, hormone secretion, and glucose transport.
Recent findings from five lines of study in different organisms using molecular
biology techniques, biochemical and pharmacological approaches, suggest a universal
docking and fusion machinery that is conserved in both prokaryotic and eukaryotic cells
(rcviewed in Sollner and Rothman, 1994). These studies are as follows: (1) cc11 frec
transport assays between Golgi stacks led to the identification of two essential general
components of the transport mechanism which are referred to as NSF and SNAPs; (2) the
subsequcnt identification of proteins that serve as receptors for SNAPs (SNAREs) on both
the vcsiclcs (v-SNAREs) and the target membranes (t-SNAREs) in the nervous system; (3)
the discovery that some SNAREs are specific proteolysis substrates of different clostridial
ncurotoxin scrotypes and the observations that cleavagc results in abrogation of
ncuroiransmittcr rcleasc highlighted the importance of SNAREs in regulatcd sccrction; (4)
thc discovery of homologues or isoforms of different docking and/or fusion proteins in
non-neural cclls of eukaryotes; (5) the existence of relatcd genc products in ycast and the
isolation of lethal ycast mutants defective in various constitutive transport steps. Results
from thesc fivc different lines of study suggest that the molecular mechanism for vesiclc
L a i è ; b r i i i g , U U L A ~ I I ~ allu I U D ~ U I ~ 13 LUIIDGL V G U LI UIAI y t a 3 ~ LU 1L la I l l l l l a13 , ILIUI CU V L I , LIIL
machinery is similar between constitutive and regulated intracellular transport.
As mentioned above, the vesicle docking and/or fusion apparatus involves general
components such as NSF and SNAPs which are both soluble proteins, as well as
membrane proteins that are localized on either the target membranes (t-SNAREs) or the
vesiclcs (v-SNARE). 1 have organized this section into four parts: part 1 will present
findings on each of the components of the docking/fusion complex; part II will introducc a
mode1 known as the "SNARE hypothesis" which was originally proposed by Rothman and
colleagues to cxplain how synaptic vesicles dock and fuse with the presynaptic plasma
membrane leading to neurotransmitter release (Rothman, 1994). Part III wiIl discuss two
proteins that may have a regulatory role in the dockinglfusion machinery by modulating the
accessibility of the SNAREs. Part IV will introduce the function of SNAREs in the
regulated exocytic process facilitating glucose transport in muscle and fat cells.
Part 1: Corn~onents of the SNARE H v ~ o t h e s i ~
Soluble Proteins
NSF - The cstabIishment of a cell-free assay by reconstituting vesicular transport betwecn
cisternae of Golgi membranes has been useful in elucidating the molecular mechanisrns
undcrlying constitutive protein transport pathways. The finding that vesicular transport
bctwecn Golgi stacks in vitro is blocked by the cysteine-alkylating agent N-ethylmaleimidc
(NEM) resulting in an accumulation of uncoated vcsicles at the acccptor Golgi cistcrnac,
suggests that a NEM-Sensitive Factor (NSF) is needed for fusion (Malhotra ct al., 1988;
Orci ct al., 1989). Here, fusion is dcfined as any stcps leading to mixing of thc donor
(vcsicles) and acceptor (Golgi slacks) cornpartments once the transport vcsiclc binds to ils
targct (Rothman, 1994). Morcover, thc blockade can bc rescucd upon addition of frcsh
LYLUJUI I I I U I C ; ~ L I I I ~ i ~ i i l ~ IY 3r IS L ~ L U J U I I L [UIILK aliu nuririiiarl, I Y O 1 ) . 1 ii~sc; L W W
observations together Ied to the purification of NSF from the cytosol (Block et al., 1988).
NSF is a homotrimer of 76 kDa subunits. Each of the subunits contains 2
homologous ATP-binding domains (Wilson e t al., 1989; Tagaya et al., 1993). ATP
hydrolysis by the first ATP-binding domain is required for NSF function in intra-Golgi
transport. Mutations of residues within nucleotide-binding motifs result in only 20-30% of
wild typc ATPase activity and no intra-Golgi transport activity, although mutated proteins
can still fonn homotrimer (reviewed in Whiteheart and Kubalek, 1995). Furthemore, only
normal trimers are active in Golgi transport; trimers with even one mutated subunit are
inactive, indicating that al1 three subunits work i n concert. The second ATP-binding
domain is needed for trirnerization since its deletion results in rnonomeric NSF. Mutations
of the nucleotide-binding motifs of this second domain result in mutant proteins supporting
up to 30% of intra-Golgi transport in vitro (Whiteheart and Kubaiek, 1995). Therefore, the
role of this second ATP-binding site, other than for trimerization of subunits, remains
unclear.
Two pieces of direct evidence that NSF is required in vesicle transport in vivo wcrc
obtained by isolating mutant yeast and flies that bear mutations in their NSF homologues.
First, in the absencc of yeast NSF homologue, Sec1 8p, uncoated vesicles accumulate at the
acccptor membrane (Malhotra et al., 1988; Orci et al., 1989). Moreover, loss of Secl8p
function has been demonstrated to be lethal (Eakle et al., 1988; Kaiser and Schekman,
1990). These results indicate that Sec 18p is required for transport vesicle fusion in yeast.
Sccond, in Drosophila, the cornutose mutant has a temperature-sensitive mutation in NSF
which lcads to a block in ncurotransmission, provides a direct functional cvidcncc for a rolc
of NSF in regulated cxocytosis (Pallanck et al., 1995). Switching thesc llies to the
rcstrictivc tcmperature results in a rapid (within 1-2 min) inhibition of neurotransmission
and paralysis, consistent with an cssential role for NSF in synaptic cxocytosis.
Studics using different cell-free systems show that NSF is required for othcr
intracellular transport events such as endosomal fusion, endoplasmic reticulum to Golgi
transport, and regulated exocytosis of synaptic vesicles. The yeast homologue of NSF,
Sec 18p, is also involved in many different fusion events with two exceptions: fusion of
haploid nuclei (karyogamy) and transport between the Golgi complex and the vacuole
(rcviewed in Whiteheart and Kubalek, 1995). Together these findings suggest that NSF is
a general component in the vesicle transport machinery. In addition, it has been observed
that plant cytosol cm fully replace animal cytosol in cell-free Golgi transport, and that yeast
Sec 18p can replace NSF in a mammalian system for cell-free Golgi transport (Wilson et
al., 1989) which strongly supports the universality of the fusion machinery in vesicle
docking.
Although there is an ample body of evidence suggesting the importance of NSF in
vesicle transport, the precise step at which NSF acts remains highly controversial.
Evidence exists in supporting its action in at least three different stages of vesicle traffic,
including a post-docking stage close to membrane fusion (Sollner et al., 1993a), a post-
docking but pre-fusion stage (O'Conner et al., 1994), or at a pre-doclung stage (Morgan
and Burgoyne, 1995a). Future experiments are needed to address the discrepancy among
thcse studics.
SNAPs
NSF itself does not bind to Golgi membranes unless a crudc cytosolic fraction is
addcd (Wcidman et al., 1989). This observation suggests the presence of another cytosolic
Factor that acts as an 'adapter' for the association of NSF with the Golgi rnembrancs.
Thrce Solublc NSF Attachmcnt Proteins, termed a-, B-, and y-SNAP (of 35, 36, and 39
kDa rcspectivcly), were purified from bovine brain extracts (Clary and Rothman, 1990b).
NSF binds only to SNAPs that arc alrcady bound to membrancs, and not to thosc ihat arc
11, U V A U L I V I I . I I L L U A l l U A V C C L Y U L A A L I L L J l A L U 1 1 1 6 V1 U A 7 1 X I 0 L U C I L k l L 1 k k k P L V l O V A 1 L I X k I L I b I L l U L U I l U
induces a confonnational change in the SNAPs which exposes the NSF binding site
(Wilson et al., 1992).
The three SNAPs have distinct tissue distribution. P-SNAP is expressed mainly in
brain whereas a- and y-SNAP have been found in many ce11 types. Since a- and b-SNAP
have 83% sequence identity (Whiteheart et al., 1993), they are proposed to represent
functionally similar isoforms. Indeed, they were found to have interchangeable roles in
many aspects of regulated exocytosis, including their ability to bind and activate the
ATPase activity of NSF (Rothman, 1994; Sudlow et al., 1996). Furthemore, exogenous
a- and P-SNAP stimulated catecholarnine release in response to ~ a 2 + from digitonin-
permeabilized chromaffin cells to the sarne extent and with essentially identicai dose-
response relationships. Comparative analysis of the role of a-, B- and y-SNAP in intra-
Golgi transport suggested that a- and P-SNAP are functionally redundant isoforms but y-
SNAP has distinct roles (Whiteheart et al., 1992; Wilson et al., 1992). This is consistent
with the high level of sequence identity between a- and P-SNAP compared to the divergent
y-SNAP, as it shows only 25% identity to a- and B-SNAP (Whiteheart et al., 1993). y-
SNAP is not essential for NSF binding to Golgi membranes, but synergizes with a-SNAP
to crcate an optimum binding site for NSF. Moreover, y-SNAP has been shown to have
minor and variable effect on catecholamine release (Morgan and Burgoyne, 1995b).
cDNAs for five SNAPs have been cloned: three bovinc forms (Clary and
Rothman, 1990b), one from Drosophilu (Ordway et al., 1994), and one from yeast (Griff
ct al., 1992). Sccl7p (of 34 kDa) is the yeast homologue (with 34% identity) to a-SNAP,
since it is a-SNAP (but not b- or y-SNAP) that can restore the fusion activity of cytosolic
cxtracts from yeast Sec17 mutants (Clary et al., 1990a). This suggcsts that ycast Sccl7p is
thc lunctional cquivalence to mammalian a-SNAP. Moreover, this finding supports oncc
again thc thcme of universality in the fusion machinery.
The functional importance of a- and P-SNAP in regulated exocytosis has been
supported by three important findings. First, exogenous a-SNAP stimulates exocytosis
following its injection into the squid giant synapse (DeBello et al., 1995). Second,
addition of recombinant a -SNAP (Chamberlain et al., 1995; Morgan and Burgoyne,
1995b; Sudlow et al., 1996) or P-SNAP (Sudlow e t al., 1996) resulted in a stimulation of
Ca2+-dependent catecholamine release from digitonin-permeabilized adrenal chromaffin
cells. Third, the importance of marnmalian a-SNAP is underscored by the requirement of
its functional equivalent Secl7p for the transit through the secretory pathway in yeast
(Pryer et al., 1992). In the absence of a functional Secl7p, transport from endoplasmic
reticulum to Golgi stops and transport vesicles accumulate (Kaiser and Schekman, 1990).
Membrane-associated Proteins
The SNARE hypothesis postulates that targeting of intracellular vesicles to specific
cornpartment is achieved through the unique interaction of membrane proteins both on the
vesicles and the target membranes. These membrane proteins, termed as SNAREs, act as
'receptors' for the general fusion particles, SNAPs and NSF, which have been shown to
be necessary for membrane fusion.
When brain extracts were solubilized with detergent, a multisubunit particlc of
NSF-SNAP-SNARE which sedimented at 20s was isolated (Wilson e t al., 1992). In
order to purify SNAREs in quantity from crude detergent exiract of bovine brain, the
formation of the 20s particle was further promoted to assemble in vitro using recombinant
NSF, a- and y-SNAP (Sollner et al., 1993b). Three membrane proteins werc identificd
which werc latcr Sound to form a stable stoichiornetric complex that scdimented at 7s in ~ h c
abscncc o î thc hsion factors NSF and SNAPs (Sollner et al., 1993a). The complcx
includcd two presynaptic plasma membrane proteins: SNAP-25 ( ~ N a p t o s o m a l -
Associated ho le in of 25 m a ) and syntaxin 1. These two proteins arc often referrcd to as -
,, V I 1 1 LI\-" "1,I"V L I I Y , U A " UUUVYlULYY .. I L I 1 L A I V C U . b V C a.. V . I . " A -..W. I L " . A " U A Y "V .."*YU L I l U L
SNAP-25 and soluble protein SNAP represent unrelated gene products which
unfortunately share similar narnes. The complex also contained a synaptic vesicle protein
VAMP (vesicle-associated membrane protein, also known as synaptobrevin), which is
often referred to as a V-SNARE. Recently, anoiher synaptic vesicle protein,
synaptotagmin, has also been described as a V-SNARE based on its ability to bind a neural-
specific form of SNAP protein, p-SNAP (Schiavo et al., 1995a) and the two t-SNAREs,
syntaxin 1 and SNAP-25 (Schiavo e t al., 1997). The four SNARE proteins are described
in detail below.
Svntaxins
Syntaxins were first identified a s two 35 kDa proteins (p35) that interact with the
synaptic vesicle protein p65 or synaptotagmin (Bennett et al., 1992). The two proteins
were termed syntaxins 1A and lB , and found to share 84% sequence identity. So far,
seven mammalian syntaxins have been described: lA, IB, and 2-6. Although the
syntaxins have distinct tissue distribution and subcellular localization, they nonetheless
exhibit a few common structural features. Al1 syntaxins are 288-301 amino acids in length
(Bennett et al., f 993; Dascher et al., 1994) except syntaxin 6 which is composed of 255
rcsidues (Bock et al., 1996). Furtherrnore, al1 members of the syntaxin farnily have a C-
terminal stretch of 17-25 highly hydrophobic residues which is of sufficient lcngth and
hydrophobicity to serve as membrane anchor (Bennett et al., 1993). As a consequence ol'
thc C-tcrminal strctch of hydrophobic residues, al1 syntaxins bchavc as type II intcgral
membranc proteins with their N-terrnini facing thc cytoplasm (Bennett et al., 1992; Bcnnctt
ct al., 1993; Bock ct al., 1996).
baCh mxnber 01 the syntaxin Iarnily contains severar domains predicted to Iorm a-
hclical coiled-coi1 structures (Inoue et al., 1992: Spring et al., 1993; Bock et al., 1996)
which are likely to be involved in protein-protein interactions (Fig. BI). One of such
coiled-coils which spans 70 residues near the C-terminal transmernbrane region (Kee et al.,
1995), also referred to as the H3 domain, displays high level of sequence similarity within
the rat syntaxin family (Bennett et al., 1993) and between yeast and rat proteins (Pelharn,
1993). in vitro binding studies of recombinant proteins revealed that this coiled-coi1 region
of syntaxin 1A (residues 199-243) and syntaxin 4 (amino acids 197-274), but not of
syntaxins 2 and 3, interacts directly with VAMPs 1 and 2 (Calakos et al., 1994). The
observation that VAMPs interact with some syntaxin members but not others supports the
hypothesis that specific SNARE interactions may contribute to vesicle targeting specificity.
The H3 domain of syntaxin IA has also been shown to bind to another t-SNARE, SNAP-
25 (Chapman et aI., 1994), and soluble protein a-SNAP (Kee et al., 1995) as shown in
Fig. BI. Moreover, the H3 domain with the transmembrane region of syntaxin 1A
(residues 194-288) binds to synaptotagmin in a Ca2+-dependent manner (Chapman et al.,
1995). Syntaxin 1A has been shown to associate with different types of ca2+ channels
including the N-type voltage-gated ~ a 2 + channel (Bennett et al., 1992; Sheng et al., 1994;
Shcng et al., 1996; Wiser et al., 1996). This interaction predorninantly involvcs thc
transmernbrane region of syntaxin (Bezprozvanny e t al., 1995; Wiser et al., 1996). In
these studies, CO-expression of syntaxin 1A with N- and L-type ca2+ channels in Xenoprls
oocytcs havc bcen shown to exert a negative regulation on both channels. Syntaxin has
also been shown to interact with the P- and Q-type ~ a 2 + channels which triggcrs rapid
rdcase of neurotransmitter at many mammalian synapses (Martin-Moutot et al., 1996).
As mentioned above, a farnily of seven syntaxin-related proteins in rat have becn
identilied and thcy share 23-84% arnino acid identity. Syntaxins 1A and 1B are expresscd
in thc ncrvous system (Bennett et al., 1992; Bennett et al., 1993; Parpura ct al., 1995), and
cnaocnne ceus inciuaing me pancreauc insuiin-sccrering p-ceus (wneeier el ai., rrvb), ana
adrcnal chromaffin cells (Bennett et al., 1993; Hodel ct al., 1994; Roth and Burgoyne,
1994; Tagaya et al., 1995; Hohne-ZeI1 and Gratzl, 1996). Syntaxins 2-6 are widely
cxpressed in different tissues which supports the hypothesis that this family of proteins
participates in vesicle traffic in various ce11 types. Unique subcellular localization of some
syntaxin isofoms may provide the targeting specificity in intracellular traffic. For instance,
irnmunofluorescent detection of transiently expressed isoforms of syntaxin suggests that
syntaxins lA, 2 and 4 are predominantly localized on the plasma membrane in COS cclls
(Bennett et al., 1993). Furthermore, in polarized exocrine acinar cells of the pancrcas,
localization of endogenous syntaxins 2 and 4 is restricted to apical and basolateral plasma
membrane respectively. Syntaxin 3 was found to be distributed in an intracellular vesicular
compartment, the zymogen granule membrane (Gaisano et al., 1996). Low et al.
demonstrated that in a different type of polarized epithelial cells, the Madin-Darby canine
kidney cells, syntaxins 2, 3, and 4 were expressed. Moreover, when these cells were
transfected with different syntaxin isoforms, syntaxins 3 and 4 were restricted to the apical
and basolateral plasma membrane respectively, whereas syntaxin 2 was found on both
membranes (Low et al., 1996). The marnmalian syntaxin proteins share 5143% similarity
with two yeast proteins, Ssolp and SsoZp, which are required for traffic betwecn the Golgi
complex and plasma membrane in yeast (Pryer et al., 1992). In addition, syntaxin 5 which
is prcscnt on the cis-Golgi has bccn demonstrated to be essential in ER to Golgi transport
(Dascher et al., 1994). This is further supported by the finding that the yeast SEDS gcnc
product, homologuc of syntaxin 5 (35% identity), is required for efficient mcmbranc
transport bctwccn ER and the Golgi complex in yeast (Hardwick and Pelham, 1992). Thc
most rccently idcntified member of the syntaxin family, syntaxin 6, was identificd bascd on
its scqucncc similarity to yeast Pep12p (56% amino acid similarity) (Bock ct al., 1996). In
ycast, Pcp 12p is neccssary for thc delivery of proteolytic enzymes îrom the Golgi complcx
W., VA-- . - - - - e V \ " - " - - 7 - - ' -Y' ----' ' W U " , ' , "-""' " J -------- to the Golgi region. It remains to be defined a t which step syntaxin 6 participates in
intraccllular vesicle traffic. Table B 1 summarizes the subcellular localization of different
syntaxin isoforms. The transport step at which each isoform functions is listed in Tablc
B2.
The function of syntaxin has been examined using various approaches.
Microinjection of anti-syntaxin 1A antibodies into PC12 ceils results in an inhibition in
Ca2+-regulated catecholamine release (Bennett et al., 1993) which suggests that syntaxin
1A is an important component of the regulated secretory machinery in PC12 cells. A
pharmacological approach to understand the function of syntaxin was also employed when
syntaxin was found to be cleaved by Botulinum neurotoxin serotype C (BONTIC) which
belongs to a large family of zinc-dependent endopeptidases (Blasi et al., 1993b; Schiavo et
al., 1995b). Fig. B2 illustrates the cleavage of SNARE proteins by different clostridial
neurotoxins. BONTIC cleaves at Lys-Ala bond of syntaxins 1A and lB, only when thcy
are inserted into a lipid bilayer. Syntaxins 2 and 3 are also cleaved by BoNT/C, while
syntaxin 4 is resistant (Blasi et al., 1993b; Schiavo et al., 199%). Cleavage of syntaxins
by BONTIC results in an inhibition of neurotransmitter release (Blasi et al., 1993 b), causes
growth cone collapse, inhibits axonal growth (Igarashi et al., 1996), and blocks Ca2+-
cvoked catecholamine release from intact and permeabilized adrenal chromaffin cclls.
Morcovcr, BoNT/C cleavagc of syntaxin has recently been shown to uncouplc the down-
segulation of presynaptic ~ a 2 + channel by G proteins (Stanley and Mirotznik, 1997). This
finding may suggest an additional new role for syntaxin in G-protein modulation of
prcsynap tic calcium channcl. Final1 y, a Drosophila mutant without syntaxin 1 A shows
subtlc axonal defccts and partial defects of CNS condensation (Schulze ct al., 1995).
Thcsc results suggcst that in addition to a role in secretion, syntaxins participatc in
phcnomcna rangin;: from mon growth to modulation of excitable channcl activity.
SNAP-25
SyNaptosomal-Associated Protein of 25 kDa (SNAP-25) is comprised of 206 -
amino acids with a calculated molecular mass of 23.315 kDa and a theoretical pI of 4.38
(Oyler et al., 1989). In contrast to the well-defined transmembrane domains of other
SNAREs, such as syntaxin, amino acid sequence analysis of SNAP-25 suggests that the
polypeptide is largely hydrophilic and lacks any stretch of hydrophobic residues necessary
for membrane insertion. Rather unexpectedly, characterization of the subcellular
localization of SNAP-25 in neurons revealed that the polypeptide is predominantly
associated with the synaptosomal membrane fraction, with undetectable levels in the
cytosol (Oyler et al., 1989). Furthermore, fractionation studies demonstrated that the
association of SNAP-25 with membranes was resistant to IM salt extraction. The protein
was released into the supernatant only after solubilizing the membranes with 1.25% Triton
X- 100 in the presence of 1M NaCl (Oyler e t al., 1989). The tight association of SNAP-25
with the presynaptic plasma membrane was later found to be achieved through a post-
translational addition of palmitic acids to one or more of a cluster of 4 cysteine rcsidues
spanning amino acids 84-92, which has been referred to as "a cysteine quartet", via
thioester linkages (Oyler et al., 1989; Hess et al., 1992). Mutant SNAP-25 with a deletion
of 12 amino acids spanning the "cysteine quartet" can no longer be labelled with radioactive
palmitate (Veit et al., 1996). Moreover, the deletion renders the protein complctely solublc.
Thcsc two findings further support the hypothesis that SNAP-25 is palrnitoylated and that
this post-translational modification of the protein is neccssary for membrane association.
SNAP-25 was also shown to be substrate for phosphorylation by protcin kinasc C
(PKC) both in vitro and in chromaffin PC12 cells (Shimazaki e t al., 1996). Thc sitc of
phosphorylation is bclieved to be Serlg7, although this remains to be confirmcd. In
addition, it was found that the amount of syntaxin CO-precipitated with SNAP-25 decrcased
with PKC phosphorylation, suggesting that phosphorylation of SNAP-25 may play a
regulatory role, more specifically by blocking the syntaxin-SNAP-25 interaction.
Cloning of the chicken gene for SNAP-25 revealed a complex organization of nine
different cxons which span more than 65 kilobases of genornic DNA (Bark, 1993).
Furthermore, two sirnilar but distinct variants of exon 5 were discovered in chicken (Bark,
1993), mouse (1. Bark and M. Wilson, unpublished), and human (Bark and Wilson,
1994). The alternative splicing of the gene gives rise to two variants: SNAP-25a and
SNAP-25b (Bark, 1993). At the amino acid level, the two alternative exon 5 sequences
differ in 9 out of 39 possible residues, resulting in two similar but distinct forms of SNAP-
25 that share 95% sequence identity (Bark, 1993). Although both isoforms retain the four
cysteine residues, the spatial organization with respect to the rest of the sequence has
changed. This may affect the efficiency of fatty acid acylation of the polypeptide. Studies
on the expression of SNAP-25 isoforms revealed devefopmental regulation: SNAP-25a is
the predominant species during embryonic and early postnatal development of mouse brain,
whereas SNAP-25b expression begins with the onset of synaptogenesis and it becomes by
far the predominant isoform in the adult brain (Bark et al., 1995). The distribution of
SNAP-25 is restricted to neural and neuroendocrine cells including neurons (Oyler ct al.,
1989), the peripheral nervous system (Duc and Catsicas, 1995), anterior pituitary cells
(Aguado et al., 1996), and pancreatic P cells (Jacobsson et al., 1994; Sadoul et al., 1995;
Wheeler et al., 1996). SNAP-25 has not been detected in other non-neural secretory cells
such as neutrophils (Brume11 et al., 1995) and pancreatic exocrine cells (Gaisano ct al.,
1 997).
Like syntaxin, SNAP-25 contains several domains that have a-helical potential, and
these prcdickd secondary structures have been implicated in protcin-protein interactions
(Fig. B 1). For instance, the amino-terminal region has been predicted to contain two
coilcd-coils (rcsidues 1-42 and 45-85), and both arc crucial dctenninants in its binding to
syntaxin (Chapman et al., 1994). SNAY-25 also contains a predicted coiled-coi1 domain al
its C-terminus (residues 160-205), which is essential for binding of VAMP (Chapman et
al., 1994). In vitro binding studies demonstrated that binding of SNAP-25 greatly
increases the affinity between neuronal expressed syntaxin and VAMP proteins, resulting
in the formation of a stable SDS-resistant cornplex (Hayashi et al., 1994; Pevsner et al.,
1994a). This suggests that SNAP-25 plays a unique role in strengthening the association
of syntaxin and VAMP, to bridge opposing membranes and thereby specify targeting and
docking of synaptic vesicles for fusion at the active zone in presynaptic nerve terminais
(Wilson et al., 1996). Recently, another V-SNARE, synaptotagmin has also been
demonstrated to interact, both in vitro and in vivo, with SNAP-25 (Schiavo et al., 1997).
Furthemore, SNAP-25, dong with syntaxin and VAMP, has also been shown to associate
with N-type calcium channel (Saisu et al., 1991; Sheng et al., 1996) in a ~a2+-dependent
manner. This finding supports the idea that synaptic vesicles are docked near presynaptic
Ca2+ channels through this interaction.
Other than mouse, human and chicken, SNAP-25 has also been reported in
goldtïsh, which has two forms of SNAP-25: SNAP-25A and SNAP-25B, due to an
ancient gene duplication in bony fish. The two forms are 9 1 % identical to each other, and
94% and 91% identical to mouse SNAP-25b (Risinger and Larhammar, 1993b). SNAP-
25 has also been identified in fruitfly Drusuphila melanogaster and ray Torpedo murmrutu
(Risinger et al., 1993a). SNAP-25 sequence is well conserved among species, as its
amino acid sequence identity between mouse and ray is 81%, and between mouse and
îi-uitlly is 61%. The product of SEC9 gene is the yeast counterpart 01 SNAP-25
(Brcnnwald et al., 1994). SEC9 was originally identified as one of the ten latc-acting SEC
gencs rcquired for post-Golgi transport (Novick et al., 1981) (Table B2). SEC9 genc
cncodes a hydrophilic 65 1-residue polypcptidc with predicted molecular mass oT 73.628
kDa. The predicted Sec9p amino acid sequence revealed no membrane-spanning domain,
consistent with the structure of SNAP-25. The optimal alignment between Sec9p and
mouse SNAP-25 protein shows 19% identity and 60% similarity in a 206-residue overlap.
Furthemore, cxpression of the SNAP-25-like domain of Sec9p was found to be sufficient
to rescue the temperature-sensitive defect in invertase secretion of yeast mutant sec9-4IS
(Brennwald et al., 1994). Subcellular fractionation studies and immunofluorescence
siaining indicate that the majority of Sec9p is localized to the plasma membrane (Table B 1).
Unlike SNAP-25, Sec9p entirely lacks cysteine residues and c m efficiently be stripped ail
the membranes by alkaline treatments but not by high salt concentrations. These data
suggest that Sec9p is a tightly associated peripheral membrane protein in yeast. Similar to
SNAP-25 which has been shown to form a complex with syntaxin and synaptobrevin,
Sec9p has also been found to associate with yeast homologues of syntaxin, Ssolp and
Sso2p, as well as with the yeast VAMP homologues, Snclp and Snc2p. This once again
supports the hypothesis that the docking and fusion apparatus is conserved from yeast to
marnmals.
Like syntaxin, SNAP-25 is a target substrate for three Botulinum neurotoxin
(BoNT) serotypes: A, E, and C (Schiavo et al., 1993; Blasi et al., 1993a; Binz et al.,
1994; Lawrence et al., 1994; Boyd et al., 1995; Pellegrini et al., 1995; Schmidt and
Bostian, 1995; Foran et al., 1996) (Fig. B2). There has been one study describing the
susceptibility of SNAP-25 to cleavage by BONTIC which was initially identified as the
cndopeptidase that targets syntaxin. However, the cleavage site of BoNTIC on SNAP-25
rcmains to bc dcfined. In contrast, the cleavage sites for BoNTIA and BoNTE were well
characterized. BoNTIA catalyses the hydrolysis of the ~ l n 1 9 7 - ~ r g 1 9 8 bond, whilc
BoNT/E cleaves the ~rglaO-1lel81 peptide linkage, generating soluble peptide fragments of
9 and 26 amino acids in length rcspectively (Schiavo et al., 1993; Binz CL al., 1994;
Lawrence ct al., 1994). Cleavage of SNAP-25 by either type A or E results in an inhibition
of ncurotransmitter release from isolated nerve terminals (Blasi et al., 1993a), a blockade in
the potassium-stimulated, calcium-dependent insulin release Irom pancreatic p cells (Boyd
ct al., 1995), as wcll as an inhibition on catecholamine secretion by adrenal chromaffin
cells (Gutierrez et al., 1995). SNAP-25 antibodies and peptides that contain the cleaved
fragment by BoNT/A at the carboxyl-terminal domain of SNAP-25 inhibit ca2+-evoked
glutamate release by synaptosomes (Mehta et al., 1996), and block ca2+-dependent
catecholamine release from digitonin-permeabilized chromaffin cells (Gutierrez et al.,
1995). Furthermore, it has recently been proposed that the step at which SNAP-25 peptide
interferes in the calcium-dependen1 secretion from permeabilized chromaffin cells is that of
vesicle docking (Gutierrez et al., 1997). Taken together, the different experimental
approaches shown above which interfered with the normal function of SNAP-25 lead to an
inhibition of regulated exocytosis. Other than a critical role in vesicle docking and fusion,
SNAP-25 has also been demonstrated to participate in axonal growth. Blocking of SNAP-
25 expression by incubating rat cortical neurons and PC12 cells with SNAP-25 antisense
oligonucleotides prevents neurite elongation and outgrowth (Osen-Sand et al., 1993).
A Coloboma (Cm) mutant mouse mode1 has been identified, which has helped to
access the impact of SNAP-25 on nervous system function. The mutation is a 1-2 CM
deletion on mouse chromosome 2 that includes the entire SNAP-25 gene (Snap), but may
encompass as many as 30-40 additional linked loci (Hess et al., 1994; Wilson et al., 1996).
When homozygous, this gene deletion is embryonically lethal; however, heterozygous mice
arc viable and exhibit phenotypic abnormalities including ocular dysmorphology, constant
hcad bobbing and profound spontaneous hyperactivity (Searle, 1966; Hess et al., 1994).
Cm/+ micc exprcss 50% levels of SNAP-25 mRNA and protein. Thc involvement of
SNAP-25 in the dysregulation of locomotor behaviour resulting from the Cm gene defect
was evaluated by genetic complementation with a Snap minigene (Sp). Thc locomotor
activity of Coloboma mutants homozygous for the Sp transgene (genotype SpISp, Cm/+)
was rescucd to virtually normal levels. The ability of a Snup transgenc to compkment thc
hyperactivity associated with the Cm gene defects supports the idea that this behavioral
abnormality results from a deficiency of SNAP-25 in promoting the vesicle releasc at
synapses (Wilson et al., 1996). Identification of the mouse mutant Coloboma as an animal
mode1 for examining the neuro-biochemical basis for hyperactivity will have important
implications in understanding hyperactive syndrome such as attention deficit hyperactivc
disorder (ADHD) which affects 3-596 of school-aged children.
V-SNAREs
VAMP
Vesicle-Associated Membrane Dotein (VAMP), also known as synaptobrevin, -
represents a family of integral membrane proteins of synaptic vesicles that were initially
discovered from the electric organ of the marine ray Torpedo californica (Trimble et al.,
1988) and subsequently from rat brain (Baumert et al., 1989; Elferink et al., 1989). So
far, three mammalian VAMP proteins have been characterized: VAMP-1, VAMP-2
(Elferink et al., 1989) and cellubrevin (McMahon et al., 1993). VAMP- i consists of 1 18
amino acids (MW = 12.760 kDa) and is 84% identical to Torpedo VAMP-1 protein
sequence. VAMP-2 is 116-residue in length (MW = 12.671 kDa) and is 77% and 75%
identical to rat and Torpedu VAMP-1 sequences respectively. Cellubrevin contains 103
arnino acids and shares 59% identity to both rat VAMP-1 and -2. Three structural domains
were found common in VAMPS and cellubrevin (Trimble et al., 1988; Elferink et al., 1989;
McMahon et al., 1993; Trimble, 1993). Each member contains an amino-terminal prolinc
rich scquence which is the most divergent region; a well-conserved hydrophilic corc which
spans about 70 amino acids; and a hydrophobic carboxyl-terminal membrane-spanning
rcgion that is composed of 22 residues. Tryptic digestion of intact and lysed synaptic
vcsicles suggests that the protein is cytoplasmically-orientcd (Trimble ct al., 1988). Likc
syntaxin and SNAP-25, al1 members of the VAMP family exhibit a coiled-coi1 rcgion
which involvcs a stretch of 30 residues within the hydrophilic core as illustrated in Fig. BI.
Thc coilcd-coi1 structure is believed to be important for the interaction of SNAP-25
(Chapman et al., 1994) and syntaxin (Calakos et al., 1994) with VAMP.
When VAMP-1 was first isolated and characterized from Torpedo, it was found to
bc expressed only in neuronal tissues such as the brain and purified electric-organ synaptic
vesicles (Trimble et al., 1988). Later searches for mammalian counterparts in rat brain
identified three related proteins. Synaptobrevin which was isolated from rat brain (Baumert
et al., 1989) is identical to VAMP-2 (Sudhof et al., 1989). VAMP-related gene products
were also cloned from bovine brain, Drosophila (Sudhof et al., 1989; Chin and Goldman,
1992; DiAntonio et al., 1993; Sweeney et al., 1995), human (Archer et al., 1990), squid
(Hunt et al., 1994), Aplysia (Yarnasaki et al., 1994), and yeast (Protopopov et al., 1993).
The data on the distribution of VAMP proteins are somewhat inconsistent. In the study
which described the cloning of rat VAMP-1 and VAMP-2, both mRNAs were detected
only in the central nervous system but not in non-neural tissues exarnined which includcd
liver, muscle, kidney and heart (Elferink et al., 1989). Subsequent studies on the
expression of VAMP-2lsynaptobrevin concluded that the protein was detected in
ncuroendocrine cells and ce11 lines such as endocrine pancreas, insulinoma cells, adrenal
medulla, PC12 cells, but not in other tissues such as muscles and exocrine pancreas
(Baumert et al., 1989). However, more recent studies have dernonstratcd that
VAMP/synaptobrevin isoforrns 1 and 2 are widely expressed in rat tissucs including brain,
kidncy, heart, and smooth rnusclc (Rossctto et al., 1996). Morcovcr, VAMP-2 has bccn
shown to bc prcsent in insulin-responsive cells such as adipocytes and skclctal musclc
(Cain ct al., 1992; Volchuk et al., 1994; Jagadish et al., 1996; Martin et al., 1996; Timmcrs
ct al., 1996), as well as secretory cclls including ncutrophils (Brumcll ct al., 1995) aiid
cxocrinc pancrcatic acinar cclls (Braun ct al., 1994; Gaisano ct al., 1994). Thc discrcpancy
may bc duc to the mcthods used for detection andor thc purity of tissue sarnplcs. Al1 in all,
IL 13 SLUL LU LVIILLUUC w a L v ~ I V I T - L 13 U U I ~ U I L U U ~ I ~ C A ~ I C ~ ~ C U , LGIIUULGVIII ulay I ~ J I L ~ L I I L a
non-neuronal VAMP isoform since it is virtually undetectable in neurons although it is
prcscnt in glial cells in the brain (McMahon et al., 1993; Chilcote et al., 1995); VAMP-1
distribution may be rcstricted to neural and endocrine cells.
Several VAMP-related genes were identified in yeast and they include SNCl (Gerst
et al., 1992), SNC2 (Protopopov et al., 1993), SLYl/SEC22 (Dascher et al., 1991;
Newman et al., 1992a), BETl and BOS1 (Newman et al., 1992b; Lian and Ferro-Novick,
1993). Snclp and Snc2p shared 79% identity to each other and 30-40% identity to
mammalian VAMPs. Both of the Snc proteins are constituents of post-Golgi transport
vesicles. Boslp, Betlp and Sec22p/Sly2p proteins al1 show limited sequence similarity
and share structural features with VAMP. These yeast proteins have been shown to be
rcquired for ER to Golgi transport (Dascher et al., 1991; Newman et al., 1992b; Lian and
Ferro-Novick, 1993). Summaries of the subcellular localization of VAMPs and the
transport step at which each VAMP protein functions are provided in Tables B1 and B2
rcspec tively .
Likc SNAP-25 and syntaxin, rnembers of the VAMP family are also cleaved by
different Clostridial neurotoxins: BoNTIB, D, F, G and tetanus toxins (Tetx) (reviewed by
Niemann et al., 1994). These toxins have been shown to cleave VAMWsynaptobrcvin at
distinct peptide bonds and thcrefore becorne important tools in dissccting thc functional
importance of VAMPs in vesicle traffic. Injection of tetanus toxin or BoNTIB into Aplysiu
ncurons has bccn dcmonstrated to block neurotransmitter rclcasc, and the blockadc is
substantially dclaycd with peptides containing the synaptobrevin-2 cleavage site (Schiavo ct
al., 1992). Microinjcction of TeTx and BoNTIB light chain into thc squid prcsynaptic
terminal clfccts an irreversible inhibition of neurotransmitter relcase (Hunt ct al., 1994). In
adrcnal chromaîlïn cells, inhibition of catecholamine rclease correlatcs with clcavagc of
synaptobrcvin and cellubrevin by BoNTIB (Foran et al., 1995). Furthcrrnorc, trcatmcnt of
permeabilized insulin-secreting cells with TeTx or BoNTB, which selectively cleave
VAMP-2 and cellubrevin, causes an inhibition of Ca2+-triggered insulin exocytosis
(Rcgazzi et al., 1995; Regazzi et al., 1996; Wheeler et al., 1996). In Drosophila, gcnctic
mutations removing synaptobrevin have not yet been reported. An alternative approach of
expressing the catalytic domain of tetanus toxin in the nervous system of transgenic fly was
used to delete the synaptobrevin protein (Sweeney et al., 1995). The loss of synaptobrevin
in such transgenic fruitfly completely eliminates synaptic transmission. Moreover, yeast
lacking both Snclp and Snc2p are defective in normal bulk secretion, accumulate large
quantity of invertase-containing post-Golgi vesicles, and display a variety of conditional
lethal phenotypes which are fully reversible upon the expression of either SNCl or SNC2
from plasmids. Disruption of either SNC2 or SNCl but not both genes was found to be
without phenotype, suggesting that the two may play functionally redundant role in post-
Golgi transport. Thus, yeast require at least one SNC gene to germinate and grow
normally (Protopopov et al., 1993).
Svnantotagmin
Synaptotagmin 1 (p65) is an intrinsic membrane protein found on the synaptic
vesicles (Matthew et al., 1981) with a domain structure that is conserved in vertebrates and
invcrtebrates (Perin et al., 1991a; Perin et al., 1991b). Nine synaptotagmins are currently
known, and they al1 have the same overall domain structure described below (recently
rcvicwcd by Sudhof and Rizo, 1996). Al1 synaptotagmins have a short intravesicular
amino terminus which varies in length between 16 to 60 amino acids and is N-
glycosylated. This is followed by a transmembrane region (22-25 residues) that contains
multiplc palmitoylated cysteines (Chapman et al., 1996). Facing the cytoplasm is a siretch
of highly charged residues which spans from 37 to 212 amino acids and this is immediatcly
Iollowcd by C2A and C2B domains, each of which is cornprised of 1 18- 123 amino acids.
The C2A and C2B domains are well-conserved regions that are homologous to thc C2
regulaiory region found in protein kinase C (Perin et al., 1990). Both C2 domains are
stabilized by ~ a 2 + , suggesting that they both bind ~ a 2 + (Davletov and Sudhof, 1994).
Furthemore, each of the C2A and C2B dornains bind two ca2+ ions with in ths i c
affinities of 60 pM and of 300-400 pM respectively (Shao et al., 1996; Sudhof and Rizo,
1996). These affinities have been shown to correlate with the ~ a 2 + dependence of
exocytosis (Heidelberger et al., 1994), suggesting that synaptotagmin I may function in a
Ca2+-regulated step of the synaptic vesicle cycle (Penn et al., 1990; Brose et al., 1992).
Native brain synaptotagmin 1 binds phospholipids in a Ca2+-dependent manner
(Brose et al., 1992), and this is mediated exclusively by the C2A domain (Davletov and
Sudhof, 1994). No binding of phospholipids to the C2 domains of native synaptotagmin I
is observed without Ca2+ (Li et al., 1995a). Synaptotagmin 1 has also been shown to bind
syntaxin in a Ca2+-dependent fashion and the interaction is rnediated by the C2A domain of
the protein (Li et al., 1995b; Kee and Scheller, 1996). In contrast, p-SNAP and SNAP-25
have been demonstrated to interact directly with the C2l3 domain of synaptotagmin and
these intcractions are not dependent on the presence of Ca2+ (Schiavo et al., 1995a;
Schiavo et al., 1997). Table B3 gives a summary of the molecules that interact with
different tagmins.
An oftcn cited evidence questioning the role of SNAREs as docking proteins comcs
[rom thc obscrvation that synaptic vesicles remain morphologically dockcd at the
prcsynaptic plasmalemma even after cleavage of SNAREs by Clostridial ncurotoxins (Hunt
ct al., 1994; Goda, 1997). As mcntioned prcviously, synaptotagmin 1 (v-SNARE) binds,
both iiî vitro and iiz vivo, to SNAP-25 (t-SNARE) (Schiavo et al., 1997). Furthcrmorc,
protcolytic clcavagc of SNAP-25 by BoNWA and BoNT/E does not comprornisc thc
binding of SNAP-25 to synaptotagmin. Since synaptotagmin is not clcavcd by any of thc
known ncurotoxins, thc interaction between synaptotagmin and SNAP-25 could bc a
poLent1ai expianarion ror vesicie aocicing omervea in roxin-rrearea presynaplic nerve
terrninals (Goda, 1997).
Different regions of the synaptotagmin protein have various degrees of sequence
conservation. The intravesicular amino-terminus, the transmembrane region and the highly
charged juxtamembrane sequence are conserved between synaptotagmin 1 and LI but varied
among other synaptotagmins (reviewed in Sudhof and Rizo, 1996). In contrast, the
sequence from C2A to C2B domains i s well conserved among a11 nine synaptotagrnins,
indicating that the function of C2 domains are conserved among al1 synaptotagmins. The
synaptotagmins not only show varying degree of sequence conservation, they also display
distinct pattern of tissue distribution. Al1 synaptotagmins are highly ennched in brain. At
least six synaptotagmins are widely expressed in non-neural tissues as well as the brain
(III, IV, VI-IX) (Hudson and Birnbaum, 1995; Li et al., 1995a; Sudhof and Rizo, 1996).
Synaptotagmin 1, II, and III are localized on synaptic vesicles. Synaptotagmin 1 is also
prcsent on large dense core granules and endocrine secretory vesicles (Matthew et al.,
198 1 ; Walch-Solimerna et al., 1993; Jacobsson et al., 1994).
Unlike other SNAREs, no known ncurotoxins cleave synaptotagrnins. Howcvcr,
other biochemical and genetic approaches have been employed to assess the functional
importance of this V-SNARE protein. Synaptotagmin 1 knockout mice were generatcd and
round to dcvelop normally until after birth but die postnatally, suggesting that
synaptotagmin 1 is csscntial for viability (Geppert et al., 1994). Therefore, in spite of the
availability of other eight synaptotagmins, the function of synaptotagmin 1 is not rcdundant.
Furthcr studies [rom thc hippocampal neurons cultured from thcse micc rcvealcd that
synaptic transmission is severely impaired in the fast phase of Ca2+-dcpcndcnt
ncurotransmittcr rclcase. Surprisingly there is no impairment of the slow asynchronous
componcnt of neurotransmitter release. Moreover, thc frequency of spontaneous miniature
rclcasc evcnts, thc sizc OS the pool of readily releasable synaptic vesiclcs, and thc ratc of
rcplenishment of this pool are also indistinguishable between wild-type and synaptotagmin
1 knockout mice (Geppert et al., 1994; Sudhof and Rizo, 1996). Therefore thesc micc
suffer from the selective loss of the fast component of ~a2+-dependent exocytosis, without
any effect on othcr synaptic vesicle functions. It was proposed that synaptotagmin 1
functioned as a Ca2+ sensor mediating Ca2+ regulation of synchronous neurotransmitter
release in hippocampal neurons. Furthermore, direct evidence that synaptotagmin is a Ca2+
sensor involved in vesicle fusion comes from Drosophila in which synaptotagmin
hypomorphic mutants lacking one of the C2 ~ a 2 + binding domains show a 50% reduction
in the ~a2+-dependence of transmission (Littleton e t al., 1994). It should be noted that
synaptotagmin is by no means the obligatory ~ a 2 + sensor for neurotransmitter release.
This i s supported by the finding, again from Drosophi la , that even though nul1
synaptotagmin mutants are early lethal (DiAntonio et al., 1993; Broadie et al., 1994), Ca2+-
dependent neurotransmitter release is still maintained, indicating that other proteins may
serve a s the ~ a 2 + sensor for release.
Fig. B1 Schematic representation of the predicted coiled-coi1 domains of SNAREs and a-SNAP which mediate protein-protein interactions (modified from Burgoyne et al., 1996).
Higher eukarvote membrane traffic Yeast membrane traffic Famil y Homolog Localization Gene Localization Syntaxin t-SNARE 1 A, lB, 2, 4 PM SSOI, S S 0 2 PM
VAMP/ Synapto- brevin
Synapto- tagniin
NSF
SNAP
V-SNARE VAMP 1 ,2
Cellu brevin
soluble NSF factor
cis-Golgi Golgi
PM
Transport vesicle
Endosorne
Transport vesicle
Cytosol
soluble a Cytosol factor P9 r cytosoi
SEDS Golgi PEP12 Vacuole
SNCl , SNC2 pans-Golgi SEC22/SLY2 ER BOSI, BETl ER
SECl 8
SECl 7
Cytosol
Cytosol
Table HZ Slmilarity between the nerve-terminal and yeast membrane trafic machinery (modified from Bennett and Scheller, 1994).
Hinher eukarvote membrane traffic Yeast membrane traffic Farnil y Homolog Transport step Gene Transport step Syntaxin t-SNARE 1 A, 16, 2, 4 Golgi -> PM S S O l , S S 0 2 Golgi ->PM
5 ER -> Golgi 6 Golgi -> ?
SNAP-25 t-SNARE SNAP-25 Golgi -> PM
VAMP/ V-SNARE VAMP 1,2 Golgi -> PM Synapto- brevin
Cellubrevin Endosorne -> ?
Synapto- V-SNARE 1 - IX tagmin
NSF soluble NSF several factor
SNAP soluble a several factor P 9 Y
SEDS PEP12
SEC9
SNCI, SNC2 SEC22/SLY2 BOSI, BETI
none
SEC1 8
SEC1 7
ER - Golgi Golgi -> vacuole
Golgi -> PM
Golgi -> PM ER -> Golgi ER -> Golgi
several
several
(from Sudhof and ~ i z 6 , 1996). -
Molecules Domain ca2+ dependence Tagmin Comments of tagmin isoform
snidied Ca2+ C2A 60 pM and 400 pM 1
Phospholipids C2A I , I I , I I I , V , V I I = 5 - 1 0 y M TV, VI, VIII, IX = no
Syntaxin C2A 1, II, V = 200 PM III, VI1 = < 1 p M XV, VI, vxn = no
Synaptotagmin C2B 250 pM
IPs InsP6 binds best; InsP4 & InsP5 bind; InsP3 does not bind.
f a l L 11. l V l U U C 1 3 l u 1 V t.blLlf; l d l >'t.L111lZ. UUCI\IIIr. d 1 1 U J? U 3 1 V l l
The secretory pathway in yeast, the constitutive protein transport between Golgi
cisternae in mammalian cells, and the highly regulated synaptic vesicle exocytosis in
mammalian synapses al1 share a similar protein machinery which underlies a diverse
spectrum of intracellular traffic. This strongly suggests that a universal mechanism for
vesicle traffic exists in most eukaryotic cells. In Part 1, 1 have introduced the major players
of the docking and fusion machinery. In fart II, 1 will focus on the mechanism by first
summarizing a general model for vesicle targeting and docking, known as the SNARE
hypothesis, as well as some recent modifications to the hypothesis. The hypothesis was
originally proposed by Rothman and colleagues to explain how synaptic vesicles target and
dock with the presynaptic plasma membrane. Lastly, 1 will end this part by introducing
an often neglected step in exocytosis, the final membrane fusion event.
Vesicle Targeting and Docking
Thc SNARE hvnothesis: a neneral vesicle targetine and dock in^ model
As mentioned in Part 1, since the specificity of the vesicle traffic machinery cannot
be duc to NSF and the SNAP proteins, it may be achieved by the specific pairing between
SNAREs on the vesicle and the target membrane (Sollner et al., 1993b; Rothman, 1994).
Indeed, the interaction among specific SNAREs has becorne the central postulate of the
SNARE hypothesis. Rothman and coworkers proposed that vesicle targeting occurs when
V-SNAREs on the donor membrane bind to t-SNAREs on the acceptor membrane.
Furthemore, Ihc hypothesis predicts that al1 eukaryotic cells should have farnilies of v- and
t-SNAREs, whose members should form cognate pairs on the basis of binding spccilicity
and should bc cssential for vesicle docking in vivo (Rothman, 1994).
Based on the SNARE hypothesis as well as experimental data including in vitro
protcin binding studies, a model for the sequential assembly and disassembly of protein
complexes, shown in Fig. B3, was proposed to correspond to the steps of docking,
activation, and fusion of synaptic vesicles (Sollner et al., 1993a; Bennett and Schcller,
1994; Rothman, 1994). In the first step, a complex between two V-SNAREs on the
vesicle, VAMP and synaptotagmin, and t-SNAREs on the plasma membrane, syntaxin and
SNAP-25, is formed, likely through coiled-coi1 interactions (Fig. B3, step a). A stable
SNARE complex consisting equimolar quantities of syntaxin, SNAP-25, and VAMP has
been isolated from detergent extract of brain membranes (Sollner e t al., 1993a). In the
same report, this SNARE complex was shown to bind to synaptotagmin, which
cosediments at 7s in a glycerol gradient. Therefore, the existence of the SNARE complex,
either freely or as a synaptotagmin-SNARE complex, implies that a V-SN-E can bind
directly to t-SNAREs, which forms a fundamental tenet of the SNARE hypothesis (Sollner
et al., 1993a). Synaptotagmin is proposed to serve as a clamp protein which prevents
fusion from occuring until a signal is given. In the case of constitutive fusion, the simplest
possibility would be that no clamp is present. Indeed, in the constitutive secretory pathway
in yeast, no homologue for synaptotagmin has been identified. The synaptic vesicle protein
synaptotagmin is believed to be a strong candidate for the role as a fusion clamp for thc
following reasons: first, it can interact with the SNARE complex; and more importantly, it
was reported that endogenous synaptotagmin bound to the native synaptic SNARE
complex can be displaced by excess amount of the soluble factor a-SNAP (Sollner et al.,
1993a). The potential cornpetition between a-SNAP and synaptotagmin for a common
binding sitc on the SNARE complex suggests a candidate mechanism for ncgativc
regulation. When neurotransmitter release is required, the clamp dissociates, possibly in
rcsponsc io incrcasc Ca2+ levels, allowing a-SNAP to bind (Fig. B3, step b). Thc
association of a-SNAP with its membrane-bound receptors allow the solublc ATPasc,
NSF, to bind (Fig. B3, step c). This is consistent with the finding that NSF only binds to
SNAPs in the presence of the integral membrane receptors, resulting in the formation of a
multisubunit protein complex with a sedimentation coefficient of 20s when detergent
extracts of Golgi membranes containing SNAP receptors are mixed with exogenous NSF
and SNAPs in the presence of EDTA (Wilson et al., 1992). Stable 20s particles can also
be formed in the prcsence of either non-hydrolysable analog of ATP, Mg-ATP-yS, or ATP
in the absence of magnesium (Wilson et al., 1992; Sollner et al., 1993a). Upon hydrolysis
of ATP by NSF, the interactions among NSF, SNAPs and the SNAP receptors are
disrupted (Fig. B3, step d). The multisubunit complex has been shown to rapidly
dissociate in the presence of Mg-ATP even at O OC, liberating NSF in a process that
requires ATP hydrolysis (Wilson et al., 1992). The potentid conformational changes
generated by ATP hydrolysis rnay either lead directly or indirectly to membrane fusion
(Fig. B3, step e). It should be emphasized that this Iast step is purely a speculation. As
Rothrnan and Wieland indicate in a recent review article, this is the simplest possibility, and
it remains unknown whether the physical fusion of bilayers following the NSF ATPase
reaction is the direct result of the action of conforrnational~y switched SNAP, SNARE
proteins, or both (without the involvement of additional proteins) (Rothman and Wieland,
1996).
VAMP
aSNAP NSF
B 1
Fig. B3 Proposed mode1 for the docking and fusion of synaptic vesicles to the presynaptic plasma membrane at a nerve terminal (from Pevsner and Scheller, 1994~). See text for details.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - , - - - - - - -
Two questions have recently prompted the reassessment of the SNARE hypothesis:
(1) what is the role of synaptotagmin in docking and fusion of synaptic vesicles? (2) is
NSF a fusion protein? Although not directly proven, the bulk of the biochemical and
genctic evidence to date indicate synaptotagmin 1 as part of the ca2+ sensor for triggering
synaptic vesicle fusion (Geppert et al., 1994; Sudhof and Rizo, 1996; Goda, 1997). In
order to trigger the final stage of the fusion reaction, synaptotagmin I is required at the site
of exocytosis (Sudhof, 1995). According to the SNARE hypothesis, this would bc
impossible since synaptotagmin is displaced from the core complex upon binding of a-
SNAP, long before fusion occurs as shown in Fig. B4, panel (a). In addition, kinetic
studies on neurotransmission suggest that the speed of ca2+-triggered exocytosis of
synaptic vesicles (<200 ps) is too fast for a complex multistep reaction (Sudhof, 1995)
Again, the SNARE hypothesis, as shown in Fig. B4, panel (a), proposes that ca2+ influx
occurs prior to the displacement of synaptotagmin by a-SNAP (Rothman, 1994).
Moreover, two reports demonstrate that vesicle fusion lags behind Ca2+ influx by only 60
p.s at physiological temperatures (Bruns and Jahn, 1995; Sabatini and Regehr, 1996). The
cxceedingly rapid induction of fusion, therefore, presents a formidable mechanical
constraint to the fusion machinery, leaving no time for sequential enzymatic reactions
(Goda, 1997), such as the hydrolysis of ATP by NSF.
After docking of synaptic vesicles at the active zone, the vesicles need to go through
ü maturation proccss that makes them competent for fast ca2+-triggercd membranc fusion
(Sudhof, 1995). Such a priming step is suggested by two observations: first, most of the
dockcd synaptic vesicles cannot be triggered to fuse by ca2+ immediately and so thcy
üppcar as not yct competcnt for fusion (Hessler ct al., 1993; Roscnmund et al., 1993);
sccond, during cxtensivc rcpetitivc stimulation, cxocytosis slows down bcforc thc numbcr
of docked vesiclcs declines (Wickelgren et al., 1985) indicating that docking is not a ratc-
limiting step, rather docked vesicles need to go through a rate-limiting priming step before
they are ready to fuse (Sudhof, 1995). Finally, the step at which NSF acts has become
highly controversial (Morgan, 1996). According to the SNARE hypothesis, NSF acts as a
fusion protein at the post-docking stage close to membrane fusion (Sollner e t al., 1993a).
However, evidence also exists to suggest a role for NSF at a post-doclung but pre-fusion
stage (O'Conner e t al., 1994). To reconcile these anomalies, an alternative model (Fig. B4
panel (b)) has been suggested (Sudhof, 1995) whereby docking occurs when synaptic
vesicle protein, VAMP (v-SNARE), forms a complex with two t-SNAREs on the
presynaptic plasma membrane, syntaxin and SNAP-25. This is followed by priming of
vesicles when SNAPs and NSF bind to the SNARE docking complex and upon ATP
hydrolysis, the core complex is disrupted which leads to hemifusion, possibly by rneans of
the amphipathic N-terminus of VAMP. Synaptotagmin then acts a s a ca2+ sensor in
completing the fusion reaction by a ~a2+-triggered interaction with syntaxin. Studies have
shown that syntaxin binding to synaptotagmin 1 is regulated by ca2+, and it requires about
250 p M ca2+ for half-maximal binding (Li et al., 1995b; Kee and Scheller, 1996). This
approximates the ca2+ requirement of synaptic vesicle exocytosis (Sudhof, 1995). The
post-docking, pre-fusion model for the role of NSF (O'Conner e t al., 1994) is consistent
with current information on the kinetic steps in exocytosis (Burgoyne and Morgan, 1995),
the central theme of which is that ~g~+-ATP-dependen t priming occurs before ca2+-
depcndent fusion (Morgan and Burgoyne, 1995a). One caveat against the new model is
that molcculcs other than synaptotagrnin have to act as a clamp to prcvent fusion occurring
randomly. Other regulators have indeed been identified and two of thern will be discusscd
in detail in part III.
As Goda pointcd out in a recent review article, now that we have gaincd much
information about "who's who" in vesicle traffic for the past several years, thc ncxt
challenge will be to sort out "who's doing what" in the field of intracellular traffic (Goda,
1997). This would require design of future experiments to establish the minimal set of
proteins required for fusion, i.e. to find out which of the protein interactions characterized
in vitro are physiologically relevant (Goda, 1997), and which ones are "red herrings".
Once the minimal set of proteins have been identified, it remains to be investigated how the
protein machinery assembly and disassembly are regulated, and more importantly, at what
stage of vesicle docking and fusion does it function. One of the major obstacle for
addressing whether particular components are required for docking and not in fusion is the
difficulty in assessing various States of docked vesicles in synaptic transmission. That is,
some docked vesicles may be functionally docked and be fusion competent, whereas others
are docked but cannot be fused (Hessler et al., 1993; Rosenmund et al., 1993).
UNDOCKED VESICLE UNDOCKED VESICLE
SyntaxinSNAP-25 SyntaxinSNAP-25
DOCKED,VESICLE DOCKEO VESlCLE SNAPs NSF
ATP
Synaptotagrnin ADP + Pi SNAPs
Synaptotagmin
PREFUStON STATE PREFUSION STATE Ir ATP
$- ADP + Pi
SNAPs NSF
SNAREs
MEMBRANE FUSION MEMBRANE FUSION
Fig. 8 4 Two proposed models for synaptic vesicle docking and fusion. (a) A model based on the SNARE hypothesis. (b) An alternative model which differs from the SNARE model in (a) as a result of IWO modifications: the action of NSF at a post-docking and prefusion stage and the role oî synapiotagmin as a Ca2+ sensor for vesicle fusion (from Morgan and Burgoyne, 199%). See text for discussion.
. . -W. . - - - ---- - -b.-.,--
Membrane fusion is ubiquitous throughout biological systems. Membrane fusion is
used to transport materials between different intracellular compartments such as the delivery
of ncwly synthesized membrane-bound polypeptides from the endoplasmic reticulum to
Golgi, then to their target organelles by constitutive exocytosis. In order to respond to
changes in their environment, cells usually undergo regulated exocytosis upon stimulation,
for example, transmitter is released to the synaptic cleft upon depolarization of the
presynaptic nerve terminal; insulin is secreted by pancreatic P cells in response to high
blood sugar. Both constitutive and regulated exocytosis require endoplasmic fusion, in
which the cytoplasm-fàcing leaflets of the membranes fuse (Monck and Fernandez, 1996).
Another type of membrane fusion is ectoplasmic, where the fusing bilayer leaflets face the
extracellular environment (Monck and Fernandez, 1996). Ectoplasmic fusion occurs
during fertilization when the sperm fuses with the egg, and is also used by enveloped
viruses to infect cells. Viral ectoplasmic fusion will not be the subject discussed here, but it
should be noted that it is unique arnong dl the cellular fusion reactions in that the proteins
involved have been identified (White, 1992; Monck and Fernandez, 1996). The extensive
studies on viral fusion have been instructive not only in Our understanding of the
mechanism in ectoplasmic fusion, but also in developing models for endoplasmic fusion
which will be the focus here.
Membrane fusion involves the merging of two membranes thereby establishing
continuity bctween the interior of the compartments. The fusion pore has bcen detccted
clectrically using capacitance measurements in patch-clamped cells (Breckenridgc and
Almcrs, 1987; Zimmcrbcrg et al., 1987; Monck and Fernandez, 1996). Thcsc studics,
mainly using mast cells to study secretory granule fusion with the plasma mcrnbrane, havc
providcd thc following picture of thc exocytotic fusion pore (Monck and Fernandez, 1996):
(1) the fusion porc is observed as an abrupt (4 ms) appearance of a small clectrical
conductance, which is equivalent to a pore size of 1-2 nm in diameter; (2) the pore
undergoes rapid fluctuations (flicker) in conductance without any distinct, stable
conductance levels; (3) during this flicker phase there is a lipid flux through the porc,
suggesting that the secretory granule membrane is under tension and that the dynamic
behavior of the pore might be explained by a changing lipid composition; (4) the flickering
of the fusion pore usually culminates in irreversible expansion of the pore to A 0 nm
diameter and release of the secretory granule contents; ( 5 ) the fusion pore sometimes
closes, leaving an intact secretory granule inside the cell; (6) the closure of the fusion pore
has an unusual, discontinuous temperature dependence, indicative of a process that
depends on lipid composition. Two main conclusions were derived from this sequence of
observations: first, the fusion pore is a predominantly lipid structure; second, the fusion-
porc formation occurs after hemifusion.
The freeze-fracture electron microscopy also revealed a likely precursor of
membrane fusion (Monck and Fernandez, 1996). Following mast ce11 stimulation, the
plasma membrane invaginated in the form of a highly curved cusp or 'dimple', which
spanned the 100 nm gap between secretory vesicles and the plasma membrane (Chandler
and Heuser, 1980). Similar dimples have been seen in other cells, although the sizes were
variablc (Ornberg and Reese, 1981; Schmidt et al., 1983), with a separation of only 5-20
nm at the presynaptic terminal of the neuromuscular junction (Heuser and Recse, 1981).
Similarly, by using atomic force microscopy, a recent report described the images of 'pits'
mcasuring 500-2000 nm in diametcr, cach of which contains 3-20 deprcssions mcasuring
100-180 nm in diametcr, observed at thc apical surface of living pancrcatic acinar cclls
(Schneidcr et al., 1997). Furthermore, the depressions widened to about 200 nm during
arnylasc sccrction and returned to their control size upon cessation of the secrctory
rcsponsc. Changcs wcre observed only in the depressions upon stimulation of sccrction.
The authors proposcd that the depressions correspond to the sites wherc thc exocytotic
fusion pores form during amylase secretion. The mast ce11 dimples measured about 100
nm at their mouths (Chandler and Heuser, 1980), and are, thus similar to the size of the
dcprcssions measured in the acinar cells (Schneider e t al., 1997). However, in mast cclls,
the dimples were observed only upon stimulation whereas the depressions of the pancreatic
acinar cells observed by atomic force microscopy were detected at resting state (Fernandez,
1997). Femandez, in his commentary, suggests that it is possible that part of what is being
imaged by atomic force microscopy i s the hard shell of the underlying cytoskeleton,
therefore the images rnay be more complex than simple topographic representation of
fusion pores (Femandez, 1997).
Observations from some freeze-fracture electron micrographs suggest that a
macromolecular scaffold of proteins brings the plasma membrane into close proximity with
the secretory granule (Monck and Fernandez, 1992; Nanavati et al., 1992; Oberhauser and
Fernandez, 1993). These results led to a scaffold model for exocytotic fusion (Monck and
Fernandez, 1992; Nanavati e t al., 1992), in which the function of the proteins in the
scaffold is to draw the plasma membrane into the dimple shape, probably as a result of an
interaction of the scaffold proteins with the head groups of the lipids (Monck and
Fcmandez, 1994; Monck and Fernandez, 1996). For ectoplasmic viral fusion, the scaffold
is composed of hemagglutinin proteins or other viral proteins (Bentz e t al., 1990; Hoekstra,
1990; White, 1992; Zimmerberg e t al., 1993). It has been suggested that the SNARE
proteins, NSF and SNAPs play a role in targeting the fusion reaction, but as yet, the
protcins involvcd in promoting thc fusion reaction rcmain unknown.
According to thc exocytotic scaffold model (Monck and Fernandcz, 1996) shown in
Fig. B5, thc sequcncc of events leading to the final membrane fusion are as follows: (i) thc
membranes are scparated by a protein scaffold of unknown identity; (ii) in responsc to the
approprialc stimulus (which may involve a rise in ca2+ or presencc of GTP-binding
protcins), the scaffold directs a dimple in the plasma membrane towards the secrctory
granule; (iii) hemifusion of the apposing leaflets occurs spontancously due to attractivc
forces that arc exposed by the membrane curvature and local tensions in the membranes;
(iv) a small pore forms in the stressed common bilayer of the hemifusion structure; at this
stage, the pore can either close or expand; (v) the fusion pore develops into the hour-glass
structure, spanning both membranes; (vi) the pore further expands to allow release of
sccretory products. An important observation provided by patch-clamp measurements of
mast cells was that a fusion pore does not always expand irreversibly but sometimes
closes, leaving an intact secretory vesicle inside the cell (Fernandez et al., 1984). Transient
fusion events have since been reported in other ce11 types including neutrophils (Lollike et
al., 1995) and adrenal chromaffin cells (Robinson et al., 1996). Likewise, some release
events observed in neurons may also correspond to release through a transient fusion porc
(Monck and Fernandez, 1996; Robinson et al., 1996), which allows discharge of the
content of vesicle and then close, obviating the need for membrane merging and subsequent
recycling (Valtorta et al., 1990).
The hemifusion step in the scaffold mode1 (Fig. B5, stage iii) is consistent with the
altcrnativc modcl to the SNARE hypothesis mentioned above. According to the modified
SNARE hypothesis, the hydrolysis of ATP by NSF results in a disruption of the corc
complex which leads to hemifusion, possibly by means of the amphipathic N-terminus of
VAMP (Sudhof, 1995). A possible candidate as a scaffold protein is synaptotagmin 1 since
it can form homo-multimers and the polymerization reaction has a ~ a 2 + depcndence (about
250 PM) (Chapman et al., 1996; Sugita et al., 1996) which approximatcs the ca2+
rcquircment of synaptic vesicle cxocytosis (Sudhof, 1995). For viral fusion, where the
scaffold is composcd of hemagglutinin proteins, the hemagglutinin proteins polymerizc as
trimcrs and the trimers assemble to form a 'collar' within which thc fusion pore forms
(Bentz ct al., 1990; White, 1992; Danieli et al., 1996). The ca2+-triggered polymcrization
of synaptotagmin 1 mirrors thc low-pH induced activation of the viral hcmagglutinin
proteins (Weber et al., 1994). This suggests synaptotagmin 1 as a likely scaffold molecule
for exocytotic fusion, similar to the way in which polymerization of hemagglutinin proteins
causes viral fusion.
(il Inactive scaffold (ii) Dimpling (iii) Hemifusion
/ ScaffoM protein
(vf) Cornpiete fusion Release of secretory products
(VI Lale fusion pore (iv) Eady fusion pore
Fig. B5 Scaffold mode1 for membrane fusion during exocytosis (from Monck and Fernandez, 1996). See text for description of each step.
Part III: Proteins Modulating the Accessibilitv of the SNAREs
An often asked question regarding the theme of universality proposed by the
SNARE hypothesis is the following: how can constitutive fusion machinery also be used
in tnggcred exocytosis? The simplest possibility is that in regulated exocytosis, a molecule
can act as a clamp which prevents the formation of the 20s fusion particle. Upon
stimulation, the clamp will be released so that SNAPs and NSF can associate with the
SNARE complex leading to membrane fusion. Synaptotagmin 1 has been proposed as a
fusion clamp since it can associate with the SNARE complex (Sollner et al., 1993a);
moreover, it cornpetes with a-SNAP for the binding to a common site on the SNARE
complex (Sollner et al., 1993a), hence a candidate mechanism for regulation of the
exocytotic process. Here, 1 will introduce two additional proteins which can serve as
potential regulators of vesicle traffic.
( I ) n-Secl / Muncl8 / rbSecl
SEC1 gene was isolated as one of the ten essential late-acting secretory (sec)
mutants that are involved in the transport between ER and the plasma membrane in ycast
(Novick et al., 1981). Slylp is a homologue of Seclp and is essential for transport
bctween endoplasmic reticulum and the Golgi in the yeast secretory pathway (Pelham,
1993; Sogaard et al., 1994). Unc-18 (Hosono et al., 1992; Gengyo-Ando e t al., 1993)
and Rop (Ras opposite protein) (Harrison et al., 1994) are Seclp homologues in
Caenorhabditis elegans and Drosophila, rcspcctively. The neural homologue of Scçlp in
highcr animals arc refcrrcd to as n-Sec1 (Pevsner et al., 1994b), Munc-18 (Mata ct al.,
1993), or rbSec-1 (Garcia et al., 1994).
An Unc-18 mutant has been shown to inhibit transrnitter release I'rom thc
prcsynaptic nerve terminais (Hosono et al., 1992). Similarly, the Rop nul1 mutant llics dic
at late cmbryonic stages and show numerous pleiotropic defects consistent with a loss of
exocytosis in a number of neuronal and non-neuronal ce11 types (Harrison et al., 1994).
Moreover, viable temperature-sensitve mutations of Rop show loss of synaptic activity in
rcsponsc to Iight stimulus, suggesting that the protein is required for synaptic transmission
(Harrison et al., 1994). Interestingly, overexpression of Rop protein likewise suppresses
synaptic transmission at the neuromuscular junctions (Schulze et al., 1994). These genetic
results suggest a negative regulatory role for vertebrate n-Sec1 in synaptic vesicle refease
(Pevsner et al., 1994b). n-Sec 1 binds to syntaxins 1, 2, and 3, but not 4 (Pevsner et al.,
1994b), and syntaxin but not SNAP-25 or VAMP binds to n-Sec1 (Pevsner et al., 1994a).
Furtherrnore, in the sarne report, n-Sec1 was shown to be absent frorn both the 7 s and 20s
particles, indicating that syntaxin can exists in at least three different States: as part of either
7 s or 20s particles, or with n-Secl. These were proposed to represent three different
intermediates in the vesicle docking and fusion pathway (Pevsner et al., 1994a). Two
alternatively spliced variants of Munc- 18, Munc- 18b and Munc- 18c, were identified in
adipocytes (Tellam et al., 1995). Munc-18b, like n-Secl, only binds to syntaxin lA, 2 and
3 but not 4, while Munc- 18c binds only to syntaxins 2 and 4 (Tellarn et al., 1997). Using
a three-way binding assay, it was shown that Munc-18c, like n-Secl, inhibits the binding
of t- and V-SNAREs, in this case, syntaxin 4 and VAMP-2 (Tellam e t al., 1997).
Thc finding that n-Sec1 inhibits binding of either VAMP or SNAP-25 to syntaxin
lcads to the developrnent of a working mode1 in which n-Sec1 associates with syntaxin and
may regulate vesicle docking by preventing SNAP-25 and VAMP binding to syntaxin. As
the vcsiclc docks, anothcr unknown factor could release n-Sec 1 from syntaxin, which is
followed by thc formation of the 7s complex (Pevsner et al., 1994a) (Fig. B3). Thc high
affinity of n-Sec1 for syntaxin (binding constant in nanomolar range), which is 1000-lold
grcatcr than that of VAMP to syntaxin, suggcsts that the transition of syntaxin [rom an n-
Sec 1-bound state to a vesiclc-bound state is highly regulated (Pevsner et al., 1994a).
A recent study demonstrated that n-Sec1 c m be phosphorylated at two Ser residues
by protein kinase C (Fujita et al., 1996). Phosphorylation prevents interaction of n-Secl
with syntaxin 1 A, and n-Sec 1 proteins that are already bound to syntaxin 1 A cannot be
phosphorylated. Thcse results imply that phosphorylation of n-Sec 1 may block its rc-
interaction with syntaxin, after it dissociates from syntaxin 1A (Fujita et al., 1996);
however, phosphorylation cannot be the mechanism to release n-Sec1 frorn syntaxin since
bound n-Sec1 cannot be phosphorylated. It remains to be determined how the interaction
between n-Sec 1 and syntaxin is regulated.
(2) Rab
Rab represents a family of more than 30 members of low molecular mass GTP-
binding proteins in mammals. Al1 Rab proteins are intrinsically hydrophilic, but are
attached to membranes via C-terminal isoprenoid (geranyl-geranyl) and fatty acid chains
(Rothman, 1994). Individual members of the Rab farnily localize to specific subcellular
compartments and are implicated at essentially al1 steps of membrane transport that have
becn studied (for recent reviews. see Pfeffer, 1992; Ferro-Novick and Novick, 1993 ;
Zcrial and Stenmark, 1993). For example, in the endocytic pathway, early and late
cndosomcs each contain a unique set of Rab proteins, Rab4 and Rab5 on early endosorncs
and Rab7 and Rab9 on late endosomes (Gruenberg and Maxfield, 1995). Synaptic vcsicles
contain at least two Rab proteins: Rab3A and 3C, which are specific for synaptic vcsiclcs
and sccrctory granulcs (Fischcr von Mollard ct al., 1994). Rab3A is the most abundant
Rab protein in thc brain, accounting for approximately 25% of the Rab GTP-binding
capacity of this tissue(Geppert et al., 1994). Most neurons express Rab3A, and a
subpopulation of ncurons also synthesize high levels of Rab3C (Li ct al., 1994). Rab
hornologucs havc also been ideniified in yeast: Ypt I p controls the endoplasmic reticulum
to Golgi transport (Segev et al., 1988; Segev, 1991), whereas Sec4p is involved in thc
transport between Golgi and the plasma membrane (Goud et al., 1988).
The findings that many of the Rab proteins are localized to specific intracellular
membrane compartments and are irnplicated in intracellular membrane regulation led to the
suggestion that they might provide an additional level of targeting specificity possibiy
through interactions with the SNAREs (Sogaard et al., 1994). In the mammalian system,
the only evidence which suggests that Rab proteins may associate with the SNARE
complex cornes from the finding that using an antibody which recognizes both SNAP-25
and Rab 3A, a complex containing syntaxin 1 and VAMP, which were detected by
Coomassie blue staining, was precipitated from bovine brain extracts (Ishizuka et al.,
1995). This observation could result from one of two situations: either the Rab 3A indeed
forms a complex with the SNAREs or that SNAP-25, but not Rab 3A, i s responsible for
coprecipitating syntaxin 1 and VAMP Therefore the apparent detection of Rab 3A as part
of the SNARE complex is due to a lack of specificity of the antibody. Using an antibody
that recognizes specifically syntaxin 1A and lB, Rab 3A was not found in the coprecipitate,
suggcsting that the latter explanation was probably true and Rab 3A is not part of the
SNARE complex. In yeast, interactions between Rab homologues and the SNAREs have
bccn well described by genetic and biochemical approaches and they are summarized bclow
(Henry et al., 1996).
Although both yeast Rab proteins, Sec4p and Yptlp are essential gene products for
transport in yeast, a number of proteins have been idcntified which when overexprcssed
can compensatc for the loss of Rab function in yeast (Dascher et al., 1991; Brennwald ci
al., 1994). Ovcrcxpression of SEC9, a yeast gcne cncoding a homologuc of the 25 kDa
synaptosomal-associated protein (SNAP-25), was found to suppress a mutation in thc
cl'l'cctor domain of Scc4p, which is a Golgi-to-plasma membrane Rab protein (Brcnnwald
ct al., 1994). Since overexprcssion of downstream components can often suppress defccts
-r""'-" r*'-"'"' \---a-, -, , Li / y . w-. ...ACT ..CI.....--. V a .a.- -CI - .y ...w*--..--- .---r .--*---
of the suppression, these results are consistent with the idea that Sec9p acts downstrcam OC
Scc4p in the exocytic pathway (Brennwald et al., 1994), although the results do not
excludc the possibility that Sec4p and Sec9p represent independent parallel pathways which
lead to the sarne effect. In the same study, the investigators could not cross-link or
coimmunoprecipitate Sec4p with SecBp, indicating that Sec4p is not part of the
corresponding SNARE complex; therefore, regulation of SNAREs by Rabs may require
the action of intermediate components (Brennwald et al., 1994).
A mutation affecting the function of Yptlp, which controls the endoplasmic-
reticulum-to-Golgi transport, inhibited the assembly of the corresponding SNARE complcx
(Lian ct al., 1994; Sogaard et al., 1994). Temperature-sensitive mutants of Ypt lp were
identified, and inactivation of Yptlp by shifting to restrictive temperature prevented the
accumulation of the SNARE-containing complex of Bos l p (v-SNARE of yeast
endoplasmic reticulum-to-Golgi transport vesicles) and Sed5p (t-SNARE of endoplasmic-
rcticulum-to-Golgi transport) (Sogaard et al., 1994). These results clearly imply that Yptlp
hnction is required for the assembly of the endoplasmic reticulum-Golgi SNARE complex.
Since Yptlp protein is not itself found in the SNARE complex, Yptlp is not likely to
contribute a core interaction necessary for the stability of this docking complex (Sogaard et
al., 1994). Though important, the requirement for Yptlp can nonetheless be by-passcd by
overexprcssing certain V-SNAREs (Dascher et al., 1991). In the absence of functional
Yptlp, thc two cndoplasmic reticulum-Golgi V-SNAREs Bos l p and Scc22p fail to form a
complex (Lian ct al., 1994), hencc, functional Yptlp promotes thc formation of thc Boslp-
Scc22p complex. Moreover, disruption of YPTl was efficiently by-passcd when SEC22
was CO-ovcrcxprcssed with B O S I . The invcstigators hypothesize that Scc22p aids thc
activity of Boslp on vesiclcs, allowing Boslp to intcract more cfficicntly with its rcccptor
or t-SNARE (proposcd to be SedSp). Sincc Yptlp does not directly intcract with Bos1 p
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promoting the oligomerization of the V-SNAREs on transport vesicles (Lian et al., 1994).
Takcn together, these observations suggested that Rab action must somehow
facilitate the assembly of the SNAREs. Because they form no part of the isolated docking
complex containing SNARE proteins, Rab proteins may therefore catalyze unique v- and t-
SNARE interactions (Rothrnan, 1994), thereby contributing an additional layer of targeting
specificity. Although highly suggestive, these studies have not yet demonstrated a direct
role for Rab proteins in SNARE complex formation. It remains to be revealed the stage at
which Rab proteins function during vesicle docking and fusion.
Part IV: Role of SNAREs in Ins~lin~Stirnulated Glucose Transuort
Glucosc is thc main encrgy sourcc for most marnmalian cclls. In thc fasting statc,
glucose is released into the circulation from the liver as a result of the breakdown of
glycogen. After a meal, the blood sugar level rises, stimulating the pancreatic 0 cells to
release insulin into the circulation. The post-prandial clearance of glucose from the
circulation is tightly regulated by insulin, which clears blood glucose in two ways: first, to
prevent the liver from releasing more glucose; second, to cause hepatocytes, muscle and fat
cells to absorb glucose from the circulation. In hepatocytes and muscle cells, glucose is
stored as glycogen, whereas in adipocytes, glucose is converted to triglycerides for long-
term storage. As the blood glucose level drops, the pancreatic B cells stop secreting
insulin, and the body's metabolism returns to basal state.
Since the lipid bilayer of the plasma membrane is impenneable to glucose, the
uptake by muscle and fat cells must be facilitated by specific transporter proteins called
GLUTs (GLUcose Dansporters). A farnily of six facilitative glucose transporters (GLUTs
1-5, and 7) has been characterized (GLUT6 is a pseudogene), which transport glucose
down its chernical gradient. The GLUT farnily is characterized by single polypeptides of
about 500 amino acids in length, each of which has 12 predicted membrane-spanning
helices (reviewed by Gould and Holman, 1993; James, 1995; Taha, 1996; Klip and
Marette, 1997). The GLUTs are expressed in a tissue-specific manner. Expression of
GLUT4, for example, is confined to ce11 types that exhibit insulin-stimulated glucose
transport, such as adipose tissue, skeletal and cardiac muscles (James et al., 1988;
Birnbaum, 1989; James et al., 1989; James, 1995). In contrast to the tissue-specificity of
GLUT4, GLUTl is expressed in many cells throughout the body, including the insulin-
responsive tissues, but at much higher levels in endothelial çells within the brain, placenta,
eyc and testis (James, 1995).
mneLiç sLuuies 01 giuwse upLaKe inw la1 ariu IIIUSL~C S U ~ ~ G S L LIiaL ~ I I S U I ~ H ~ I I L I C ~ S C S
the V,, of glucose transport (Bimbaum, 1989; Gould and Holman, 1993; Stephens and
Pilch, 1995). In 1980, two independent studies by Cushman and Wardzala, and Suzuki
and Kono, first demonstrated an increase in glucose transporter number at the ce11 surface
in response to insulin. Glucose transporter distribution was assessed in plasma membrane
fraction and rnicrosornes of rat fat cells that were isolated before and after exposure to
insulin (procedure described by Simpson et aI., 1983). Cushman and Wardzala used a
labeled fungal metabolite, cytochalasin B, which binds directly to glucose transporters, to
assess the number of transporters in the membrane fractions (Cushman and Wardzala,
1980). Suzuki and Kono measured glucose uptake in isolated membrane fractions from rat
adipose cells (Suzuki and Kono, 1980). Both groups reached the same conclusion, that in
unstimulated adipose cells most of the glucose transporters (now known to be
predominantly GLUT4) are associated with an intracellular (microsomal) membrane
compartment. Upon exposure to insulin, the transporters are 'recruited' from an
intracellular site to the ce11 surface. These results led to the transporter recruitment
hypothesis, which postulates that an increase in the number of functional transporters on
the surface is the major mechanism for insulin stimulation of glucose transport in both fat
and muscle tissues (Stephens and Pilch, 1995). Therefore, when the insulin level is low,
the hypothesis predicts that most of the glucose transporters are sequestered intracellularly.
Whcn thc levcl of insulin is high, these transporters are mobilized from the intraccllular
compartment to the ce11 surface to mediate the influx of glucose.
The cloning and characterization of different isoforms of glucose transporters as
well as the availability of isoforrn-specific antibodies, allow re-examination of the insulin-
stimulalcd glucose uptake by rat adipocytes. Immunoblotting and photolabeling studies
showcd that native rat adipocytes cxpress glucose transporter proteins, GLUTf and
GLUT4. Thc level of GLUTl is about 5-10% that of GLUT4 (Oka et al., 1988; Zorzano
-.- --., * - --, ---------- -- --., - - - -, -------- -.-- - a----, - - - V,. --- -- --Y ------- ---mm---
abundancc, GLUT4 is by far thc prcdominant spccics rcsponsiblc for thc 20-30-fold
increase in glucose transport as a result of exposure of fat cells to insulin (Whitesell and
Gliemann, 1979: Taylor and Holman, 1981; May and Mikulecky, 1982; Simpson and
Cushman, 1986; Holman et al., 1990). In unstimulated adipocytes, more than 95% of
GLUT4 is located intracellularly (Rice et al., 1996) whereas more than 50% of GLUTl
resides at the plasma membrane, with the rest being sequestered inside adipocytes (Kandror
et al., 1995). The ubiquitous distribution of GLUTl in many tissues and its preferential
localization at the ce11 surface in unstimulated rat adipocytes suggests its function in
maintaining the growth and metabolism of a cell. GLUT4, on the other hand, is expressed
only in insulin-responsive celIs and the majority of GLUT4 is sequestered intracellularly in
unstimulated cells. These two characteristics of the distribution of GLUT4 together with
the observations that insulin causes an increase in the ceII surface transporter number
(Cushman and Wardzala, 1980; Suzuki and Kono, 1980) strongly suggest that insulin-
stimulated glucose influx is rnediated predominantly, if not entirely, by the translocation of
GLUT4 to the plasma membrane. It should be noted that the intracellular GLUTl pool,
although less significant, is also translocated to the ce11 surface after insulin administration,
just likc the GLUT4-containing vesicles (Zorzano et al., 1989; Kandror et al., 1995;
Kandror and Pilch, 1996).
Skcletal muscle is the major site for whole body insulin-stimulated glucose
disposal, whercas adipocytes contribute to a relatively minor degrec in normal individuals
(Kraegcn et al., 1985); thcrefore, effort has been made to charactcrizc thc mcchanism of the
insulin-depcndent glucose transport activation in this tissue. Despite considerablc technical
difficultics in the fractionation of skeletal muscle, it has been shown that the major
molccular mechanism of insulin action, namely the recruitment of GLUT4 protcin to the
LLIl r > U L L U b b 13 L 1 1 b JUlIIb Ill L U I . UllU L L l U ù b l b \ A \ l l p b L CU., A / U 1 , U U U b L L b L W., I / / W , l l l L J l L l L l U l l
ct al., 1990; Rodnick et al., 1992; Kandror and Pilch, 1996).
The mechanism by which insulin stimulates glucose uptake by fat and muscle has
been studicd vigorously due to its clinical relevance in diabetes mellitus. There are two
major categories of the disease: Insulin-Dependent Diabetes Mellitus (IDDM) and Non-
Insulin-Dependent Diabetes Mellitus (NIDDM). The majority of diabetic patients surfer -
from NIDDM. It has been shown that NIDDM patients have a normal complement of
GLUT4, at least in muscle, which is the major target tissue for insulin-stimulated glucose
disposa1 (Kahn, 1992; Livingstone et al., 1996). Therefore defects in translocation of
GLUT4 to the ce11 surface in response to insulin may underlie the insulin resistance
observed in these patients (Rice et al., 1996). There are two phenomena in the
translocation of GLUTs by insulin: first, the signalling pathway responsible for activating
the mobiiization of the intracellularly-sequestered glucose transporter; second, the targeting
and docking of GLUT4-containing vesicles with the plasma membrane in response to
insulin.
Thc insulin signalling pathway starts whcn insulin binds to its rcccptor on thc cc11
surface. The insulin receptor is composed of two extraceliular a-subunits that are linked to
two membrane-spanning P-subunits by disulfide bonds (reviewed by White and Khan,
1994; Taha, 1996). The binding of insulin to the a-subuni t causes an auto-
phosphorylation of the P-subunit on tyrosinc rcsidues, which lcads to an activation of thc
tyrosine kinase of the P-subunit. This is followed by phosphorylation of several substrates
on tyrosinc rcsiducs, of which IRS-1 (Insulin Receptor subs t ra te -1) is thc bcst
characterizcd. IRS- 1 is a cytoplasmic protein with multiple tyrosine phosphorylation sitcs
that, Sollowing insulin stimulation, serves as docking sites for intracellular molecules that
contain specific recognition domains, termed SH2 (Src Hornology 2) domains (Kahn,
1995). Protein-protcin interactions mediated by specific phosphotyrosincs and SH2
U U L I I ~ I I I ~ LVIII I LUC vao13 VI I l l a l l y V I LIIG I I I L G I Q L L I V I I ~ I I I LIIC J ~ ~ I I ~ I I I I I ~ paulway \ vv IIILL QLIU
Khan, 1994; Seaman et al., 1996). A strong SH2-domain-containing candidate protein
directly involved in the GLUT4 translocation pathway is the enzyme Phosphatidyl~nositol
3 -kinase (PI 3-kinase). Pharmacological agents such as the fungal metabolite, -
wortrnannin, and a synthetic inhibitor of PI 3-kinase, LY 294002, which effectively
abolish the activity of the enzyme, also potently block the translocation of GLUTl and
GLUT4 to the plasma membrane (Cheatharn et al., 1994; Clarke et al., 1994; Tsakiridis et
al., 1995), and glucose transport (Cheatham et al., 1994; Okada et al., 1994; Tsakiridis et
al., 1995) upon exposure of insulin to muscle and fat cells. PI 3-kinase contains a
regulatory subunit of 8 5 kDa (p85) whose two SH2 domains interact with the
phosphotyrosines of IRS-1, as well as a 110-kDa catalytic subunit (pl 10) which
phosphorylates inositol lipids on the D-3 position, converting ~hosphat idyl~os i to l (PI),
PI-4-monophosphate and PI-4,s-bisphosphate to PI-3-monophosphate, PI-3,4-
bisphosphate and PI-3,4,5-trisphosphate respectively (for recent review, see Taha, 1996;
Klip and Marette, 1997). The physiological significance of the production of D-3-
phosphorylated phosphoinositides remains to be determined.
Although necessary, the activity of PI 3-kinase is not sufficient to stimulate GLUT4
translocation in response to insulin. This is suggested by the observation that other growth
factors such as Platelet-oerived Growth Factor (PDGF) and interleukin 4, which are potent
activators of PI 3-kinase, do not stimulate glucose transport (Isakoff et al., 1995; Sale et
al., 1995). Sincc insulin promotes the association of activated PI 3-kinasc with
intraccllular membranes, whereas PDGF causes association of the kinase with the plasma
mcmbranc (Navc et al., 1996; Ricort et al., 1996), it was proposed that the unique
localization of PI 3-kinase activated by insulin in part determines its effect on the
translocation o î glucose transporters (Klip and Marette, 1997). This hypothcsis is
supported by a reccnt finding that after a short exposure of insulin to fat cells,
immunoisolated intracellular GLUT4 vesicles contain PI 3-kinase-iRS- 1 complex (Heller-
Harrison et al., 1996). From this observation, it is speculated that arriva1 of PI 3-kinase to
the GLUT4-containing vesicles primes them for translocation (Klip and Marette, 1997).
Future experiments are needed to address the consequences after priming of GLUT4
vesicles. Ultimately, the insulin-signalling pathway leads to the mobilization of the
GLUT4 proteins from an intracellular compartment to the ce11 surface.
The second site of regulation in the process of transporter recruitrnent is the
targeting and docking of GLUT4-containing vesicles with the plasma membrane. The
SNARE hypothesis, which predicts that al1 eukaryotic cells should have families of v-and t-
SNAREs, to form cognate pairs on the basis of binding specificity (Rothman, 1994),
allows easy testing for the relevance of the mode1 in insulin-regulated GLUT4 traffic.
Several SNARE proteins have since been identified in muscle and fat cells after the initial
proposal of the SNARE hypothesis, and they are summarized below.
V - S N A R E s
VAMPS
In an attempt to search for similarities between the insulin-regulated exocytosis of
GLUT4 and transmitter release at synapses, Cain et al. screened the GLUT4-containing
vesicles isolated from rat adipocytes for proteins that were found on synaptic vesicles by
immunoblotting (Cain et al., 1992). Two proteins immunologically related to VAMPs
wcre found. In response to insulin, the amount of VAMPs in the intracellular membranc
compartment reduced with a concomitant increase in the plasma membrane. The same
cffect was observed on the distribution of GLUT4 protein in response to insulin (Cain ct
al., 1992). Subsequent studies have confirrned that the two VAMP-related immunoreactive
species are VAMP-2 and cellubrevin/VAMP-3 that are present on the GLUT4 vesicles
(Volchuk et al., 1995; Tamori et al., 1996), and in the intracellular membrane fraction of
3T3-Ll adipocytes (Jagadish et al., 1996; Martin et al., 1996) and rat adipocytes (Timmers
ct al., 1996). VAMP-2 and ccllubrcvin havc also bcen reported to bc prcscnt in rat skclctal
muscle and cultured L6 myotubes, and they are found to be preferentially enriched in the
intracellular membrane compartment (Volchuk et al., 1994). The functional importance of
the VAMPS in insulin-regulated GLUT4 exocytosis is assessed by incubating
permeabiIized 3T3-L1 adipocytes with BoNTID (Cheatham et ai., 1996) or BoNTIB
(Tamori et al., 1996) that cleave VAMP-2 and cellubrevin. Cleavage results in an
inhibition of insulin-stimulated translocation of GLUT4-containing vesicles to the plasma
membrane and of glucose uptake (Cheatham et al., 1996; Tamori et al., 1996).
Furthemore, incubation of perrneabilized adipocytes with recombinant soluble VAMP-2
protein, which interferes with the association of native membrane-bound VAMP-2 with
other SNARE proteins that are necessary for vesicle docking to occur, also effectively
blocks the insulin-dependent translocation of GLUT4 to the ce11 surface (Cheatham et al.,
1996).
Svnaptotagmins
Several synaptotagmin isoforms (tagmins) are expressed ubiquitously in different
tissues, of which synaptotagmin IX (originally reported as synaptotagmin-5) was identified
as a nonncuronal isoform by screening a rat adipose tissue library with a synaptotagmin 1
cDNA probe (Hudson and Birnbaum, 1995). Synaptotagmin IX mRNA was detected in
brain, adipose and cardiac tissues. In neurons, synaptotagmin 1 serves as a Ca2+ sensor
for ncurotransmitter release (Sudhof and Rizo, 1996). Although therc was no direct
information on thc Ca2+-binding properties of synaptotagmin IX, it was shown that, unlikc
synaptotagmin 1 which demonstrates ~a2+-dependent binding of phospholipids,
synaptotagmin IX lacks this property (Hudson and Birnbaum, 1995). In musclc and k t
cclls, it is gcncrally believed that Ca2+ does not play a significant role in insulin-stimulatcd
LCLI UILILIGIIL VL È ; I U L V ~ C L L C U L ~ ~ U I L C ~ ~ , L L I C I C ~ U ~ C ~ ~ I I ~ ~ L U L Q ~ I I I I 1~ 13 u l u l ~ u y LU a L L aa a LQ- -
sensor in these cells. The investigators could no1 characterize the subcellular localization ol'
synaptotagmin IX, which would be important in deterrnining its role in GLUT4
translocation, due to its low level of expression and the insensitivity of antisera (Hudson
and Birnbaum, 1995). Furthennore synaptotagmin IX mRNA was found in adipose and
cardiac tissues, but not in skeletal muscle, suggesting that either skeletal muscle expresses
an additional isoform of synaptotagmin, or synaptotagmin IX is unlikely to be important in
insulin-regulated glucose transport (Hudson and B irnbaum, 1995).
t - S N A R E S
Syntaxins
Two isoforms of syntaxin, syntaxins 2 and 4, have been detected in 3T3-L1
adipocytes (Jagadish et al., 1996; Volchuk et al., 1996; Tellam et al., 1997), rat adipose
cells (Timmers et al., 1996), rat skeletal muscle and cultured L6 muscle cells (Sumitani et
al., 1995). Both syntaxins 2 and 4 are enriched in the plasma membrane fraction. Insulin
causes intracellular syntaxin 4 (Volchuk et al., 1996; Tellam et al., 1997), but not syntaxin
2 (Volchuk et al., 1996) to redistribute to the plasma membrane similar to GLUT4. The
prcsence of syntaxins which are supposingly t-SNARE molecules in the intracellular
compartment suggest that the proteins are involved in traffic other than insulin-stimulated
GLUT4 exocytosis. Introducing anti-syntaxin 4 antibodies to 3T3-L1 adipocytes by either
using permeabilized cells or microinjecting into intact cells results in a significant reduction
in insulin-dependent stimulation of glucose transport and recruitment of GLUT4 proteins to
the cc11 surface (Volchuk et al., 1996; Tellam et al., 1997). These results clearly indicate a
rolc of syntaxin 4 in the recruitment of GLUT4 and insulin-dependent stimulation oC
glucose uptake.
I L I U L U V I I L I L I Y I i L W V I I VI L w . V A L I Y & L . V V I U V A L w A u 1 1 W 1 V A U I l l L l J , A I A U I A Y A W U LUlY Y ) 111 d A d Y1
adipocytes (Tcllarn c t al., 1995) suggests a role for these cytosolic protcins in modulating
the accessibility of syntaxins. Munc- 18b, like the neuronal Munc- 18/nsec 1, binds to
syntaxins 1 A, 2, and 3, while Munc 18c binds to syntaxins 2 and 4 (Tellam et al., 1997).
The interaction between Munc- 18c and syntaxin 4 is of particular interest since syntaxin 4,
other than the neural-specific syntaxin 1, is the only syntaxin that interacts with VAMP-2
(Calakos et al., 1994). Using a three-way binding assay, it was shown that Munc-18c
inhibited the interaction between syntaxin 4 and VAMP-2 (Tellam et al., 1997). It is
tempting to propose that in an unstimulated cell, the majority of syntaxin 4 is associated
with Munc- 18c; upon insulin stimulation, Munc-18c dissociates from syntaxin 4 thereby
allowing VAMP-2 on the GLUT4 vesicles to bind to syntaxin 4 on the plasma membrane.
Future studies will have to validate this hypothesis.
SNAP-25
Unlike the SNARE proteins mentioned above where isoforms are found in non-
neuronal tissues, the expression of SNAP-25 is restricted to neural and neuro-endocrine
cells only. According to the SNARE hypothesis, functional equivalents of SNAP-25
should exist outside the nervous system to allow intracellular vesicle traffic to occur in non-
neural tissues. The search for non-neural equivalent of SNAP-25 will be the focus of the
remainder of this Thesis.
The SNARE hypothesis has been proposed as a universal docking and fusion
mechanism for intracellular traffic in eukaryotic cells. The mode1 postulates that vesicle
targeting occurs when vesicle-associated membrane protein, VAMPIsynaptobrevin (v-
SNARE), binds to two proteins on the target membrane, synaptosomal-associated protein
of 25 kDa (SNAP-25) and syntaxin (t-SNAREs). According to the SNARE hypothesis,
eukaryotic ceIls should have farnilies of v- and t-SNAREs whose members f o m cognate
pairs that define the targeting specificity. Indeed, homologues of these proteins have been
identified, some of which are neural-specific, such as VAMP- 1 and syntaxin 1, whereas
some are ubiquitously expressed. The expression of the two alternatively spliced variants
of SNAP-25, SNAP-25a and SNAP-25b, is restricted to neural and neuro-endocrine cells
only. According to the SNARE hypothesis, functional equivalent of SNAP-25 rnust exist
outside the nervous system to allow vesicle traffic in non-neural tissues. Therefore thc aim
of this Thesis is to search for a non-neural equivalent of SNAP-25, as well as to examine
its tissue distribution and subcelIular distribution in non-neural cells.
INTRODUCTION
As discussed in previous sections, the SNARE hypothesis of vesicle traffic predicts
that vesicle targeting occurs when V-SNAREs on the vesicle and t-SNAREs on the target
membrane form a complex which is necessary for vesicle fusion. Moreover, the model
postulates that targeting specificity is deterrnined by the interaction between particular
members of v- and t-SNARE families. In the nervous system, VAMP-1 acts as a v-
SNARE, which binds to two t-SNARE proteins: syntaxin 1 and SNAP-25. Several
isoforms of VAMP and syntaxin have been identified outside the nervous systern;
however, the distribution of two alternatively-spliced variants of SNAP-25, SNAP-25a and
SNAP-25b, is restricted to neural and neuroendocrine cells. If the SNARE hypothesis
were to be a general model for vesicle docking and membrane fusion, functional
equivalents of SNAP-25 should be found in non-neuroendocrine cells. This has prompted
a search for a SNAP-25 like protein as well as to examine the tissues distribution and
subcellular localization of this novel protein in non-neural ceils.
Materials
IMAGE Consortium expressed-sequence tag (EST) cDNA #384468 (GenbankTM
accession number H82169) was obtained from Research Genetics Inc. Human melanoma
cDNA library, mouse melanocyte melan-b and human melanoma MeWo ce11 lines were
generously provided by Dr. N. Lassam (University of Toronto, Toronto, ON). SNAP-25
monoclonal antibody SM18 1 (here called aSN25mAb) was obtained from Sternberger
Monoclonals Inc. Anti al Na+/K+ ATPase monoclonal antibody 6H was kindly provided
by Dr. M. Caplan (Yale University, New Haven, CT). GLUT4 polyclonal antibody was
from East Acres Biologicals. An affinity purified antiserum (aSN25pAb) was raised
against residues 33-206 of SNAP-25 was a kind gift from Dr. M. Wilson (University of
New Mexico, New Mexico) (Schiavo et al., 1993). D-MEM, calf and fetal bovine sera,
and other tissue culture reagents were purchased from GIBCOIBRL. Enhanced
chemiluminescence detection kit was obtained from Amersham.
Methods
RNA Isolation and Northern Blot Analysis
Total RNA was extracted from tissues and cultured cells using the single-step
method of isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
(Chomczynski and Sacchi, 1987). Northern blot analysis was performed according to the
protocol described in (Sambrook et al., 1989). Total RNA was separated by
elcctrophoresis through a denaturing agarose gel containing 1.2% (vlv) forrnaldehyde and
was then transferred to nylon membranes. RNA was fixed to the membranes by baking for
2 h at 80 OC in vacuum. The membranes were prehybridized with denatured, fragrnenied
salmon sperm DNA at a concentration of 200 pglrnl in prehybridization solution (6X
35~12, lun uennarar s reagem, u.370 3 ~ ~ 3 ) . lnls IS wiiwweu DY nymuizawn ur LW--rj
dCTP-labelled cDNA probe at 42 OC in hybridization solution (6X SSPE, 0.5% SDS, 100
pglml salmon sperm DNA, 5% dextran sulfate) in the presence (high stringency) or
absence (low stringency) of 50% formamide. Blots were washed for 20 min at room
temperature in 1X SSC and 0.1% SDS. This was followed by 3 washes of 20 min each at
65 OC in 0.1X SSC and 0.5% SDS. The RNA of interest was detected by
au toradiography.
cDNA Cloning and Sequencing
The database of expressed sequence tags (ESTs) was searched by comparing its
translated contents to the SNAP-25 amino acid sequence. Translated content of one clone
(GenbankTM accession number H82169) was found to be 79% similar and 64% identical to
the last 78 residues of SNAP-25. The EST clone labeled with [ C X - ~ ~ P I ~ C T P was used to
screen approximately 500,000 plaques from an UNI-ZAP XR human melanorna cDNA
library. Twelve positive plaques were identified and their cDNA inserts were subcloned
into Bluescript SIC(-) phagemids (Stratagene) which after infection with helper
bac tcriophages, M 1 3KO7, yielded antisense DNA strand. Single-stranded DNA was then
used for sequencing using the Sequenase version 2.0 kit (United States Biochemical).
Translation of nucleotide sequences was done with the aid of TRANSLATE
algorithm and sequence alignments were performed using the GAP algorithm (Group,
1996). Similar residues are determined using the following convention: K and R; D and E;
S and T; F and Y; 1, L, V, and M. Percent Identity and Percent Sirnilarity are the respective
percentages of symbols that actually match and similar. Symbols that arc across from gaps
were ignored.
Bacterla1 Expression oj- SNAP-23 And SNAP-25 Proteins
Thc amplified fragment of SNAP-23, with BamHl and EcoRl restriction sites
cngineered at the 5' and 3'ends respectively, was subcloned into pGEX-2T vector and used
to transform Escherichia coli cells. Transformants were selected using ampicillin
resistance. Expression of GST fusion protein of SNAP-23 was induced with 1 m M
isopropyl-1-thio-P-D-galactoside (IPTG). Fusion protein was purified by binding to
glutathione-agarose beads and subsequent elution using excess amount of reduced
glutathione (10 mM). GST fusion protein of SNAP-25 was generated using the same
method. The cDNA insert of EST clone (H82169) was isolated and subcloned into the
PET-32a expression vector to generate recombinant SNAP-23 protein.
Generation of SNAP-23 Antisera
Two rabbit antisera specific to SNAP-23 were raised. Antibody aSN23.Cl16 was
produced against a recombinant protein encoding carboxyl-terminal 1 16 residues of SNAP-
23 (amino acids 96-21 1) that was liberated from thioredoxin by thrombin cleavage. The
serum was affinity purified on a column of Affi-gel 15 to which the GST SNAP-23 fusion
protein described above was coupled. Antibody aSN23.Cl2 was raised against a peptide
corresponding to carboxy-terminal residues 200-21 1 of SNAP-23 (H2N -
IANARAKKLIDS-COOH) coupled to keyhole limpet hemacyanin (Biotechnology Service
Ccnter, Toronto, ON). An affinity column was produced by coupling the samc pcptidc to
SulfoLink Gel (Pierce Chemical Company). Antibodics bound to affinity colurnns werc
clutcd with 20 m M glycine and 0.2 M NaCl, pH 2.5 and the eluates were neutralized with
0. I M tris, pH 8.5.
Ce11 culture
PC12 rat adrenal pheochromocytoma cells were grown in D-MEM with 10% fetal
bovine serum. Murine melanocyte ce11 line, melan-b, and human melanoma MeWo cells
were grown in D-MEM supplemented with 2% and 10% fetal bovine serum respectively.
A rat muscle ce11 line, L6, was grown in a-MEM containing 2% fetal bovine serum.
Myoblasts were allowed to fuse and differentiate into myotubes upon fusion (Mitsumoto
and Klip, 1992). Mouse 3T3-L1 fibroblastic pre-adipocytes (herein referred to as 3T3-LI
fibroblasts) were grown in D-MEM supplemented with 10% caIf serum. Cells were
allowed to reach confluence, and two days after confluence, monolayers were incubated for
48 h with differentiation medium (D-MEM containing 10% fetal bovine serum, 115 mglml
isobutylmethylxanthine, 390 nglml dexarnethansone, and 10 mg/ml insulin) (Student et al.,
1980). Monolayers were incubated with D-MEM containing 10% fetal bovine serum and
10 mglml insulin for another 48 h. Cells were maintained in D-MEM with 10% fetal
bovine serum. Al1 media were supplemented with 100 U/ml of penicillin G sodium and 10
mg/ml streptomycin sulfate. For culturing human melanoma cells, 250 ndml arnphotericin
B was also included.
Membrane isolation
Total membranes (TM) were prepared from confluent cells. A11 procedures were
carricd out at 4 OC. Monolayers were washed with homogenization buffer (20 mM Hepes,
255 mM sucrose, and 1 m M EDTA, pH 7.4), scraped with a rubber policeman in
homogenization buffer containing protease inhibitors (0.2 m M phenylmethylsulfonyl
lluoride, 1 mM leupeptin, 1 mM pepstatin, and 10 mM E-64), and homogcnizcd with 10
strokcs using a cc11 cracker. Homogenatcs were centrifuged at 1,000 xg for 5 min. Thc
rcsultant postnuclear supernatants were centrifuged at 245,000 xg Sor 90 min to yicld total
mcmbrancs. The supernatants represented the cytosolic fraction.
I I O O U U U U L l U U A 6 U 1 1 0 V V W A W U A U U U W L W U 1 1 U l l L l l L A W U UllU l l l W l A l U l U l L U V V T U A V p l U p U L V U
according to the protocol described (Nagamatsu et al., 1992). Al1 steps were carried out at
4 OC. Tissues were homogenized in a buffer containing 10 mM TrisIHC1 (pH 7.4), 1 mM
EGTA, 0.25 mM sucrose, 1 mM phenylmethylsulfonyl fluoride and 1 mM leupeptin, using
a Potter homogenizer (20 strokes, 2,000 rpm). The homogenate was centrifuged at 900 xg
for 10 min to remove insoluble materials. The supernatant was further centrifuged at
1 10,000 xg for 75 min. The pellet contained total membranes.
Subcellular Fractionation of 3T3-LI Adipocytes
Confluent 3T3-L1 adipocytes (7- 10 days post-differentiation) were sub-fractionated
as described previously (Piper et al., 1991). Al1 procedures were carried out at 4 OC.
Monolayers were rinsed and scraped in homogenization buffer containing protease
inhibitors. Cells were homogenized by passing them through a ce11 cracking device as
described above. The homogenate was centrifuged at 19,000 xg for 20 min to yield a pellet
P l and supematant SI. The pellet Pl was resuspended in homogenization buffer, laycrcd
on top of a 1.12 M sucrose cushion and centrifuged at 100,000 xg for 1 h. The material at
the intcrface was collected and pelleted by centrifugation at 40,000 xg for 20 min to yield
the plasma membrane (PM). The supernatant S 1 was centrifuged at 41,000 xg for 20 min
and the rcsulting supernatant S 2 was subjected to further centrifugation at 195,000 xg for
75 min to pellet the light density microsomes (LDM). The supematant was collected as thc
cytosolic fraction (CYT).
lrnrnurioblotting
Protcin samples were solubilized in Laemmli sample buffcr (Laemmli, 1970) and
separated by SDS-PAGE (Sambrook et al., 1989). Proteins werc electrophorcticdly
translerrcd from gels to polyvinylidene difluoride membranes. These membranes wcm
"*Y.,...#.. V . V a... b"" " "" " ," \ ", ' / "" . A." "" "" "*"-"'a" "' 'J"'"r"-'- "-"-A-- '-"""
Protcins wcrc detected with the following primary antibodies: anti al Na+/K+ ATPase
monoclonal (1 :25O); anti SNAP-25 monoclonal aSN25mAb (SM1 8 1, 1 :2,000); anti
GLUT4 polyclonal (1: 1,000): anti SNAP-25 polyclonal (aSN25pAb, 1:200) which was
raised to residues 33-206 of SNAP-25b; affinity-purified SNAP-23 polyclonal antisera
aSN23.C 1 16 and aSN23.Cl2 were used at 4 nglml and 0.12 l g /ml , respectively.
aSN25pAb was preincubated with bacterial GST protein followed by incubation with
glutathione linked to agarose beads to remove any antibodies that may cross-react with the
GST portion of SNAP-23 or SNAP-25 fusion proteins before immunoblotting. Enhanced
cherniluminescence was used as the method of detection.
Isolation of SNAP-23 from Human Melanoma cDNA Library
In preliminary experiments using a full-length SNAP-25 cDNA probe, 1 was unable
to detect signals corresponding to SNAP-25 in total RNA from 3T3-L1 fibroblasts or
adipocytes using high and low stnngency hybridization conditions as shown in the left and
right panels of Fig. 1 respectively (Wong et al., 1996). In addition, immunoblotting of
proteins from 3T3-L1 adipocytes with the monoclonal antibody specific to SNAP-25
(aSN25mAb) failed to give a positive result. However, when plasma membranes prepared
from 3T3-Ll adipocytes were probed with a polyclonal SNAP-25 antiserum (aSN25pAb)
raised to near full-length SNAP-25 (Schiavo et al., 1993), a polypeptide that migrated at 29
kDa was detected (Fig. 2, left panel). The detection was specific since it was prevented
upon preincubation of the antiserum with recombinant SNAP-25 protein (Fig. 2, right
panel). These results imply that an immunoreactive species related to SNAP-25 existed in
3T3-L1 cells and prompted us to search for such a gene product.
Through a search of the GenbankTM database, Dr. Tnmblc idcntificd a human EST
clone which encoded a protein predicted to share 64% sequence identity with the C-terminal
78 amino acids of SNAP-25 (Wong et al., 1997). The cDNA (approximately 1.3 Kb) was
iïrst used as a probe to screen for expression in different ce11 lines and tissues at the RNA
lcvcl. Of thc many RNA samples tcstcd, an mRNA species of approxirnately 2.5 Kb from
human mclanoma reacted strongly with the probe. This is not surprising since thc cDNA
inscrt ol' thc EST clone was originally isolated from human melanocytc, and al1 thc other
RNA sarnples screened were isolated from rodent tissues. The strong dctcction at RNA
lcvcl suggested a high probability of cloning a gene product from human mclanoma cclls,
which formcd thc basis of screening a human melanoma cDNA library. Approximatcly
500,000 plaques wcre screened using the same cDNA probe and twelvc positive plüqucs
were isolarea. Ar me rime wnen rne rweive ciones were Deing seyuenwu, a paper appcarw
which described the identification of a SNAP-25 related gene product, SNAP-23, in human
neutrophils (Ravichandran et al., 1996). Sequences of the 5' end of the twelve clones
revealed four that were homologous to regions of the SNAP-23 sequence. The longest one
(approximately 2.0 Kb) began at nucleotide 50, 43 base pairs upstream from the putative
initiation codon of SNAP-23. Comparison between the size of the melanoma clone (2.0
Kb) and the coding region of SNAP-23 (633 bp) indicated that our clone contained the
cntire coding region of SNAP-23. To amplify the coding region of Our clone, PCR
technique was employed. Sequence analysis of the PCR product revealed that it was almost
identical to SNAP-23 with the exception of two nucleotides: nt 396 (A to G ) and nt 404 (T
to C) (Fig. 3, bolded letters). The latter nucleotide change results in a different amino acid,
Val"5->Ala (Fig. 3, bolded letter, and see "Discussion"). The two nucleotide
substitutions were not likely to result from errors of PCR (see "Discussion").
The human rnelanoma SNAP-23 polypeptide contains 21 1 amino acids with a
calculated molecular mass of 23.382 kDa and a theoretical pI of 4.89. Using the GAP
algorithm (Genetic Computer Group, 1996) for optimal alignment between amino acid
sequences of SNAP-23 and SNAP-25, the two polypeptides were found to be 63%
idcntical and 74% similar to each other (Fig. 4). SNAP-23 exhibits structural
characteristics that are similar to SNAP-25. Using the prograrn COILS version 2.1 (Lupas
ct al., 1991; Lupas, 1996), amino acids 45-79 are predicted to form coiled-coi1 with
probability greater than 80% when scanning the SNAP-23 sequence with both a weightcd
and unwcighted MTIDK matrix. This stretch of amino acids corresponds to one of thc
thrcc regions in SNAP-25 which have been predicted to form coiled-coi1 structures. Thc
scgmcnts of SNAP-23 that correspond to the other two coiled-coi1 forming regions of
SNAP-25, howevcr, are not likely to form coiled-çoils as prcdictcd by thc COILS
algorithm. Anothcr structural feature of SNAP-23 that resembles SNAP-25 is thc clustcr
d \ U r d
whereas SNAP-25 has four, al1 of which locate in the middle portion of the two
polypeptides. At least one of the four cysteine residues in SNAP-25 has been shown to bc
palmitoylated, which is believed to be the mechanism responsibk for the tight association
of SNAP-25 protein with membrane (Oyler et al., 1989; Hess et al., 1992). The presencc
of a similar cluster of cysteine residues in SNAP-23 may therefore serve the sarne function.
Generation of Two SNAP-23 S ~ e c i f i c Antibodies
Two antisera to the C-terminal portion of SNAP-23 were generated. Antibody
aSN23.Cl16 was raised against a truncated portion of the protein (amino acids 96-21 1)
and antibody aSN23.C 12 was directed against a peptide corresponding to residues 200-
21 1 of SNAP-23. Equal amounts (1 pg) of GST SNAP-23 and GST SNAP-25 fusion
proteins were irnmunoblotted to test the specificity of the two affinity-purified antisera.
Both of these antisera detected only SNAP-23 fusion protein but not SNAP-25 (Fig. 5, top
two panels). Moreover, no cross-reactivity to the GST portion of the recombinant protein
was detected and no immunoreactivity was detected with the preimmune sera (data no1
shown). Further characterization of the two antibodies used in preliminary studies is
shown in the bottom two panels of Fig. 5. The polyclonal SNAP-25 antiserum,
aSN25pAb, cross-reacted to both SNAP-23 and SNAP-25, whereas the monoclonal
SNAP-25 antibody, aSN25mAb, was specifiç for SNAP-25.
Ex~ression of SNAP-23 rotei in in different ceIl lines and tissues
Ravichandran et al. demonstrated that SNAP-23 is expressed in a variety of non-
ncuronal tissucs at the RNA level (Ravichandran et al., 1996). However, thc endogcnous
expression of the protein was not studied. Therefore I examined the expression of SNAP-
23 protein in various ce11 lines and tissues. SNAP-23 protein was detected using
adipocytes, melan-b melanocytes, rat L6 myotubes, and human melanoma MeWo. Low
levels were detected in PC12 neuroendocrine cells (Fig. 6A, top panel). The protein
dctected by the aSN23.Cl2 antiserum migrated at approximately 29 kDa. The
immunodetection of SNAP-23 was specific since it was blocked by preincubating the
antiserum with excess amount of the peptide (Fig. 6A, middle panel). In contrast to the
widespread distri bu lion of SNAP-23, SNAP-25 showed a very restricted expression,
limited only to the neuroendocrine PC 12 ceils (Fig. 6A, bottom panel).
As shown above for ce11 lines, SNAP-23 was also widely distributed in different
tissues from rats and mice (Fig. 6B). The protein was found to be abundant in lung, liver,
spleen and kidney, but relatively less concentrated in rat skeletal muscle and mouse heart.
Upon longer exposure, a low Ievel of SNAP-23 could also be detected in the mouse brain
(not shown).
Subcellular localization of SNAP-23 in 3T3-Li cells
The subcellular distribution of SNAP-23 is not known, but like SNAP-25, it lacks
a stretch of hydrophobic residues necessary to forrn a transrnembrane domain. In ordcr to
dctermine the localization of SNAP-23 in 3T3-L1 fibroblasts we used subcellular
fractionation. The results in Fig. 7A show that SNAP-23 was exclusively found in the
membrane fraction (TM) with no detectable arnount found in the cytosolic fraction (CYT).
Since SNAP-23 also contains a cluster of cysteine residues, it may associate with the
mcmbranc by palmitoylation of one or more of thc cysteines. 3T3-L1 fibroblasts can bc
differentiatcd into adipocytes, hence allowing me to study the protein at IWO stagcs of
diflcrcntiation. Protcins that arc involved in intracellular traffic have bcen shown to bc
cxprcssed at diffcrent lcvels following differentiation into adipocytes. For cxarnplc,
ccllubrevin, a member of the synaptobrevin family, is expressed prcdominantly in
- -
abundant at the fibroblast stage (Volchuk and Klip, unpublished observation). As can be
seen in Fig. 7B, there was no significant change in the Ievel of expression of SNAP-23
upon differentiation, suggesting that the protein is necessary in both fibroblasts and
adipocytes.
The presence of SNAP-23 in 3T3-L1 adipocytes provided us with an opportunity to
further study the subcellular distribution of the protein. This is because the fractionation
protocol for 3T3-L1 adipocytes has been well-established (Piper et al., 199 1). By this
procedure, the plasma membrane (PM), light density microsomes (LDM), and cytosol
(CYT) were readily purified. The al subunit of Na+/K+ ATPase is a useful marker for the
PM. This protein was detected almost exclusively in the PM and not in the LDM or CYT
of 3T3-L1 adipocytes (top panel of Fig. 7C). The LDM contained the majority of the
GLUT4 glucose transporter which locates intracellularly in unstimulated cells (Fig. 7C,
middle panel). The majority of SNAP-23 was found to be associated with the PM (Fig.
7C, bottom panel). Occasionally, a small amount of SNAP-23 was also detected in the
LDM (data not shown).
Subcellular distribution of SNAP-23 in non-neural secretorv cells
The presence of SNAP-23 and its subcellular distribution was also examined in
non-neural secretory cells such as white blood cells, specifically human neutrophils and the
pancrcatic acinar cells. Thesc studies were done in collaboration with Dr. S. Grinstcin and
Dr. H. Y. Gaisano respectively. To search for the presence of SNAP-23 in different typcs
of white blood cells, platelets, lymphocytes, monocytes and neutrophils were isolated from
heparinized blood from hcalthy individuals by John Brumell in Dr. Grinstein's laboratory.
Lysatcs wcrc prcparcd l'rom thcsc cells and protcins, and 1 uscd thcm for imrnunoblot
analysis using the SNAP-23 antibody, aSN23.Cl2 (Fig. 8). Consistent with thc
ODservarions maae apove, ~ A Y - L ~ was no[ aereccea in Drain microsomes DUL was
cxpresscd in 3T3-L1 adipocytes (Fig. 8, lanes 1 and 2). The protein was also detectcd in
abundance in lymphocytes, monocytes and neutrophils (Fig . 8). This observation had
prompted us to look further at the subcellular distribution of SNAP-23 in neutrophils. The
three types of granules (primary, secondary and tertiary), the secretory vesicles and plasma
membrane fraction, as well as the cytosol were obtained from Dr. N. Borregaard
(Rigshospitalet, Denmark) following fractionation of human neutrophils (Kjeldsen et al.,
1993). Equal amount of protein (25 pg) from each fraction was subjected to imrnunoblot
analysis and a representative blot was shown in Fig. 9. SNAP-23 was predominantly
associated with the secretory vesicle/plasma membrane fraction with barely detectable levels
in secondary and tertiary granules (Fig. 9). The positive detection of SNAP-23 in granules
may result from contamination of the granular fractions with secretory vesicles and plasma
membrane. Preliminary results using separated secretory vesicles and plasma membranes
indicate that SNAP-23 is associated pnmarily with the plasma membranes but not secretory
vesicles (data not shown).
The second non-neural secretory ce11 type which was tested for the presence of
SNAP-23 was pancreatic acinar cells. In order to determine the presence and subcellular
distribution of SNAP-23 andlor SNAP-25 in pancreatic acinar cells, purified plasma
membrane (PM) and zymogen granule membrane (ZGM) were isolated from rat pancreas in
Dr. Gaisano's laboratory. To avoid contamination of SNAP-25, which is abundant in
endocrine pancreas, with the exocrine pancreatic acini, streptozotozin-treated rats were
used. This treatment specifically destroyed the P-cells of the endocrine pancreas. SNAP-
23 was detected with SNAP-23 peptide antibody aSN23.Cl2, and as expected, in
membranes from 3T3-L1 fibroblasts (3T3) but not in brain microsomes (Fig. 10, first
row). Moreover, SNAP-23 was also detected in PM and ZGM of pancreatic acinar cclls.
Thc immunodetection was specific for SNAP-23 since preincubation of the antibody with
Y,."""" U I I . V U . I C " L V U A Y I .A .A -4 *""IV.. y'"""' ".." "' w"-'- "' U" -""-a'-' \- -0- - - 7
second row); however, the bands disappeared when the antibody was preincubated with
GST-SNAP-23 fusion protein (Fig. 10, third row). The presence of SNAP-25 was also
examined in the purified fractions of pancreatic acinar cells. An affinity-purified antibody
that was raised against the full-length fusion protein of SNAP-25 was used for the
immunodetection. The antibody reacted strongly with SNAP-25 present in rat brain and
gave a weaker signal in PM and ZGM from pancreatic acini, and membranes from 3T3-LI
fibroblasts (Fig. 10, fourth row). Preincubation of SNAP-25 antibody with GST-SNAP-
23 fusion protein had no effect on the band in rat brain, but abolished the signal in PM,
ZGM, and 3T3 (Fig. 10, fifth row). Al1 the signal disappeared when the antibody was
preincubated with GST-SNAP-25 fusion protein (Fig. 10, bottom row). These results
suggest that an immunoreactive species that was similar but not identical to SNAP-25 was
present in pancreatic acinar cells. Furthermore, the SNAP-25 antibody cross-reacted with
both SNAP-23 and SNAP-25 proteins. Taking together al1 the observations, SNAP-23
was present in both the plasma membrane and the zymogen granule membrane of
pancreatic acinar cells.
High stringency Low stringency
Fig. 1. A SNAP-25 cDNA probe did not hybridize with RNA from 3T3-L1 cells. Twenty micrograms of total RNA from brain, 3T3-LI adipocytes (3T3-L1 A), and fibroblasts (3T3-Ll F) was analyzed by Northern blot analysis. Hybridization was canied out under both high (left panel) and low (right panel) stringency conditions with a full- length SNAP-25 cDNA probe. 28s and 18s referred to the locations of the nbosomal RNAs. An arrow indicated SNAP-25 mRNA and its expected size is 2.2 Kb.
(-) GST SNAP-25 (+) GST SNAP-25
Fig. 2. Immunodetection of a SNAP-25 l ike protein in membranes from 3T3-L1 adipocytes. Two micrograms of brain microsomes, equal amounts (30 pg) of plasma membranes (3T3-L1 A PM) and light density microsomes (3T3-L1 A LDM) from 3T3-L1 adipocytes were subjected to SDS-PAGE and imrnunoblotted with either an affinity purified antisenim raised to residues 33-206 of SNAP-25, aSN25pAb (left panel) or antiserum that was preincubated with excess amount of GST fusion protein of SNAP-25 (right panel).
-
M D N L S S E E I Q Q R A H Q I T D E S -
L E S T R R I L G L A L E S Q D A G I K -
T I T M L D E Q K E Q L N R I E E G L D -
G L C V C P C N R T K N F E S G K P - Y K -
T T W G D G G E N S P C N V V S K Q P G -
Fig. 3. Nucleotide sequence of human melanoma SNAP-23 and its predicted amino acid sequence. The amino acid sequence of human melanoma SNAP-23 is represented in single-letter code deduced from the nucleotidc sequence shown above. Thc bold letters represent an amino acid and nucleotides that are difièreni from the reccntly identilïed SNAP-23 in human neutrophils (Ravichandran et al., 1996). The two nucleotidc changes arc at nt 396 (A to G ) and nt 404 (T to C). The latter dilfércncc rcsults in a changc in srnino acid, va1135->~la.
S NAP-23 1 , , , , . , MCXVLSSEEIQQ~QITDESLES~ILGLAIESÇDAGZ. KTITM 4 4
SNAP-25 I I I l : ! I I 1: I I I I I I I I I~I I II IlII:!: I
1 MAEDADMRN . ELEEMQRRADQWESLESTRRMLQLVEESKDAGIRTLVM 4 9
Fig. 4. Cornparison of amino acid sequences of human melanoma SNAP-23 and SNAP-25B. The two sequences were aligned using the GAP algorithm (Group, 1996). A pipe character ( I ) indicates identical arnino acids. Similar residues are indicated by a colon ( : ) using the following convention: K and R; D and E; S and T; F and Y; 1, L, V, and M. The two sequences are 63% identical and 74% sirnilar.
Fig. 5. Characterization of SNAP-23 and SNAP-25 antibodies. Immunoblot analysis was used to test the specificity of four antibodies to either SNAP-23 or SNAP-25 GST fusion proteins. One microgram of each fusion protein was subjected to SDS-PAGE and immunoblotted with the following antibodies: aSN23.Cl16 affinity purified antiserum raised to the C-terminal 116 amino acids of SNAP-23; aSN23.Cl2 affinity purified antiserum raised to the C-terminal 12 amino acids of SNAP-23; aSN25pAb polyclonal antiserum raised to residues 33-206 of SNAP-25b; and aSN25mAb (SM1 81) monoclonal antibody to SNAP-25.
Fig. 6. Expression of SNAP-23 in cell lines and tissues. (A) Immunoblot of total membranes prepared from various ce11 lines of different species. Twenty rnicrograms of proteins from rat neuroendocrine PC12 cells, rat L6 myotubes (L6 Mt), mouse 3T3-L1 fibroblasts (3T3-L1 F) and 3T3-L1 adipocytes (3T3-L1 A), mouse melanocytes (melan-b), and human MeWo mclanoma ce11 line were resolved by SDS-PAGE and probed for SNAP-23 with aSN23 .Cl2 antibody (0.12 pg/mI) (top). The spccificity of immunodetection was confirmed by preincubation of aSN23.C 12 antiscrum with excess arnount peptide (0.2 pgfml) (middle). Detection of SNAP-25 by the monoclonal antibody aSN25mAb (SM1 8 1) was restricted to neuroendocrine PC12 cells (bottom). (B) Immunoblotting of total membranes from rat skeletal muscle (Sk. musçlc) and other tissues fiom mousc with aSN23.C 12 antibody.
aSN23.CI 2 + SNAP-23 peptide 29 -
. . . .
. . . . . . . . '
TM CYT
,,n, PM LDM CYT
Fig. 7. Localization of SNAP-23 in 3T3-L1 cells. (A ) Twenty micrograms of total membranes (TM) and cytosolic fraction CYT) from 3T3-LI fibroblasts (3T3-L1 F) were subjected to imrnunoblotting using the aSN23.Cl2 antibody. (B) Twenty micrograms of total membranes (TM) from 3T3-Ll fibroblasts (3T3-L1 F) and adipocytes (3T3-L1 A) were imrnunoblotted for SNAP-23 using aSN23.Cl2 antibody. (C) To study the subcellular distibution of SNAP-23 in 3T3-L1 adipocytes (3T3-L1 A), cells were subfraclionated and equal amounts (15 pg) of plasma membrane (PM), light density microsomes (LDM), and cytosolic fraction (CYT) were separated by SDS-PAGE and prohcd for al subunit of Na+/K+ ATPase with 6H monoclonal antibody, GLUT4 with a polyclonal antiscrum, and SNAP-23 with aSN23.Cl2 antibody.
Fig. 8. Expression of SNAP-23 in different types of white blood cells. Isolation of different white blood ce11 types from fresh heparinized blood of human was perfomed by John Brume11 in Dr. S. Grinstein's laboratory. Thirty micrograrns of protein from mouse brain microsornes and 3T3-L1 adipoctye total membranes were used as negative and positive control for SNAP-23. Lysates prepared from 106 cells of platelet, lymphocyte, monocyte and neutrophil were subjected to irnmunoblot detection for the presence of SNAP-23 using aSN23.Cl2 antibody.
Fig. 9. Subcellular distribution of SNAP-23 in human neutrophils. Fractionation of human neutrophils were done in Dr. N. Borregaard's laboratory and the following fractions were obtained: primary, secondary and tertiary granules; secretory vesicles and plasma membrane fraction (svlpm); and cytosol. Twenty-five micrograms of protein from each of the fractions were subjected to 12% SDS-PAGE. The presence of SNAP-23 were detected using the SNAP-23 antibody, aSN23.C 12.
Fig. 10. Distribution of SNAP-23 in pancreatic acinar cells. Highly purified pancreatic acinar plasma membranes (PM, 10 p g of protein) and zymogen granule membrane (ZYM, 10 pg of protein) from diabetic (streptozotocin-treated) rats, crude brain homogenates (rat brain, 5 pg of protein) and 3T3-L1 fibroblast microsomes (3T3, 10 pg of protein) were prepared, electrophoresed on a 15% SDS-polyacrylamide gel and blotted on Immobion-P transfer membranes. These were then immunoblotted with the following antibodies: top row - aSN23.CL2 SNAP-23 antibody; second row - a S N 2 3 . C l 2 antibody prcincubated with 1 pg of GST SNAP-25 fusion protein; third row - aSN23.Cl2 antibody preincubated with 1 pg of GST SNAP-23 fusion protein; fourth row - SNAP-25 antibody (Anti-SNAP-25) that was raised against the full-length fusion protein of SNAP- 25; firth row - Anti-SNAP-25 preincubated with I pg of GST-SNAP-23; and botiom row - Anti-SNAP-25 prcincubated with 1 pg of GST-SNAP-25.
DISCUSSION
SNA P-23 is a S N A P-25-related gene product
1 have described here the identification of a novel protein composed of 21 1 amino
acids, which is 63% identical and 74% similar to SNAP-25. At the time when 1 was
sequencing al1 the potential clones resulting from screening a human melanoma cDNA
library, a paper was published describing the identification of a SNAP-25-related protein,
SNAP-23. One of our clones contained the entire coding region of SNAP-23, and
alignment of the coding region of the two SNAP-23 sequences revealed only two
nucleotide substitutions which are highlighted in Fig. 3. The change at nt 396 gives rise to
synonymous codons that specify the same amino acid. However the change at nt 404
results in a different arnino acid, from Val to Ala. Both Val and Ala are hydrophobic amino
acids, and so is the corresponding amino acid Ile in SNAP-25 (Fig. 4), therefore these
amino acid changes are not likely to drastically alter the secondary structure of these
proteins. The two nucleotide changes that were observed could not be attributed to errors
in Our sequencing since the exact changes are documented in a recent paper which describes
the identification of two isoforms of SNAP-23 in human neutrophils (Mollinedo and Lazo,
1997). One of the isoforms, SNAP-23A, contains the exact nucleotide substitutions as
observed in the melanoma clone. The other isoform, SNAP-23B, has a deletion of 159
nucleotides in the middle portion of SNAP-23 from nt 266 to nt 424, and is predicted to
encode a protein containing 158 residues (discussed later). The nucleotide diffcrenccs
observcd in both SNAP-23A and the melanoma SNAP-23 clone can be attrihuted to cithcr
polymorphism among humans caused by spontaneous mutations that arise randomly in thc
nucleotide sequences of genes or to sequencing errors made by Ravichandran et al. in the
original SNAP-23 sequence (Ravichandran et al., 1996).
Other than its high level of sequence similarity to SNAP-25, SNAP-23 also exhibits
several features that are characteristic of SNAP-25. According to the SNARE hypothesis,
SNAP-25 and syntaxin on the target membrane (t-SNARE) form a complex with
VAMP/synaptobrevin on the vesicle membrane which is crucial for mediating vcsiclc
Kusion with the target membrane. Binary interactions arnong the three SNAREs have been
characterized in vitro. Furthermore, a detergent-insoluble temary complex of these three
proteins has been isolated from presynaptic nerve terminais (Sollner et al., 1993b).
Ravichandran et al. characterized the affinity of the in vitro translated SNAP-23 protein for
syntaxin isoforms 1-4 as well as VAMPS 1 and 2, and found them to be similar to that
observed for SNAP-25 (Ravichandran et al., 1996). The interactions among the SNARE
proteins are believed to be mediated through coiled-coils. SNAP-25 has three regions with
a high probability of coiled-coi1 formation. Using the COILS version 2.1, the three regions
that are predicted to form coiled-coils with probability higher than 80% are residues 1-39,
49-80, and 166-202 (Lupas et al., 1991; Lupas, 1996). SNAP-23 has only one region
with high probability of forming coiled-coi1 and it spans residues 45-79 (> 80%
probability) as dctermined by the COILS algorithm (Lupas et al., 1991 ; Lupas, 1996).
Sincc the coiled-coi1 domains in SNAP-25 are not completely conserved in SNAP-23, the
association of SNAP-23 with syntaxin and VAMP isoforms is predicted to be diffcrent
from that of SNAP-25.
A second structural feature of SNAP-25 that is conserved in SNAP-23 is the cluster
of cystcinc residucs located in the middle portion of the protein spanning rcsiducs 79-87
(Fig. 3). SNAP-25 protein is an intrinsically hydrophilic polypeptide since its amino acid
scqucncc lacks a strctch of hydrophobic residues that are characteristic of transmembranc
rcgions. Howcver, fractionation of hippocampal extracts has revealed that SNAP-25 is
tightly-associalcd with the synaptosomal mcmbrane fraction and it cannot bc cxtractcd by
conccntratcd salt washes unless dctergent is added (Oyler et al., 1989). The association of
SNAP-25 protein with membranes is probably via palrnitoylation of one or more of the
four cysteine residues which span arnino acids 84-92 (Hess et al., 1992). It has been
shown that deletion of a stretch of 12 amino acids encompassing the four cysteine residues
renders the deletion mutant of SNAP-25 completely soluble and resistant to [%il palmitic
acid labeling (Veit et al., 1996). Since SNAP-23 also contains a similar cluster of cysteine
residues, it may associate with the membrane by a similar mechanism. Interestingly,
SNAP-23 contains five cysteine residues that are in the same arrangement as the Torpedo
SNAP-25 sequence (Risinger et al., 1993a). In contrast, the marnmalian SNAP-25a and
SNAP-25b have each a substitution of one of the cysteines, breaking the cluster
arrangement. The extra cysteine residue found in SNAP-23 and Torpedo SNAP-25 may
define an ability to associate with specific proteins andor affect its ability and efficiency of
acylation.
The functional importance of SNAP-25 is highlighted by the discovery that the
protein is a proteolytic substrate of Clostridiurn botulinum neurotoxin types A, E (Schiavo
et al., 1993; Blasi et al., 1993a; Binz et al., 1994; Lawrence et al., 1994; Boyd et al., 1995;
Pellcgrini et al., 1995; Schmidt and Bostian, 1995), and C l (Foran et al., 1996). Cleavage
of SNAP-25 by BoNT/A or B O N T E results in an inhibition of neurotransmitter release
from isolated nerve terminals (Blasi et ai., 1993a), a blockade of stimulated insulin release
from pancreatic p cells (Boyd et al., 1995), and an abrogation in catecholamine secretion
by adrcnal chromaffin cells (Lawrence et al., 1994). Sequence alignment of SNAP-23 and
SNAP-25 proteins reveals that the two amino acids which constitute the proteolytic
clcavage site for BoNTIE but not those for BoNTIA are conserved in SNAP-23 (Fig. 4).
Thercl'ore we predict that SNAP-23 may be protected from proteolytic cleavagc by
BoNTIA. BoNT/E may bc a useful tool to discern whether SNAP-23 plays a role in
cxocytosis. Indeed, we have shown that both SNAP-23 and SNAP-25 are prcsent in
PC 12 cclls; in these cells, exocytosis of large dense core vesicles is inhibited completely by
BoNT/E but not BoNT/A, although both toxins effectively cleaved SNAP-25 (Banerjee et
al., 1996). The presence of SNAP-23 and its potential insensitivity to proteolysis by
BoNT/A may explain the incomplete inhibition in the peptide hormone secretion observed
in PC12 cells. However, it remains to be proven that BoNTE acts on SNAP-23, and
indeed the residues flanking the cleavage site in SNAP-25 are not reproduced in SNAP-23.
Expression of SNAP-23 in diffeerent tissues and cells
Preliminary experiments using a polyclonal antibody, aSN25pAb, which was
raised against a recombinant protein encompassing residues 33-206 of SNAP-25, detected
a protein that migrated at 29 kDa in the plasma membrane fraction of 3T3-L1 adipocytes.
The detection was specific since it was prevented upon preincubation of the antiserum with
recombinant SNAP-25 protein (Fig. 2). However, immunoblotting of proteins from 3T3-
L l adipocytes with a monoclond antibody specific to SNAP-25 (aSN25mAb) failed to
detect the signal. Furthemore, using a full-length SNAP-25 cDNA as probe, RNA blot
analysis was unable to detect a signal corresponding to SNAP-25 in 3T3-Ll adipocytes
under both high and low stringency hybridization conditions (Fig. 1). Taking together al1
these observations, we concluded that an immunoreactive species related to SNAP-25
cxists in 3T3-L1 cells. These results also imply that the polycional SNAP-25 antiscrum,
aSN25pAb, cross-reacted to both neural-specific SNAP-25 and the related protein
expresscd in 3T3-L1 adipocytes. Indeed, aSN25pAb reacts with both SNAP-23 and
SNAP-25 fusion protcins (Fig. 5 , third panel), whereas the monoclonal aSN25mAb is
specific for SNAP-25 (Fig. 5, bottom panel). These data indicate that the immunoreactivc
protein detected by aSN25pAb in 3T3-Ll adipocytes was most likely SNAP-23 but not
SNAP-25.
In contrast to Our inability to detect SNAP-25 in 3T3-L1 adipocytes, L6 muscle
cells and rat skeletal muscle (Volchuk et al., 1994), Jagadish et al. report the detection of
SNAP-25a cDNA in fat tissues and SNAP-25b cDNA in skeletal muscle by polymerase
chain reaction which amplifies DNA template using isofom-specific primers (Jagadish et
al., 1996). Polymerase chain reaction is a very sensitive technique in detecting low level of
DNA template, however, presence of trace arnounts of DNAs that c m serve as template can
also contaminate the reaction mixture and give false positive detection. The detection of
SNAP-25 cDNA in fat and muscle tissues could potentially result from the presence of
nerve terrninals, which are enriched in SNAP-25, in the biological material. Alternatively,
the amount of SNAP-25 protein in Our fat and muscle ceIl lines may be below the detection
level of Our antibodies.
Unlike SNAP-25 whose distribution is restricted to neural and neuroendocrine
cells, SNAP-23 protein was found to be expressed by al1 non-neuronal ce11 types and
tissues tested (Fig. 6). Interestingly, the immunoreactive bands from cultured L6
myotubes (L6 Mt, Fig. 6A) and heart (Fig. 6B) appear as a doublet. There are three
potential explanations for this observation: first, the doublet can represent SNAP-23
protein in two phosphorylation States; second, the doublet c m indicate the presence of two
isoforms of SNAP-23 of similar size in muscle cells; third, the antibody may cross-react
with a SNAP-23 related gene product of similar size which remains to be identified.
Subcellular localization of SNAP-23 in non-neural cells
To investigate the subcellular distribution of endogenous SNAP-23 protein, total
membrane and cytosolic fractions were obtained from undifferentiated cultured fibroblastic
cclls. Fig. 7A shows a representative immunoblot where SNAP-23 associated entirely
with membranes (3T3-L1 F TM) and was undetectable in the soluble fraction (CYT). The
distribution of SNAP-23 is reminiscent of SNAP-25 in which the majority of thc protein is
bound tightly to the synaptosomal membrane (Oyler et al., 1989). As statcd above, it is
plausible that SNAP-23 associates with the membrane by a similar acylation mechanism,
although this needs to be confirmed experimentally.
The finding that SNAP-23 protein was expressed in differentiated 3T3-L1
adipocytes (Fig. 7B) allowed further characterization of the subcellular localization of the
protein in these cells. Fig. 7C demonstrates clearly the preferential localization of SNAP-
23 at the plasma membrane. This is consistent with the idea of it being a t-SNARE protein
dong with syntaxin 4 which has d s o been detected at the ce11 surface of 3T3-LI adipocytes
(Volchuk et al., 1996). Preliminary immunofluorescence studies carried out by Dr. P. J.
Bilan using Our affinity-purified SNAP-23 peptide antibody aSN23.CI2, showed a signal
not only at the perimeter of 3T3-L1 adipocytes but also intracellular staining. This appears
to contradict the exclusive distribution of SNAP-23 in the plasma membrane fraction of
3T3-L1 adipocytes indicated by the immunoblot analysis as shown in Fig. 7C. The
apparent inconsistency between the two methods of detection might be explained by the
existence SNAP-23B. The SNAP-23B cDNA contains a deletion of 159 nucleotides which
results in a truncated SNAP-23 protein: arnino acids 89- 142 are replaced by a Ser residue
which joins the N- and C-terminal fragments of SNAP-23 (Mollinedo and Lazo, 1997).
SNAP-23B contains 158 residues and the predicted molecular mass is 17.8 kDa. The level
of expression of SNAP-23B protein in cells remains to be deterrnined. An immunoblot of
subcellular fractions of 3T3-L 1 adipocytes using aSN23 .C 12, which should detect both
the iull-length SNAP-23 (SNAP-23A) and SNAP-2313, showed a positive detection of a
protein which migrated at about 19 kDa in the cytosolic fraction but not associated with
membranes (data not shown). Although this observation needs to be confirmed by future
cxpcriments, the presence of an isoform of SNAP-23 which is mainly soluble couId
explain the intracellular staining of SNAP-23 observed in the immunofluorcscence images
OC 3T3-LI adipocytes. Alternatively, the intracellular staining could be attributcd to non-
specilïc binding of the antibody to cellular components.
To investigate whether the SNARE hypothesis can indeed serve as a general mode1
for intracellular vesicle traffic, the expression of SNAP-23, a non-neural equivalent of
SNAP-25 (t-SNARE), was investigated in two additional non-neural secretory ce11 types:
white blood cells and pancreatic acinar cells. In contrat to 3T3-L1 adipocytes in which
insulin causes incorporation of vesicle membrane-bound glucose transporters to the ce11
surface, white blood cells and pancreatic acinar cells secret digestive enzymes via
exocytosis of the luminal content of granules to the extracellular mellieu in response to
stimuli. Four types of white blood cells were screened for the presence of SNAP-23.
Lymphocytes, monocytes, neutrophils and platelets, al1 were found to express SNAP-23
(Fig. 8). The subcellular localization of SNAP-23 in neutrophils was examined further.
Polymorphonuclear neutrophils protect the body against invading organisms such as
bacteria, by attacking and destroying them. The initial step of the process involves
phagocytosis of the infectious agent. Once the foreign particle was phagocytosed,
lysosomes and oîher granules dock and fuse with the phagocytic vesicle (plasma membrane
vesicle located intracellularly) and subsequently fuse with it, thereby allowing release of
digestive enzymes and bactericidal agents into the phagosome (Guyton, 1992). The
SNARE proteins syntaxin 4 and VAMP-2, but not SNAP-25, have been detected in
ncutrophils (Brume11 et al., 1995). Fractionation of neutrophils yields the cytosolic, and
threc granule fractions (primary, secondary, and tertiary) as well as a membrane fraction
containing sccretory vesicles and plasma membrane (svlpm). Immunoblot of these
ilactions indicated that SNAP-23 is almost exclusively associated with sv/pm (Fig. 9).
Upon further separation of secretory vesicles from plasma membrane, results ïrom one
cxperiment indicated that SNAP-23 was detected only in the plasma membrane fraction
(data not shown). The localization of SNAP-23 at the plasma membrane is suggestive that
the protein acts as a t-SNARE. Previous studies have shown that syntaxin 4 is localized
exclusively at the plasma membrane of neutrophils, whereas VAMP-2 is detected on the
secretory vesicle and tertiary granule (Brume11 et al., 1995). The identification of SNAP-
23 in neutrophils suggest that SNAP-23 and syntaxin 4 are likely to act as t-SNAREs, with
VAMP-2 being the V-SNARE. Indeed upon activating neutrophils with ca2+ ionophore,
VAMP-2 is found to redistribute to the plasma membrane (Brume11 et al., 1995). Granular
secretion of bactericidal agents into the phagosome could be achieved by interaction of
SNAP-23 and syntaxin 4 on the phagosomal membrane (plasma membrane) with VAMP-2
on the granule, iollowed by vesicle fusion with the phagosome.
Unlike adipocytes and white blood cells, pancreatic acinar cells are polarized
epithelial cells. The plasma membrane of the acinar cells is organized into two discrete
regions: apical and basolateral. The extracellular spaces are segregated by tight junctions
which are essential to maintain the functional integrity of the exocrine pancreas. Pancreatic
acinar cells undergo regulated secretion of digestive enzymes which are stored in zymogen
granules (ZG), upon stimulation with molecules such as acetylcholine, gastrin and
cholecystokinin (Guyton, 1992). Previous studies have identified VAMP-2 (Gaisano et
al., 1994) and syntaxin isoforrns 1-4 in pancreatic acinar cells (Gaisano et al., 1996). In a
collaborative study with Dr. Gaisano, the subcellular distribution of SNAP-23 was
examined in pancreatic acinar cells. Surprisingly SNAP-23 was detected by
immunoblotting in both the plasma membrane (PM) and zymogen granular membrane
(ZGM) fractions (Fig. 10, top panel). The detection was specific since it was inhibited
upon preincubation of SNAP-23 peptide antibody, aSN23.Cl2, with SNAP-23 fusion
protcin (Fig. 10, third panel), and was not affected by preincubation of thc antibody with
cxccss amount of SNAP-25 fusion protein. (Fig. 10, second panel). The prescncc of
SNAP-23 in the ZGM was not likely to be caused by contamination of this fraction with
plasma membrane since the intensities of both signals were almost identical. The presence
of SNAP-23 on the ZGM could mean that the ZG is the target for fusion of other
iniracellular vesicles. This idea is supported by the observation of ZG-ZG fusion (Scheele
et al., 1987). Of the four isoforms of syntaxin present in the pancreatic acini, syntaxin 3
was present on ZG (Gaisano et al., 1996). Therefore, syntaxin 3 dong with SNAP-23
could act as t-SNARE on ZG. VAMP-2 is the only V-SNARE found to be localized on
ZG. In vitro binding studies have demonstrated that VAMP-i and -2 bind only to syntaxin
1 and 4 but not 2 and 3 (Calakos et al., 1994) , whereas SNAP-23 can bind to al1 four
isoforrns of syntaxin (Ravichandran et al., 1996). Therefore ZG-ZG fusion can be
achieved either by binding of VAMP-2 to SNAP-23 or a yet-to-be-identified V-SNARE
which can interact with both syntaxin 3 and SNAP-23.
The results presented in this chapter described the identification of SNAP-23 - a
novei isoform of SNAP-25. In contrast to SNAP-25 which is expressed only in neural and
neuroendocrine cells, SNAP-23 is distributed in a variety of cells and tissues. Subcellular
fractionation of 3T3-L1 adipocytes, human neutrophils, and pancreatic acinar cells
demonstrated that SNAP-23 is localized predominantly at the plasma membrane which is
suggestive of its role in being a t-SNARE dong with syntaxin 4 on the plasma membrane.
The SNARE hypothesis was initially proposed to explain how synaptic vesicles
dock and fuse with the presynaptic plasma membrane, allowing transmitter release at the
nerve terminals. Homologues of proteins responsible for this highly regulated exocytic
process at the nerve terminals are found to be necessary for the constitutive exocytosis in
yeast. Isoforms of syntaxin and VAMP, which are key components of the protein
machinery for vesicle transport, have also been identified in mammalian non-neural tissues
and cells. Furthermore, the functions of syntaxin and VAMP have been implicated in
regulated exocytosis in both neural and non-neural cells. From these data emerges the
theme of universality of the SNARE hypothesis which predicts that al1 eukaryotic cells have
families of v- and t-SNAREs, whose members forrn cognate pairs that define binding
specificity that is essential for vesicle docking and fusion in vivo (Rothman, 1994).
SNAP-25 is a crucial component of the protein machinery important for vesicle docking
and fusion. Prior to my M.Sc. project, expression of SNAP-25 and its function had only
been characterized in the nervous system and neuroendocrine cells. According to the
SNARE hypothesis, functional equivalents of SNAP-25 must exist outside the nervous
system to allow regulated exocytosis to occur in non-neural tissues.
The aim of this Thesis was to search for a non-neural equivaient of SNAP-25 that
can explain regulated exocytosis in cells outside the nervous system. The search led to the
identification of a novel polypeptide which shares 63% identity to SNAP-25. This novel
protcin was found to bc idcntical to SNAP-23 which was publishcd at thc samc timc whcn
thc sequenccs of our cDNA clones resulted from screening a human melanoma cDNA
library werc being analyzed. SNAP-23 is expressed in a variety of tissues and ce11 lincs
including non-neural cells such as adipocytes, muscles, neutrophils and pancrcatic acinar
cclls as well as neuroendocrine PC12 cells. This ubiquitous distribution of SNAP-23 was
- ---- . -- --- - ---- J -- ---- r-- - - - - 7 - -
expression of SNAP-23 at the mRNA level (Ravichandran et al., 1996). Fractionation
studies of murine 3T3-Ll adipocytes and human neutrophils suggest that SNAP-23 is
exclusively associated with the plasma membrane. SNAP-23, along with syntaxin 4 which
is primarily localized at the plasma membrane of 3T3-L1 adipocytes (Volchuk et al., 1996)
and human neutrophils (Brumell et al., 1995), may act as a t-SNARE. The corresponding
V-SNARE, both on the GLUT4 vesicles in 3T3-L1 adipocytes (Volchuk et al., 1995) and
in secretory vesicles and tertiary granules of human neutrophils (Brumell et al., 1995), is
VAMP-2. In rat pancreatic acinar cells, SNAP-23 is found on the plasma membrane as
well as the zymogen granular membrane. This observation indicates that some of the
SNAP-23 protein acts as t-SNARE on the plasma membrane. In addition, rat pancreatic
acinar cells possess another pool of SNAP-23 that is localized intracellularly, suggesting
that zymogen granule may act as the target membrane for vesicle fusion. Now that a new
player for vesicle traffic has been identified, future work is needed to establish the
functional importance of the protein in regulated exocytosis underlying insulin-stimulated
translocation of GLUT4-containing vesicles in 3T3-L1 adipocytes and secrction in human
ncutrophils and rat pancreatic acinar cells.
One of the important tools in discerning the function of SNAP-25 is one of the most
potent ncurotoxins known to mankind: Botulinum neurotoxin (BoNT). Both BoNT types
A and E cleave SNAP-25 at distinct sites as indicated in Fig. 4. The two residues
composing the BoNT/E cleavage site, ~ r g l 8 0 - 11e181, are conserved in SNAP-23.
However. the BoNTIA cleavage site is not conserved since Gln197 in SNAP-25 is replaced
by Ala in SNAP-23; therefore, SNAP-23 is unlikely to be a substrate for proteolysis by
BoNT/A. I t will be important to test the susceptibility of SNAP-23 to cleavagc by
BoNT/E. Since the cleaved product of SNAP-23 will lack the C-terminal 25 residucs
which contains the epitope of thc SNAP-23 peptide antibody aSN23.C 12, immunoblotting
- - -- - - - - - . - - - - . - - - - - . . - - - - - - - - - - - - - - . - - - u - - - - - - - d
intact SNAP-23 that is resistent to cleavage.
Different experiments can be used to establish a role of SNAP-23 in non-neural
cells. In 3T3-L 1 adipocytes where insulin stimulates the translocation of intracellular pool
of GLUT4 proteins, SNAP-23 antibodies (aSN23.Cl2) and BoNT/E can either be
microinjected into intact cells or incubated with permeabilized adipocytes to interfere with
the normal function of SNAP-23 prior to insulin treatment. The inhibition can be measured
by either arrivai of GLUT4 protein on the ce11 surface detected with GLUT4 antibody or, in
the case of permeabilized adipocytes, insulin-stimulated glucose transport using
radioactively-labelled 2-deoxyglucose. Furthermore, C-terminal SNAP-25 peptide
fragments of 12-mer and 20-mer in length have been used to inhibit the regulated
exocytosis in adrenal chromaffin cells and synaptosornes (Gutierrez et al., 1995; Mehta et
al., 1996; Gutierrez et al., 1997). The C-terminus of SNAP-25 has been suggested to be
crucial for binding to VAMP-1. If similar interaction occurs between SNAP-23 and
VAMP-2, incubation of electroporated neutrophils with excess arnount of C-terminal 12-
mer of SNAP-23 may interfere with the secretion of granules. The peptide is predicted to
bind to most of the native VAMP proteins, thereby preventing the normal v- and t-SNARE
interactions necessary for vesicle fusion. In rat pancreatic acinar cells, the function of
SNAP-23 can be assessed by the effect that introducing SNAP-23 antibody or BoNT/E
into permeabilized cells may have on the ~a2+-stimulated enzyme secretion.
As stated earlier, two isoforms of SNAP-23 were recently identified in human
neutrophils (Mollinedo and Lazo, 1997). Preliminary results suggested that the SNAP-23
peptide antiserum, which should detect both SNAP-23A and SNAP-23B, reacted with a
polypeptide that migrates at 19 kDa in the cytosolic fraction of 3T3-L1 adipocytes and the
dctcrgcnt-solubilizcd lysate of human neutrophils. If SNAP-23B protein werc indccd
expressed by these cells, it would be important to study its interaction with syntaxin 4 (t-
" A . ' . I . Y , Ull" 1 a A . 1 ) 1 \. U A . * a*.-/, " 1 l l Y W U.., " I I I W 1 V . I V Y UV"" . . V U 11. & * I V V L .* .a.-
interactions would suggest a new regulatory role of SNAP-23B in vesicle traffic in
adipocytes and neutrophils. It is easy to envisage SNAP-23B as a fusion clamp in the non-
stimulated state, for example, if it has binding capacity for either syntaxin 4 o r VAMP-2 but
not both. Future study c m be designed to investigate the in vitro binding properties of
SNAP-23B with the other SNARE proteins.
Recent work by Wang et al. described the identification of a SNAP-25 related
protein, syndet, in murine 3T3-L1 adipocytes (Wang et al., 1997). An important question
arises immediately - is syndet a mouse isoform of SNAP-23? The similarities and
differences between syndet and SNAP-23 are discussed here. Syndet is 58% similar to
SNAP-25, a value which is much lower than the 74% similarity shared between SNAP-23
and SNAP-25. The mouse syndet and human SNAP-23 amino acid sequences share only
88% identity, unlike mouse and human isoforms of SNAP-25 which are completely
identical. This may suggest the two proteins represent distinct polypeptides rather than
being species-specific isoforms of the same protein. Furthenore, the three regions that are
highly probable in coiled-coils formation in SNAP-25 (two at the amino-terminus and one
at the carboxyl-terminus) are not conserved in either SNAP-23 or syndet. Using the
COTLS algorithm, only the second coiled-coi1 is conserved in SNAP-23, whereas for
syndet, both the sccond and third coiled-coi1 regions are conserved. The coiled-coi1
domains may have significant influence upon the interactions among SNARE proteins.
Sincc the affinity of syndet for other SNARE proteins has not been dctcrmincd
experimentally, in vitro binding studies of recombinant proteins will scrve this purpose as
well as help in defining syndet as a SNAP-25 related protein as a result of its interactions
with VAMP or syntaxin. Moreover, neither of the first residues constituting the BoNTIE
and BoNTIA clcavagc sitcs of SNAP-25 arc conscrvcd in syndct, whcreas in SNAP-23 thc
site for BoNTIE is conserved. The first residue of the cleavage sitc for BoNTIE in syndet
L U D U I L J 111 U UUIIÙbl V U L A V U U I L l l l l U U U L U D U U O C l L U L l W l l - L L W l l l 1 %A6 V I U A - 1 A 1 hJ LW Y J O V A
syndet. Ii is not known whether the presence of a basic amino acid is sufficient for
cleavage by for BoNTIE. The potential difference in susceptibilities of SNAP-23 and
syndet to BONTE cleavage will be important in differentiating the functional importance of
cach protein in 3T3-LI adipocytes.
One structural characteristic that is conserved between syndet and SNAP-23 is the
arrangement of cysteine residues within the cysteine-rich region. SNAP-23 and syndet
behave as integral membrane protein even though both proteins lack a stretch of
hydrophobic residues characteristic of trammembrane region. The two proteins probably
associate with the membrane via palmitoylation - a post-translational modification
responsible for the tight association of SNAP-25 with the synaptosomal membrane (Hess
ct al., 1992). Future experiments are needed to test this hypothesis by demonstrating
SNAP-23 and syndet are indeed palmitoylated.
In an attempt to detect expression of mRNA encoding SNAP-23, we have used a
cDNA containing the entire coding region of human melanoma SNAP-23 as a probc for
total RNA prepared from different murine tissues and cells including 3T3-Ll adipocytes as
wcll as human melanoma. The probe only reacted positively with RNA from human
mclanoma. The lack of detection of SNAP-23 mRNA in murine tissues and cells with a
human cDNA probe could bc due to species differences. Future Northern blot analysis
using lower levcIs of stringency for hybridization may allow detection of SNAP-23 at thc
nucleotide lcvcl.
Thc identification of SNAP-23 and syndet supports the themc of univcrsality
proposed by the SNARE hypothcsis by suggesting the existence of SNAP-25 rclatcd genc
products outside the nervous system. Now that new players for intracellular traffic havc
becn identified, the ncxt task will involve characterization of the interactions among thc
playcrs as wcll as dctemining the role of each in vesicle traffic. Future knowledgc in thc
iïcld of membrane traffic will provide a better understanding in mechanism underlying
constitutive exocytic process which is crucial in maintaining the integrity of subcellular
compartments. In addition, the information will provide insights regarding regulated
cxocytosis such as the insulin-stimulated docking and fusion of glucose transporter,
GLUT4-containing vesicles with the plasma membrane. A better understanding of the
mechanism of GLUT4 traffic will have important implication in identifying the cellular
defects in diabetes mellitus, since it is generally believed that one of defects in non-insulin
dependent diabetes mellitus is ineffective translocation of GLUT4 vesicle to the ce11 surface
in response to insulin.
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Amira Klip. 1996.
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Tissue Distribution of SNAP-23 and Its Subcellular Localization in 3T3-LI Cells
Peggy P. C . Wong,* Nicholas Daneman," Allen Volchuk,* Norman Lassam,t Michael C . Wilson,$ Amira Klip,**l and William S. Trimble* *Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario, M5G 1x8, Canada;
a ?Toronto-Bayview Regional Cancer Centre, Toronto, Ontario, Canada; and $Department of Biochemistry, University of New Mexico, School of Medicine, Albuquerque, New Mexico
Received November 21, 1996
The SNARE hypothesis of vesicular traffic proposes that three proteins, VAMP/synaptobrevin, syntaxin, and SNAP-25, constitute a complex that docks the vesi- cle at the target membrane. VAMP and syntaxin iso- forms have been identified outside the nervous system, and a cDNA to a SNAP-25 related protein, SNAP-23, was recently identified in human lymphocytes. Here we report the generation of isoform-specific antibodies to SNAP-23 cloned from human melanoma cells, and their use in detecting the expression and localization of the endogenous SNAP-23 protein in several tissues and ce11 lines. SNAP-23 was readily detected in liver, lung, kidney, and spleen, to a lesser extent in muscle and heart, and was almost undetectable in brain. The protein was also abundant in fibroblast, muscle, and fat ceil lines, but relatively less enriched in neuroendo- crine PC12 cells. SNAP-23 abundance did not change during differentiation of 3T3-Ll fibroblasts into adipo- cytes. In both, SNAP-23 was membrane-bound and be- low detectable levels in the cytosolic fraction. Subcel- Iular fractionation of 3T3-Ll adipocytes revealed that the majority of the protein was associated with plasma membranes. These findings support the conclusion that a tripartite SNARE complex exists outside of the nervous system, and suggest that SNAP-23 may play a role in vesicle traffic in most cell types. s iw7 Academic
Pretin
target for the binding and action of soluble fusion com- ponents NSF and SNAP (1). Furthemore, the specific- ity of vesicle targeting is thought to be mediated by the correct association of v- and t-SNARE isoforms. In the nervous system the vesicle-associated membrane pro- tein VAMP acts as a V-SNARE and binds to the t- SNARE membrane proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) to form a stable tripartite complex. Multiple isoforms of VAMP and syntaxin have been identified, and sever.11 of these are known to be expressed outside the nervous system. However, until very recently, only two akmatively spliced f o m s of SNAP-25 were known to c:xist, and their expression was found to be restricted t , the ner- vous system (2, 3) and neuroendocrine cel!a such a s pancreatic islets of Langerhans (4-6), adrenal chromaf- fin cells (7), and anterior pituitary cells (8). If the basic tenets of the SNARE hypothesis are correct, and if this ternary cassette of proteins also functions to mediate membrane fusion in other ce11 types, then SNAP-25- like molecules should be present in non-neuronal cells undergoing regulated membrane traffic events.
Recently, Ravichandran e t al. (9) described the cDNA cloning of a novel isoform of SNAP-25, named SNAP- 23, and observed expression of its mRNA in several non-neuronal tissues. To examine the presence and dis- tribution of SNAP-23 a t the protein level, we have iso- lated a full length cDNA clone from human melanoma cDNA library and produced antibodies specific for
The binding of vesicles to, and their fusion with, tar- SNAP-23. u&ng these reagents we demoitrate the
get membranes is a key step in intracellular traffic in presence of this protein in a variety of tissues and ce11
al1 eukaryotic cells. The SNARE hypothesis predicts lines. In contrast, we were unable to detect SNAP-25 outside neuronaVneuroendocrine cells. Further, we that a combination of vesicular (v-SNARE) and target show that SNAP-23 is localized at the plasma mem- (t-SNARE1 membrane proteins interact to form the brane in the 3T3-L1 adipocyte tell line, where it may SNARE (SNAP-receptor) complex which serves as the likely act as a t-SNARE along with syntaxin 4. Thcse results support the generality of the SNARE hypothe-
' To whorn correspondencc çhould be addrcssed. Fax: (416) 813- sis and suggest that the V-/~-SNAR& ternary com~lex 5028. may be found in most ce11 types.
0006-291)(/97 $25.00 Copyright 0 1997 by Acctdernic 1'1vss AI1 n ~ l t t s n i rcprnductian in an! forrii rc.r;wvc-d
MATERIAL AND METHOGS
Maierials. IMAGE Consortium expressed-sequence h g (EST) cDNA #384468 ( ~ e n ~ a n k ~ ~ accession number H82l69) was obtained from Research Genetics. SNAP-25 monoclonal antibody SM1 81 (here cailed aSN25mAb) was obtained from Stemberger Monoclonals Inc. Anti al Na'IK'ATPaçe monoclonal antibody 6H was kindly provided by Dr. M. Caplan (Yale University, New Haven, CT). GLUT4 poly- clonal antibody was from East Acres Biologicais. An affinity punfied antiserum (aSN25pAb) was raised against residues 33-206 of SNAP- 25b isoform (10).
Cloning and bacterial expression of SNAP-23 and SNAP-25. An EST clone (see Results) labeled with [a-91dCTF' was used to screen approximately 500,000 plaques from a UNI-ZAP XR human mela- noma cDNA library. Twelve positive plaques were identified and their cDNA inserts were subcloned into Bluescript plasmids (Stra- tagene) for single-stranded DNA sequencing using the Sequenase version 2.0 kit (United States Biochemicai). Sequences of the 5' end of the twelve clones revealed one which began a t nucleotide 50.43 base pairs upstream from the putative initiation codon reported for the recently published SNAP-23 cDNA (9). The coding sequence of this clone was amplified by poiyrnerase c h a h reaction (PCR) using primers (nucleotides 93-110, 714-731) which flanked the coding re- gion. This amplified fragment was then subdoned into pGEX-2T vector. The entire sequence was confirmed by autornated sequence analysis (Biotechnology Service Centre, Toronto, ON) and found to be identical to human SNAP-23 cDNA (9). GST fusion proteins of SNAP-23 and SNAP-25 were expressed and purified from Esche- richia coli (11). The cDNA insert of the EST done was isolated and subcloned into the PET-32a expression vector (Novagen).
Gerreratiorz of SNAP-23 antisera. Two rabbit antisera specific ta SNAP-23 were raised. Antibody aSN23.Cl16 was produced against a recombinant protein encoding carboxy-terminal 116 residues of SNAP-23 (amino acids 96-211) that was liberated from thioredoxin by thrombin deavage. The serum was affinity purified on a column of Ani-gel 15 to which the GST SNAP-23 fusion protein described above was coupled. Antibody aSN23.CX2 was raised against a pep- tide corresponding to carboxy-terminal residues 200-211 of SNAP- 23 (H2N-IANARAKKLIDS-COOH) coupled to keyhole limpet hema- cyanin (Biotechnology Service Centre, Toronto, ON). An affinity col- umn was produced by coupling the same peptide to SulfoLink Gel (Pierce Chemical Company). Antibodies bound to afiinity colurnns were eluted with 20 mM glycine and 0.2 M NaCI, pH 2.5 and the eluates were neutralized with 0.1 M Tris, pH 8.5.
Ce12 culture. The following ce11 lines were grown: r a t adrenal pheochromocytoma PC12, munne melanocyte melan-b, and human melanoma MeWo. hlyoblasts of the ra t muscle ce11 line, L6, were allowed to fuse and differentiate into rnyotubes upon fusion (12, 13). Mouse 3T3-L1 fibroblasts were differentiated into adipocytes as de- scribed previously ( 14).
hlcntbrane isolation arid ir~~rnurzoblotting. Total membranes (TM) wcre preparcd from conflucnt cells. Ail procedures were carried out a t 4 OC. Moiiolayers were washed with homogenization buffer (20 m M Hepes, 255 rnM sucrose, and 1 mM EDTA, pH 7.4), scraped with a rubbcr policeman in homogenization buffer containing proteasc inhibitors (0.2 mM phenylmetliyIsulfony1 fluoride, 1 p M leupeptin, 1 ph1 pepstaiin, and 10 PM E-64) and homogenized with 10 strokes usiiig a ceIl cracker. Honiogcnates were ccntrifuged a t 1,000 xg for 3 niin. T h rcsultant postriuclear supernatants wcre centrifuged at 245,000 xg for. 90 miri to yicld total membranes. The supcrnatants rcpresented the cytosolic fraction. Confluent 3T3-L1 adipocytes (7- 10 days post-diffcrcntintio~~) wcre sub-fractionated a s dcscribed pre- viously (7 ) . Organs were dissectcd from mice and niicrosomes were prcp:wed nccording to the protocol dcscribed (15). Protein samples wciv solubilizcd in Lacmnlli sample bufïcr (16) and scparated by SDS-PAGE (17). Proteins were dctected with the following primary antibodics: anti c u l Na'K' ATPasc monoclona1 (1:250), anti SNAP-
25 monoclonal nSN25rnAb (SM1 81, 1:2000), anti GLUT4 polyclonal (1:1000), anti SNAP-25 polyclonal (aSN25pAb, 1:200), affinity-puri- fied SNAP-23 polyclonal antisera aSN23.Cl16 and aSN23.Cl2 were used a t 4 ng/ml and 0.12 mglm1 respectively. aSNS5pAb was preincu- bated with bacterial GST protein followed by incubation with gluta- thione linked to agarose beads to remove any antibodies that may cross-react with the GST portion of SNAP-23 or SNAP-25 fusion proteins before immunoblotting. Enhanced chemiluminescence was used as the method for detection.
RESULTS AND DISCUSSION
In prelirninary experiments using a full-length SNAP-25 cDNA probe, we were unable to detect signals corresponding to SNAP-25 in total RNA from 3T3-L1 fibroblasts or adipocytes under high or low stringency conditions ( 18). I n addition, immunoblotting of proteins from 3T3-L1 adipocytes with the monoclonal antibody specific to SNAP-25 (aSN25mAb) also failed to give a positive result. However, when plasma membranes prepared from 3T3-L1 adipocytes were probed with a polyclonal SNAP-25 antiserum (aSN25pAb) raised to near full-length of the protein (IO), a polypeptide that migrated at 29 M)a was detected. The detection was specific since it was prevented upon preincubation of the antiserum with recombinant SNAP-25 protein (18). These results irnplied that an immunoreactive species related to SNAP-25 existed in 3T3-LI cells and prompted us to search for such a gene product.
Through a search of the GenbankTM database we identified a human EST clone whieh encoded a protein predicted to share 64% sequence identity with the C- terminal 78 amino acids of SNAP-25. Using this cDNA as probe, we screened approximately 500,000 plaques of a human melanoma cDNA library. Twelve positive plaques were isolated, the longest of which contained a cDNA of approximately 2.0 I(b. Sequence analysis of this clone revealed that it was identical to the recently described SNAP-25-related gene product SNAP-23 tg), and contained the entire coding sequence.
We have generated two antisera to the C-terminal portion of SNAP-23. Antibody aSN23.Cl16 was raised against a truncated portion of the protein (amino acids 96-211) and antibody aSN23.Cl2 was directed against a peptide corresponding to residues 200-211 of SNAP- 23. Equal amounts (1 pg) of GST SNAP-23 and GST SNAP-25 fusion proteins were immunoblotted to test the specificity of the two affinity-purified antisera. 130th of these antisera detected only SN@-23 fusion protein but not SNAP-25 (Fig. 1, top two panels). Moreover, no cross-reactivity to the GST portion of the recombinant proteins was detected and no immunoreactivity was detected with the preimmune sera (data not shown). Further characterization of the two antibodies used in preliminary studies (18) is shown in the bottom two panels of Fig. 1. The polyclonal SNAP-25 antiserum, aSN25pAb, cross-reacts to both SNAP-23 and SNAP- 25, whereas the monoclonal SNAP-25 antibody, aSN2-
FIG. 1. Characterization of SNAP-23 and SNAP-25 antibodies. Immunoblot analysis was used to tes t the specificity of four antibod- ies to either SNAP-23 or SNAP-25 GST fusion proteins. One micro- gram of each fusion protein was subjeded to SDS-PAGE and immu- noblotted with t he followingantibodies: cuSN23.Cl16 affinity purified antiserum raised to the G k r m i n a l 116 amino acids of SNAP-23; aSN23.Cl2 f f i n i t y purified antiserum raised to the C-terminal 12 amino acids of SNAP-23; aSN25pAb polyclonal antiserum raised t o residues 33-206 of SNAP-25; and aSN25mAb (SM1 81) monoclonal antibody to SNAP-25.
5mAb, is specific for SNAP-25. These data indicate that the immunoreactive protein detected by aSN25pAb in 3T3-LI adipocytes in previous studies was most likely SNAP-23 and not SNAP-25 (18).
The predicted amino acid sequence of SNAP-23 is 59% identical and 72% similar to SNAP-25b isoform (9). Northern bIot analysis demonstrates a wide tissue distribution of SNAP-23 mRNA (9), however endoge- nous expression of the protein has not been exarnined. Here we report the expression of SNAP-23 protein in various ce11 lines and tissues. SNAP-23 protein was detected using aSN23.C 12 in both non-neuroendocrine ce11 lines such as mouse 3T3-L1 fibroblasts, 3T3-L1 adi- pocytes, melan-b melanocytes, rat L6 myotubes, and human melanoma MeWo cells a s well a s lower levels in PC12 neuroendocrine cells (Fig. 2A, top panel). The protein detected by the aSN23.Cl2 antiserum migrates at approximately 29 kDa. The immunodetection of SNAP-23 was specific since i t was blocked by preincu- bating the antiserum with excess amount of the peptide (Fig. 2A, rniddle panel). In contrast to the widespread distribution of SNAP-23, SNAP-25 showed a very re- stricted expression, limited only to the neuroendocrine PC12 cells (Fig. 2A, bottorn panel).
In contrast to Our inability to detect SNAP-25 in 3T3- L1 adipocytes and L6 muscle cells, Jagadish e t al. (19) recently reported the expression of SNAP-25a in fat tissue and SNAP-25b in skeletal muscle. The detection of SNAP-25 in the fat and muscle tissues could poten- tially result from the presence of nerve terminals in the biological material. Alternatively, the amount of SNAP-25 protein in Our fat and muscle ce11 lines may be below the detection Ievel of Our antibodies.
As shown above for ce11 lines, SNAP-23 was also widely distributed in different tissues from rats and
mice (Fig. 2B) . The protein was found to be abundant in lung, liver, spleen and kidney, but relatively less concentrated i n r a t skeletal muscle and mouse heart. Upon longer exposure of the immunoblot to autoradiog- raphy, a low level of SNAP-23 could also be detected in the mouse brain (result not shown).
The subcellular distribution of SNAP-23 is not known but, like SNAP-25, it lacks a stretch of hy- drophobic residues necessary to fonn a transmembrane domain. In the case of SNAP-25, the majority of the protein is tightly associated with synaptosomal mem- branes (20) probably via palmitoylation of one or more of the four cysteine residues clustered in the middle of the protein (21). I t has recently been shown that dele- tion of a stretch of 12 amino acids encompassing the 4 cysteine residues renders the deletion mutant of SNAP-25 completely soluble and resistant to C3H] pal-
aSN23.CI2 + SNAP-23 peptide
FIG. 2. Expression of SNAP-23 in cc11 lines and tissues. (A) Im- munoblot of total membranes prepared from various ce11 lines of different species. Twenty micrograms of proteins from ra t neuroendo- crine PC12 cells, r a t L6 myotubcs (L6 Mt), mouse 3T3-LI fibroblasts (3T3-L1 FI and 3T3-L1 adipocytes (3T3-L1 A), mouse melanocytes (melan-b), and human MeWo melunoma ce11 line were resolved by SDS-PAGE and prohed for SNAP-23 with aSN23.Cl2 antiscmm (0.12 pg/ml) (top). The specificity of immunodetection was confirmed by preincubation of aSN23.CI2 antiserum with excess amount of peptide (0.2 pglml) (middle). Dctection of SNAP-25 by t he monoclonal antibody aSN25mAb (SM1 81) wus rcstricted to neuroendocrinc PC12 cells (bottom). (B) Imiriunoblotting of total membranes from ra t skeIetal muscle (Sk. muscle) a n d othcr tissues from mouse with aSN23.Cl2 antiscrum.
3T3-LI F TM CYT --
kDa
FIG. 3. Localization of SNAP-23 in 3T3-L1 cells. (A) 'lùlenty micmgrams of total membranes (TM) and cytosolic fraction (CYT) from 3T3-L1 fibroblasts (3T3-L1 F) were subjected to immunoblotting using the aSN23.Cl2 antiserum. (6) Twenty micrograms of total membranes (TM) from 3T3-L1 fibroblasts (3T3-L1 F) and adipocytes (3T3-L1 A) were immunoblotted for SNAP-23 using aSN23.Cl2 antiserum. ( C ) To study the subceilular distribution of SNAP-23 in 3T3-L1 adipocytes (3T3-L1 A), cells were subfractionakd and equal amounts (15 pg) of plasma membrane (PM), light density microsomes (LDM), and cytosolic fraction (CYT) were separated by SDS-PAGE and probed for al subunit of Na+/K+ ATPase with 6H monoclonal antibody, GLUT4 with a polyclonal antiserum, and SNAP-23 with aSN23.Cl2 antiserum.
mitic acid labeling (22). In order to determine the local- ization of SNAP-23 in 3T3-Ll fibroblasts we used sub- cellular fractionation. The results in Fig. 3A show that SNAP-23 is exclusively found in the membrane fraction (TM) with no detectable amount found in the cytosolic &action (CYT). Since SNAP-23 also contains a cluster of cysteine residues, it may associate with the mem- brane by a sirnilar mechanism. Xnterestingly, SNAP- 23 contains 5 cysteine residues in this cluster, as is the case for Torpedo SNAP-25 (23). In contrast, the marnmalian SNAP-25a and SNAP-25b have each a substitution of one of the cysteines, breaking the clus- ter arrangement. The extra cysteine residue found in SNAP-23 and Torpedo SNAP-25 may define a n abibty to associate with specific proteins.
3T3-LI fibroblasts can be differentiated into adipo- cytes, hence allowing us to study the protein a t two stages of differentiation. Proteins that are involved in intracellular traffic have been s h o w to be expressed a t different levels following differentiation into adipo- cytes. For example, cellubrevin, a member of the synap- tobrevin family, is expressed predorninantly in differ- entiated 3T3-LI adipocytes (24). In contrast, syntaxin 4 is already abundant a t the fibroblast stage (Volchuk and Klip, unpublished). As can be seen in Fig. 3B, there was no significant change in the level of expression of SNAP-23 upon differentiation, suggesting that the protein is necessary in both fibroblasts and adipocytes.
The detection of SNAP-23 in 3T3-L1 adipocytes pro- vided us with an oppoitunity to further study the sub- cellular distribution of the protein in plasma mem- brane (PM), light density microsomes (LDM), and cyto- sol (CYT). In contrast to fi broblasts, the fractionation protocol for 3T3-LI adipocytes has been well estab- lished (25). The c u l subunit of Nai/K' ATPase is a use- ful marker for the PM. This protein was detected al- most exclusively in the P M and not in the LDM or CYT of 3T3-L1 adipocytes (top panel of Fig. 3C). The LDM contains the majority of the GLUT4 glucose trans- porter which locates intracellularly in unstimulated
cells (Fig.ôC, rniddle panel). The majority of SNAP-23 was found to be associated with the PM (Fig. 3C, bot- tom panel). Occasionally, a small amount of SNAP-23 was also detected in the LDM (not shown).
The functional importance of SNAP-25 in synaptic vesicle traffic has been established by the discovery that it is the substrate for botulinum neurotoxins A and E (26, 27). These toxins are endopeptidases and potent inhibitors of neurotransmitter release. Botuli- num neurotoxins A and E cleave SNAP-25 a t distinct peptide bonds ( 10). Sequence alignment of SNAP-23 and SNAP-25 proteins reveals that the two amino acids which constitute the proteolytic cleavage site for botuli- num neurotoxin type E, but not those for type A, are conserved in SNAP-23. Therefore we predict tha t SNAP-23 rnay be protected from proteolyhc cleavage by botulinum neurotoxin A. Botulinum toxin E may be a useful b o l to discern whether SNAP-23 plays a role in exocytosis. Indeed, we have shown that both SNAP- 23 and SNAP-25 are present in PC12 cells; i n these celIs, exocytosis of large dense core vesicles is iahibited completely by botulinum neurotoxin E but not A, al- though both toxins effectively cleaved SNAP-25 (28). The presence of SNAP-23 and its potential insensitivity to proteolysis by botulinum neurotoxin A may explain the incomplete inhibition in the peptide hormone secre- tion observed in PC12 cells.
The detection of SNAP-23 in non-neuronal cells com- pletes the tenet of the SNARE hypothesis, that func- tional homologues of SNAP-25, syntaxin-l and VAMP exist at al1 steps of vesicle docking/fusion in non-neu- ronal cells. Since muscle and 3T3-L1 cells express syn- taxin 4 (29, 30) and VAMP-2 (31), it is tempting to speculate that SNAP-23 could potentiate the binding of syntaxin 4 to VAMP-2 and in this way contribute to the formation of a stable complex that would facilitate exocytic activity in these tissues.
ACKNOWLEDGMENTS We thank Dr. Michncl Cnplnn for the anti-Nai/K+-ATPase anti-
body, Dr. L. Liu foi. hclp in the afiinity purification of antisera, and
Dr. P.J. Bilan for critical reading of the manascnpt. This work was 15. Nagamatsu, S., Kornhauscr, J. M., Burant, D. P., Seine,, S., supported by a g a n t from the Juvenile Diabetes Foundation to A.K. Mayo, K. E., and Bell, G. 1. (1992) J. Biol. Chem. 267, 462-472. and W.S.T. 16. Laemmli, U. K. (1970) Nature 227,680-685.
17. Sarnbrook, J. , Fritsch, E. F., and Maniatis, T. (1989) Molecular
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SNAP-23 IS LOCATED IN THE BASOLATERAL PLASMA MEMBRANE OF
RAT PANCREATIC ACINAR CELLS
Herbert Y. Gaisano', Laura Shed, Peggy P. C . wong2, Amira IUipL3, William S. ~rirnble".
From the D e p m e n t s of Medicine1, Biochemistry2 and Physiologg, University of Toronto, The Toronto Hospital, Toronto, Ontario MSS 1 ~ 8 ' , and the Division of Cell Biology of the Hospiral for Sick Children Research Institute, Toronto, Ontario, M5G 1x8'.
Please address correspondence to: Herbert Y. Gaisano Room 7226 Medical Sciences Building University of Toronto Toronto, Ontario, Canada M5S 1A8 Tel. (416) 97s-1526 Fax. (4 16) 978-8765 E-mail. [email protected]
Kunning title: SNAP-23 in rat pancreatic acinar cells
The SNARE hypothesis proposes that specificity of exocytotic fusion processes is regulated
by the appropriate interactions of the vesicle (v-) SNARE VAMWsynaptobrevin and the target
membrane (t-) SNAREs syntaxin and SNAP-25. Recently we demonstrated that VAMP-2 was
present on the pancreatic acinar rymogen granule membrane while syntaxin-2, 4 and -3 were
localized to the apical plasma membrane, basolateral membrane and zymogen granule membrane,
respectively. However, other studies had suggested that SNAP-25 was not expressed in non-
neuronal-cells such as the achar cell. We therefore began to search for SNAP-25 like proteins
in exocrine pancreatic tissue. Using an isofom-specific antibody generated to the first novel
SNAP-25-like protein reported outside the nervous system, SNAP-23, we now show by
immunoblotting that achar cells express SNAP-23, and this t-SNARE is located on zymogen
granule membranes and the plasma membrane. Using laser confocal immunofluoresence
microscopy we show that the plasma membrane component of SNAP-23 is distinctiy locaiized
to the basolateral plasma membrane of acinar cells. This colocalization of t-SNAREs SNAP-23
and svntaxin-4 to the basolateral' plasma membrane, coupled with their ability to form a ternary
complex with the V-SNARE, VAMP-2, suggest that this SNARE complex may serve as the
rnolecular substrate for basolateral membrane fusion of secretory granules, currently believed to
be a phenonlenon occuring in acute pancreatitis.
Key words: esocytosis / pancreatitis / SNAREs
The pancreatic acinar cell has long been used as a mode1 ce11 type for the smdy of
exocytosis in non-excitable cells (1). Through intensive study, the kinetics and morphology of
complex constitutive and regulated exocytotic pathways have been defined in these highly
polarized cells (2). Regilated exocytosis of zymogen granules (ZG) in response to low,
physiologie concentrations of secretagogues, occurs at a limited apical portion of the acinar cell,
which constitutes only 5-10% of the total PM surface (1,3). Enzyrnatic ZG contents are then
delivered io the collecting ducts of the paocreas and subsequently emptied into the intestinal
lumen to digest nutrients. In contrast, exposure of acinar cells to supraphysiologic secretagogue
concentrations either in vipo or in vivo appears to cause a blockade of apical exocytosis, and
aberrant ZG fusion events including intragranular fbsions and fusion with the basolateral plasma
membrane (4). in vivo, this basolateral ZG secretion ,resuhs in expedenta l conditions which
appear analogous to clinicai pancreatitis (4) where serum levels of pancreatic enzymes are
elevated. However, the mechanisrns controlling normal exocrine secretion, and their possible
dysfunction during pancreatitis, remain to be determined.
Recent studies by us and others suggest that regdation of ZG fusion may follow the same
basic principles as described in the SNARE hypothesis. This general mode1 was derived to
explain mechanisms controlling neurotransrnitter reIease, and extrapolated to cover most
membrane transport processes in virtually al! cells (5). The SNARE hypothesis predicts that
cytosolic NEM-sensitive factors (NSF) and soluble NSF attachment proteins (SNAPs), bind to
(t-SNAREs), to mediate the docking and fusion o f the two membranes (5). In the nervous system
the vesicle-associated membrane proteins (VAMPS) on the synaptic vesicle, along with syntaxin
1 and synaptosomal-associated membrane protein of 25kDa (SNAP-25) on the synaptic plasma
membrane, fgrms a stable cornplex which acts as the receptor (SNARE) for the soluble factors
(reviewed in 6). It is hypothesized that specific v- and t-SNARE combinations may control the
accuracy of vesicle-target membrane interactions. The presence of multiple isoforms of VAMP
(7) and syntaxin proteins (S), and the specificity of their interactions (9), support this tenet.
~oweve< SNAP-25 is expressed predominantly in the nervous systern and in neuroendocrine cells
(10) leaving non-neural cells without a cntical SNARE component. Recently, Ravichandran et
al. ( 1 1) reported the cDNA cloning of a novel isofom of SNAP-25, called SNAP-23, which is
broadly expressed in non-neuronal tissues, hcluding the pancreas. ht ibodies specific for
SNAP-23 detect a single protein species associated with the plasma membrane which is abundant
in most non-neural but not neural ceIls (12). This identification provides a complete cassette of
SN= proteins in virtualiy al[ ceII types.
To deterrnine if exocrine granule secretion is controlled by mechanisms similar to those
involved in neurotransmitter release, we besan to search for expression of SNARE homologs in
pancreatic acinar cells. lnit ialIy we demonstrated that VAMP isoform-3 was an integral
component of the zymosen ganule membrane and cleava~e of this protein by tetanus toxin
resulted in inhibition of calcium-evoked digestive enzyme secretion ( 1 3,14). More reccntly we
have demonstrated that acinar cells express four isoforms of syntaxin with syntaxin isoforn-i-2
4 on the basolateral plasma membrane of the pancreatic acinar cell ( 1 5). Taken in the context of
the SNARE hypothesis, their restricted appearance at these membrane compartments indicates that
each of these syntaxins rnay serve as a distinct exocytotic target for each site (6).
In the present study, we demonstrate that acinar cells also express SNAP-23. Using an
affinity purified antibody directed to the C-terminus of SNAP-23 (1 S), we show that SNAP-23,
but not SNAP-25, cm be detected in the acinar cells while endocrine celis of the pancreas express
SNAP-25 only. We also show that SNAP-23, like syntaxin-4, is specifically localized to the
basolaterd plasma membrane of the acinar cell. The basolateral location of SNAP-23, dong with
that of syntaxin 4, suggests that these proteins do not participate in apical secretion, but they may
be învolved in normal or aberrant secretory events occurring at the basolateral surface.
Anfibody Generation. Rabbit polyclonal antibodies to VAMP-2, syntaxins-2 through -4
and SNAP-23 were generated, subjected to affinity purification and their specificity validated as
we have previously reported (12- 15). Anti-SNAP-23 specific antibody was generated against a
peptide correspondhg to the carboxy-terminal residues 200-21 1 of SN* 23, called aSN23.C 12,
and affinity purified on columns to which the peptide had been cuupled (12). Antibody raised
against SNAP-25, kindly provided by M.K. Benne& was generated against the bacterially-
expressed-full length fusion protein of SNAP-25 and affinity-punfied using full-length fusion
protein immobilized on Affigel beads (Bio-Rad, Richmond, CA). The specificity of these
antibodies was detemined by immunoblotting against the recombinant full-length fusion proteins
and a g a k t native tissues (see Figures 1 through 3) wtuch showed that while the SNAP-23
antibody is specific only for SNAP-23, the SNAP-25 antibody cross-reacts with b o t . SNAP-23
and SNAP-25.
SubceiIular Membrane .PreparaZions and immunoblotling. Pancreatic achar ZG
membranes and plasma membranes were prepared from excised pancreata of Sprague-Dawiey rats
(250-300 g) made diabetic, as described (13-15), by treatment with stretozotocin which oblirerates
p-cells. Total membranes were prepared from 3T3-L 1 fibroblasts as previously reported ( 1 2).
SDS-PAGE and immunoblots were performed as described (13) using prirnary antibodies diluied
to 1: 1000. Detection of the antigen on the blot was by enhanced ctieniiluminescence (ELL,
Aniershani Corp.). Specificity was determined by preincubating the antibodies with IO bl excess
ternperature prior to use as in Figure 1.
hmunofluorescence Confocal Microscopy. T hese studies were performed as previous 1 y
described (l4,l5). Briefly, pancreata from a diabetic or normal rat that have been perfused in
vivo with 4% paraformaldehyde in physiological saline and frozen in liquid nitrogen, were
ernbedded in Tissue Tek OCT. 5 - p n cryotstat sections were generated, nnsed in PBS and
postfixed in 4% paraformaldehyde, rinsed again with PBS, and then blocked with 5% normal goat
serum wifh 0.1% saponin for 30 min. The tissue on the glass slides was then incubated ar room
temperature for 1 hr with the primary antibodies: rabbit anti-SNAP-23 (1 :50), rabbit anti-SNAP-
25 (1:50), guinea pig anti-insulin (1:100, a gift from R. A. Pedersen, University of British
Columbia, Vancouver, Canada). Specificity was detennined by preincubation of the primary
antibody with 10 M excess ( 4 0 ~rg'rnl) of full length recombinant fusion proteins of the
appropnate isoforms for 1 hr at room temperature prior to use. These were then rinsed with
0.1% saponin, and then treated with appropriate FITC- or rhodamine-IabeIed secondary antibody
(1 hr, room temperature): goat ati-rabbit (1 500) and goat anti-guinea pig ( 1 :500). Phalloidin
conjugated to FITC (Molecular Probes, Eugene, OR) was added during the secondary antibody
step at 1 U/slide. The slides were then mounted with fading retarder; 0.1% p-Phenylenediarnine
in glycerof, and exarnined using a Iaser scanning confocal imaging system (Cari Zeiss,
Thomwood, NY).
S M - 2 3 is preseni on pancreatic plasma membrane and zymogen granule mem brunes.
To determine the presence and subcelluIar distribution of SNAP-23 andor SNAP-25
immunoreactive proteins in the pancreatic acinar cell, we isolated preparations of highly purified
pancreatic acinar ZGM and PM for imrnunobiot analyses (Figure 1). To avoid possible
contamination with islet membranes which we (16) and others (17) have reported to be abundant
in SNAP-25, membranes were isolated from streptozotocin-treated rats as previously described
(13-1 5). ~ h e s e were then compared to a homogenate of rat brain, which is abundant in SNAP-
25 (18), and 3T3-LI fibroblast total membranes, which are abundant in SNAP-23 (12). The top
row shows that SNAP-23, which migrated at about 30kDa, is abundant in both pancreatic PMs
and ZGMs as in the 3T3-LI fibroblast membranes, but is virtually absent in the rat brain. The
SNAP-23 signal was not affected by preincubation with excess GST-SNAP-25 (1 pg) (second
row) but was blocked when preincubated with GST SNAP-23 (third row), c o n f ~ n g the
specificity of the SNAP-23 antibody. The SNAP-25 antibody gave a strong immunoreactive
signal in the rat brain and a weaker signal in the pancreatic PM, ZGM and 3T3-L1 membranes
(fourth row). GST-SNAP-23 had no effect on the SNAP-25 signal in rat brain but significandy
inhibited the s i s a l in the pancreatic membranes and 3T3-L1 membranes (row 5), whereas GST
SNAP-25 completely blocked al1 of the signais from this antibody (row 6). Furthemore, a
monoclonal antibody (SM1 81, Stemberger Monoclonal Inc.) which reco~nizes brain and islet
SNAP-25 failed to recognize the acinar isoforms. However, this antibody gave a strong cross-
reaction to high molecular weight material within the ZG (data not shown) and was not used
pancreatic PM and ZGM, and 3T3-L1 membranes, was actually SNM-23 to which the
SNAP-25 antibody cross-reacted.
The SNAP-23 on the ZGM is tightly associated with that membrane since it was resistant
to washing with NaBr (0.25 M, pH 11) which removes peripherally adhering proteins. It is
unlikely that the ZGM signal could be due to contamination with PMs since the ZGMs were
prepared from a highly purified ZG fraction (13-15), and the Coomassie blue staining of the
ZGM a d PM fractions showed the membrane-protein profiles to be distinct (data not shown).
Furthemore, a slight contamination with PM wouid not explain the intensity of the ZGM signal
which is equivalent to that on the PM.
SNAP-23 and SNM-25 are present in pancreatic acinar cel2.s and islets, respectively. The
pancreas provides an ideal opportunity to compare the endocrine and exocrine tissues side-by-side
for the presence of SNAP-25 and SNAP-23 (Figure 2). We therefore performed double label
immunofluorescence microscopy on pancreatic sections taken from normal (non-diabetic) rats
using affinity-purified SNAP-23 and SNAP-25 antibodies individuafly along with antibodies
specific to rat insulin raised in winea pigs. In the islets, insulin (Figure 2b) was restricted to
cells in which SNAP-25 was abundant and localized to the ceIl membrane (Figure 2a). ln
contrast, SNAP-23 antibody strongly stained the plasma membrane of every acinar ce11 (Figure
2c), but minirnally if at al1 in the insuiin-positive islets (Figure 2d). Therefore, unlike VPj'viP
and syntaxin proteins which are expressed in both the endocrine and exocrine pancreas ( 14- 161,
respectively.
S W - 2 3 is located on the basolateral plasma membrane of the pancreatic acinar cell.
To more precisely map the subcellular location of SNAP-23 in acinar cells we have performed
double label immunofluoresence microscopy on sections from the pancreas of diabetic rats with
the SNAP-25 and SNAP-23 antibodies along with phalloidin, which labels actin filaments near
the apical surface of the acinar ce11 (1 5,19). As shown in Figure 3% the SNAP-23 antibody
labels thé basal and lateral plasma membrane of every acinar ce11 but not the apical portions
which are labeled by phalloidin ( F i g r e 3b). hti-SNAP-23 also Save weak and inconsistent
labelling of interna1 stnictures which may include the ZGs or other intracellular membranes. The
SNAP-23 signal was completely blocked by GST-SNN-23 (Figure 3c) but was not affected by
preincubation of the antibody with GST-SNAP-25 (Figure 3d). A similar pattern was detected
with the SNAP-25 antibody (Figure 3e) when the laser intensity was significantly increased,
although this could not be seen at intensities used for SNAP-23 (See Figure 2a). The SN*-25
signal on the acinar celI was completely blocked by preincubation o f the antibody with GST-
SNAP-25 (not shown), as well as with GST-SNAP-23 (Figure 39. The GST-SNAP-23, however,
did not block the SNAP-25 signal of a nerve fiber straddling the basal surface of an acinus
(arrowhead in f). Again, this confirms that the only imrnunoreactivity detected by the SN*-25
antibody in exocrine acinar cells wa.s SNAP-23. The ZG membrane SNAP-23 signal in Our
immunofluorescence study was not consistently seen, in conuast to our irnmunoblotting study.
This may be due to preferential sensitivity o f the ZGM-bound SNAP-23 to the aldehyde fixatives
ZGM SNAP-23.
SNAP-23 is a recently described, widely expressed isoform of the neuronal t-SNARE
SNAP-25 (1 1,12). SNAP-23 is 59% identical to SNAP-25 at the amino acid Ievel and its rnR.NA
and protein are expressed in non-neuronal tissues (1 1,12). To determine which of these two
proteins are present in pancreatic acinar cells, we used polyclonal antibodies specific for SNAP-
25 (1 5,16), and SNAP-23 ( 12) in Western blotting and immunocytochemical snidies. The
antibodies raised against SNAP-25 recognized proteins in the rat brain, pancreatic islet, and
exocrine acinar celi. However, the latter signal appears to be the result of cross-reaction since
it could be fully blocked by pre-incubation of the antibodies with recombinant SNAP-23 proteins.
In contrat, anti-SNAP-23 antibodies gave strong signais for acinar ce11 membrane preparations
and littie if any signal for neural membranes, and this signal was not sensitive to blocking by
preincubation with GST-SNAP-25 but was eliminated by GST-SNAP-23. Together with a
previous study which failed to detect SNAP-25 in the acinar cell using an antibody specific to
the carboxyl terminus of SNAP-25 (17), our results show that SNAP-23 is the predorninant
isoform and that SNAP-25 is not expressed in these cells.
The pancreas is a tissue which allows sirnultanmus assessrnent of protein expression in
both endocrine and exocrine cells. We observed that SNAP-23 was expressed in the exocrine but
not endocrine cells, the reciprocol of that seen for SNAP-25. hterestingly, the other SNARE
proteins VAMP-2 and syntaxin-1 appear to be expressed in both ce11 types (13,15-17) albeit at
much higher levels in endocrine cells. This suggests that the SNAP-2YSNA.P-25 cornponent of
of this difference will await fùrthei characterization of the precise role of SNAP-23 in membraiie
fusion.
Within the pancreatic acinar d l , SNAP-23 is present in abundance on the PM and on the
ZGM. On the PM, the SNAP-23 was highly enriched on the basoiateral membranes in the acinar
cells and was excluded from the apical surface. These subcellular locations overlap with the
patterns we previously obtained for syntawin-3 (ZGM) and syntaxin4 (basolateral PM) in acinar
celis (15): Although SNAP-23 can form binary interactions with al1 four of the syntaxin isofoms
in vitro, it is interesting to see that it CO-localizes with only these two, and that no CO-localization
was seen with the apicaliy locaiized syntaxin-2. S W - 2 3 is therefore unlikely to be localized
soiely by its association with the syntaxins, but wheher it is targeted by association with other
proteins or carries its own targeting signals is not known.
The appearance of SNAP-23 at the basolateral surface respresents the first evidcnce for
regional localization of this n e w t-SNARE isoform within polarized cells and a r g e s that this
protein may participate, along with syntaxin-4 and a member of the VAMP family to mediate
basolateral membrane fusion events. In vitro studies have shown that VAMP-1 and VAMP-2 cm
bind to syntaxin-1 or syntaxin-4 with high affinity, but not with syntaxin-2 or -3, su_ogesting
specificity of vSNARE-tSNARE interactions (9). SNAI?-25 and SNAP-23, on the other hand,
do not appear to contribute to this specificity and are capable of binding to each of the syntaxin
and VAMP isoforms (9, l l ) . Given the presence of VAMP-2 on the ZGM, a complete cassette
rarely occurs under normal cirrumstances, this mechanism must be under additional levels of
control. Interestingly, in response to high agonist concentrations, interstitial pancreatitis c m result
from the inappropriate release o f zymogens to the basolateral surfaces (4,20). Whether such
events result from a failure of such control mechanisms, o r entry of the vesicles into minor
constitutive exocytosis pathways operating through the basolateral surfaces (2 1) is not known.
The functional significance of SNAP-23 localized to the ZGM is not ctear, but vesicular
localizati6ns have been previously detected for both syntaxin-l and SNAP-25 on synaptic vesicles
(22) and on the chromaffin granule membrane (23,24). Within the context of the SNARE
hypothesis, ZGM localized SNAP-23 could be explained if the ZGM itself were to act as a target
membrane for fusion (1 5). This idea is supported by previous studies demonstrating ZG-ZG
fusions in vivo (4) and in vitro (25). Such fusions between Golgi-derived progranules may be
necessary for the formation of immature and mature ZGs (26), while fusions between mature
granules could be important to promote wmpound exocytosis. If SNAP-23 forms SNARE
complexes with syntaxin-3, the V-SN- with which they associate remains to be identified.
Finally, the location of SNAP-23 to the basolateral membranes, coupled with the
observation that syntaxin isoforms were also distinctly localized within these cells, suggests that
apical secretion is quite distinct from basolaterai secretion. Since the only identified V-SNARE
on the ZGM, VAMP-2, does not form stable complexes with the apical t-SNARE syntaxin-2 in
vitro, it is possible that additional isoforms of SNAP-25 and VAMP niay exist co provide a
secretion may occur by m e a s other than those explained in the SNARE hypothesis. It has been
shown in Madin-Darby canine kidney cells that vesicular transport from the tram-Golgi network
to the basolateral plasma membrane is dependent on NSF and a S N A P , and is sensitive to tetanus
and botulinum neurotoxins which cleave VAMP-2 (27). In contrast, apical secretion in MDCK
cells is independent of NSF, a S N A P and insensitive to tetanus toxin (27). In Our studies, we
have found that while Ca2'-evoked amylase secretion from isolared, permeabilized acinar cells
can be inhibited by tetanus toxin cleavage of VAMP-2, this inhibition is always partial (13). We
are currefitly investigating the possibility that the permeabilized preparation may exhibit both
apical and basolateral secretion with only the latter being sensitive to teranus toxin. If so, this
may provide a valuable mode1 system to examine the mechanisms conuolling normal granule
targeting specificity and to develop compounds which couid be effective at blocking the
basolateral secretion associated with pancreatitis. ,
In s u m m q , we have demonstrated that SNAP-23 is the predominant isofonn of the
SNAP-25 t-SN- family expressed in the exocrine cells of the pancreas. Furthemore, we have
demonstrated that it is localized abundantly on the basolateral surface of these cells where it could
interact with syntaxin4 and the ZGM V-SNARE VAMP-2 to participare in the aberrant secretory
events associated with experimental pancreatitis. More work is required to identify the apical and
vesicular proteins responsibie for regulated apical secretion in acinar cells.
supplying the anti-SNAP-25 and anti-insuIin antibodies, respectively. This work was supported
by grants from the Medical Research Council of Canada to WST and HYG, the Fraser Elliott
Foundation to HYG, and the Juvenile Diabetes Foundation to AK and WST. HYG 1s a recipient
of an American Gastroenterology Association Foundation/Industry Research SchoIar Award.
Foonzotes.
'The ab breviations used are: VAMP-2, vesicle associated membrane pro tein isoform 2; ZG,
zymogen granule; ZGM, zymogen grande membrane(s); SNAP-25 (and 23), synaptosomal
associated protein of 25 kDa (and fi ma); PM, plasma rnembrane(s); SNARE, soluble NSF-
attachment protein (SNAP) receptor; V-SNARE, vesicle-SNARE; t-SNARE, target-SNARE; SDS-
PAGE, sodium dodecyl sulfate-~olyacrylamide gel electrophoresis.
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Figure 1, SNAP-23 is present on pancreatic acinar plasma membranes and zymogen
granule membranes. Highly purified pancreatic acinar plasma membranes (PM, 10 pg of
proteidlane) and zymogen granule membranes (ZGM, 10 pg of proteidlane) from diabetic
(streptozotocin-treated) rats, cmde brain homogenates (rat brain, 5 pg of proteidlane) and 3T3-
L l fibroblast microsornes (3T3-LI, 10 pg of protein/lane) were prepared, electrophoresed on a
15% SDS-polyacrylamide gel and blotted on Immobilon-P transfer membranes (MiIlipore) as
describe& in Methodr. These were the immunoblotted with: Top row - SNAP-23 antibody;
second row - SNAP-23 antibody preincubated with GST-SNAP-25 (1 pg); third row - SNAP-23
antibody preincubated with GST-SNAP-23 (1 pg); fourth row - SNAP-25 antibody; fa row -
SNAP-25 antibody preincubated with GST-SNAP-23; and bottom row - SNAP-25 antibody
preincubated with GST-SNAP-25.
Figure 2, SNAP-23 is present in pancreatic acinar ce11 and SNAP-25 is present in
pancreatic islets. Laser scanning confocal microscopy was used to disthguished the distribution
of SNAP-25 (in A) and SNAP-23 (in C) in normal (non-diabetic) pancreatic tissue from a rat.
These tissue sections were double labeled with guinea-pig anti-insulin antibody to indicate the
location of the islets (in B and D).
Figure 3. Subcellular location of SNAP-23 to the basolateral plasma membrane of
pancreatic acinar cells by laser confocal microscopy. Pancreatic sections from diabetic rats
.. -
with either GST-SW-23 (C,F) or GST-SNAP-25 (D) recombinant proteins were peGormed
prior to immunostaining to demonstrate the specificity of the irnmunostaining. SNAP-25 in a
nerve fiber (arrowhead in F) was not blocked by GST-SNAP-23. Double labelling with FITC-
phdloidin (B) was perfonned to indicate the apical portion of the achar cells (arrows in A and
W.
Exercise increases the plasma membrane content of the Na+-K+ pump and its rnRNA in rat skeletal muscles
TlKEODOROS TSAKIRIDIS, PEGGY P. C. WONG, ZHI LIU, CAROL D. RODGERS, MLADEN VRAMC, AND AMIRA ICLIP Diviswn of Cell Biology, The Hospital for Si& Chddren, Toronto M5G m8; Departments of Physwlogy a d Medicine, University of Toronto, Toronto M5S iA8; a d School of Physical and Health Education, University of Toronto, Toronto, Ontario M5S iAI, Canada
T8akiridis, Theodoros, Peggy P. C. Wong, Zhi Liu, Carol D. Rodgers, a d e n Vranic, and Amira Xiip. Exercise increases the plasma membrane content of the Na+-K+ pump and its mRNA in rat skeletal muscles. J. Appl. Physiol. 80(2): 699-705, 1996.-Muscle fiberri adapt to ionic challenges of exercise by increasing the plasma membrane Na+-K+ pump activity. Chronic exeruse training has ben shown to increase the total amount of Na+-K+ pumps present in skeletal muscle. However, the mechanism of adaptation of the Na+-K+ pump to an acute bout of exercïse has not been determined, and it is not known whether it involves alter- ations in the content of plasma membrane pump subunits. Here we examine the effect of 1 h of trearimill running (20 m/min, 1û% grade) on the subcellular distribution and expres- sion of Na+-K+ pump subunits in rat skeletal muscles. Red type I and IIa (red-I/IIa) and white type IIa and iIb (white-IIa/ IIb) hindlimb muscles h m resting and exercised fernale Sprague-Dawley rats were removed for subcellular fraction- ation. By homogenization and gradient centrifugation, crude membrane8 and purified plasma membranes were isolated and subjecteà to gel electrophoresis and immunoblotting by using pump subunit-specific antibodies. Furthemore, mRNA was ieolated h m s p e a c red type I (red-1) and white type IIb (white-IKb) muscles and subjected to Northern blotting by using subunit-spdc probes. In both red-I/IIa and white-IIa/ IIb muscles, exercise signilïcantly raised the plasma mem- brane content of the al-subunit of the pump by 64 a 24 and 55 + 2296, respectively (P < 0.05), and elevated the a=- polypeptide by 43 + 22 and 94 2 39%, respectively (P < 0.05). No eigniscant effect of exercise could be detected on the amount of these subunits in an interna1 membrane &action or in total membranes. In addition, exercise significantly in- creaaed the al-subunit mRNA in red-I muscle (by 50 i 7%; P < 0.05) and the p2-subunit mRNA in white-IIb muscles (by 64 + 1996; P < 0.01), but the (Y*- and PI-mRNA levels were un&ected in this t h e period. We conclude that increased presence of al- and a2-polypeptides at the plasma membrane and subsequent elevation of the al- and Pz-subunit niRNAs may be mechanisms by which acute exercise regulates the Na+-K+ pump of skeletal muscle. sodium-potassium-adenosinetriphosphatase; sodium-potas- sium pump; red muscles; white muscles; muscle fiber type; contraction; messenger ribonucleic acid
THE NA+-K+ ADENOSINETRIPHOSPHATASE (ATPase) or Na+-K+ pump [EC 3.6.1.371 is a transmembrane pro- tein complex tha t functions a t the ce11 surface to main-
tain the intracellular Na+ and Kr concentrations hy simultaneously pumping Na+ out of the cell and K+ into t h e cyt-oplasm. In resting skeletal muscle, the plasma membrane Na+-K+ pump operates a t only a small fraction of its total maximal capacity (5). During con- tinuous muscle contraction, the rates of Na+ infiux and K+ efflux increase markedly and plasma K+ i-ises because of multiple depolarization-repolarization cycles (5). Na+-K+ pump activity rises in an attempt to deal with these changes in ionic gradients (5). However, the mechanism of this activation of the piiinp has not been determined. The adivity of the Na+-K+ pump c m be elevated in part by substrate mass action, i.e., by a rise i n the levels of intracellular Na+ (5, 10). However, stimulation of Na+ efflux and Rb+ infiux can be ob- served in isolated skeletal muscles even before any significant increase occurs in intracellular Na+ or extra- cellular K+ concentrations (11). Cell-surface pump activ- ity can be increased through either an elevation in the rate of the activity of each pump or a gain in the number of pump units at the plasma membrane. The latter could take place through recruitment of units from other intracellular compartments or increased stability of the surface pumps (i.e., reduced degrada- tion rate). None of these events have been explored to explain the regdation of the Na+-K+ pump in skeletal muscle by exercise.
The purpose of this study was to analyze the biochemi- cal mechanisms employed by the muscle to regdate the pump components during a single bout of exercise. For biochemical studies, substantial amounts of tissue are typically required to generate membranes and deter- mine pump content. Skeletal muscle is a heterogeneous tissue, giving rise to the possibility that each fiber type may respond to exercise differently. Indeed, muscle fibers contrast in their contractile, electrophysiological, and metabolic characteristics (3). Ln mammals three main categories of muscle fiber types have been de- scribed: type 1 (red, slow twitch, oxidative), type Ha (red/white, fast twitch, oxidative and glycolytic), and type IIb (white, fast twitch, glycolytic) (3).
I t is believed that the minimal functional unit of Na+-K+ pump is an a-p-subunit heterodimer (13, 23). The a-subunit (110 kDa) has the catalytic activity of the enzyme and contains binding sites for al1 its substrates
0161-7567196 $5.00 Copyright O 1996 the Amenciin Physiologicol Society 699
(Na+, K+, ATF', and Mg2+), but its enzymatic activity relies on its association with a glycosylated P-subunit (50 ma) . Two a- (cri and arz) and two 8- (pl and p2) isoforms are expressed in rat muscle (13). We have previously observed a differential segregation of pump subunits in red and white rat skelehl muscles (17). The al- and a2-polypeptides are expressed in al1 muscle fibers (17). In contrast, the j31-polypeptide is expressed primarily in muscles rich in type I and IIa fibers but is absent from muscles composed primarily of type IIIb, w h e m is found in muscles composed primarily of type IIb and is absent from muscles composed mostly of type 1 fibers. It is not known whether type Da fibers express both p-isoforms.
Whereas there are no muscles of pure fiber type composition, the rat offers the opportunity to study a t the biochemical level red muscles composed mainly of type 1 and Ila fibers (red-I/IIa) or white muscles composed mainly of type IIa and Iib (white-IIa/ïIb) h. Small quantities of muscle (of -2 g) of each type can be isolated h m rat hindlimbs for subcellular fiactionation proce- dures to yield crude membranes, plasma membranes, and internat membranes. M h e r m o r e , single muscles of mainly type 1 or type 1% fibers an be isolated for &A studies.
The objectives of the present study were 1) to deter- mine the effect of exercise on the amount of each subunit of the pump in isolated plasma membranes h m red-I/IIa or white-IIa/IIb muscles and2) to exam- ine whether a single bout of exercise can also initiate changes in pump subunit mR.NA expression. We found that 1 h of treadmiU running (20 m/min, 10% grade) increases the plasma membrane levels of al- .and a2-polypeptides of the pump in both red-I/IIa and white-Ila/IIb muscles. Furthemore, the exercise bout elevates the a,- and P2-mRNA content in red-I and white-IIb muscles, respectively.
METHODS
Materials. Monoclonal antibodies anti-Na+-K+-ATPaseq (Mck-1) and anti-Na+-K+-ATPase- Ncb-2) were kind gift~ of Dr. K Sweadner (Boston, MA) (23). Polyclonal antitiNa+- K+- ATPase-Pl and anti-Na+-K+-ATPase-B2, raised against peptides of the extracellular domains of the proteins, were kindly donated by Dr. P. Martin-Vasallo (Tenerife, Spain). The specificity of the these antibodies in skeletal muscles was recently examined (20a). The fuil-length cDNA probes were kind giRs of Dr. J. Lingrell (Cincinnati, OH). Restriction enzymes for the cDNA probes Nad, StuI, ScaI, NheI, NcoI and SspI were obtained from Promega (Madison, WI). The materials and chernicals for sodium dodecyl sulfate-polyacryl- amide gel electrophoresis and immunoblotting were obtained from BioRad (Mississauga, ON, Canada) All other chemicals were obtained from Sigma Chernicai (St. Louis, MO).
Animals and muscle preparation. The week before the day of the exercise bout, 12 female Sprague-Dawley rats weighing 250 i 25 g were accustomed to treadmill running by one 5-min running period once every 2 days (no d g was done the day before the experimental exercise bout). On the day of the experiment, animals were separated randomly into two groups, exercise and resting control (6 animals per group). The exercise group performed 1 h of treadmiil running at 20 &min up a 10% grade. Imrnediately afkr exercise or rest,
animals were Idled by decapitation, and hindlirnb muscles were rapidly excised, Çozen in iiquid N2, and stored at -80°C until use. For membrane fiactionation we separated red muscles composed mainly of type 1 and IIa oxidative and glycolytic (red-IIIIa) fibers (soleus, peroneals, vaatus interme- dius, and red portions of gastrocnemius, rectus femoris, vastus lateralis, and semimembranosus) and white muscles composed mainly of type IIa and IIb (white-IIaIIIb) glycolytic fibers (extensor digitonun longus, flexor hallicus longus, tensor fasciae latae, vastus medialis, biceps femoris cranid, biceps femoris caudal, and white portions of gastrocnemius, tibialis anterior, redus femoris, and vastus lateralis) (1). For extraction of total RNA, we used soleus (89% type 1) as a representative red (red-1) muscle, and as representative white (white-IIb) muscles, we used a pool of tensor fasciae latae (96% I n ) and white portions of the rectus femoris (99% 1%) and the vastus lateraiis (89% 1%) (1). Membrane fmctwnation. The day of the subcellular fiaction-
atim experiment h z e n muscles were removed h m the -80°C and w m thawed to 0°C. Subcellular membranes were isolated fiom both red-IIIIa and white-IIaIIïb muscles aRer a protocol that has been previously established (9,191 and was recently adjusted for s m d amounts of tissue (7). SmaUer amounts of wbite-IIaIIIb muscles were obtained fimm each animai com- pared with red-I/na muscles (1.43 i 0.18 vs. 2.9 & 0.13 g for white-IIaIIIb and red-1 IIIa, respectively). For this reason, white-IIaIIIb muscles h m control or exercised rats were pooled in pairs within each p u p to allow for the performance of three independent subceliular hctionation and immune- blotting experiments. Membranes isolated by this pmcedure have been previously characterized through both enzymatic and immunologie approaches (9, 19). Briefiy, a series of differential centrifugation steps yields a crude membrane fraction (CM). Subfkactionation of CM on discontinuou su- crose gradients yields plasma membranes (PM; 25% sucrose fraction), which con- plasma membrane markers but not transverse tubules, and an interna1 membrane &action (IM; 35% sucrose fiaction) depleted of ceil surface markers and of sarcoplasmic reticulum, which contains the intracellular pool of insulin-sensitive GLUT-4 glucose transporters and 012-
subunits of the Na+-K+-ATPase (16,19). Imrnunoblotting. Immunoblotting was performed as previ-
ously describecl (17, 21) and also described briefly here (see Fig. 1). Quantitation of immunoreactive bands was done with a Molecular Dynamics PhosphorIrnager system (Sunnyvale, CA). To secure accurate estimation of the distribution of pump subunits, the amount of protein transferred to the polyvinylidene difluoride membranes was also quantitated. Afbr immunoblotting, membranes were stained with h t o - gold (British BioCell International, Cardiff, Wales) and scanned with a Discovery Series (Protein Database) DNA scanner equipped with one-dimension gel-analysis software (ver. 1.3, Huntington Station, NY) as described earlier (15). The immunoblot density values were normalized relative to the detected protein in each lane.
RNA extraction and Northern blotting. Total RNA was extracted fkom the soleus (red-1) and the pool of three white-IIb muscles (tensor fasciae latae and white parts of rectus femons and vastus lateralis) by using the single-step RNA isolation with acid guanidinium thiocyanate-phenol- chloroform extraction (4). Northern blotting was carried out essentidy as described earlier (17). The al-cDNA probe was generated aRer restriction of the fu21-length al-cDNA with Nad and StuI (to give a probe comprising nucleotides 89-
full-length aa-cDNA with ScaI and Nhel (to give a probe comprishg nucleotides 121-502). The &-cDNA probe was obtained by incubating the full-length pl-cDNA with restric- tion enzymes NcoI and SspI (to give a probe oomprising nucleotides 459-1,554), and the full-length &cDNA (nucleo- tides 1-1,331) was used. These selected sequences do not cross-read with the alternative isofom transcript (i.e., al vs. a2 or pl vs. Pz) (17). Radioactive labeling of cDNA probes was performed by the random primer method (14). After hybridi- zation, nytron membranes were subjected to three successive 10-min washes with lx sodium chloride (150 mM)-sodium citrate (15 mM) bufTer, pH 7.0, and 0.1% sodium dodecyl sulfate at 65OC before PhosphorIrnager analysis.
Statistical ardysis. Statistical analysis was done with paired and unpaired Student's t-test, as indicated.
Simi3a.r amounts of red-I/IIa or white-IXa/IIb muscle tissue from control and exercised rat hindlimbs were isolated and used for subcellular fractionation. The amounts of protein yielded in the PM, IM, and CM isolated by the fractionation procedure were also simi- lar between the control and exercise groups (Table 1).
Effect of exercise on the content and distribution of pump subunits in red-l/Ia muscles. Figure 1A shows representative immunoblots of five independent experi- ments investigating the effect of exercise on the distri- bution of al-, a2- and PI-subunits in PM, IM, and CM isolated h m red-I/IIa muscles. Exercise significantly increased the content of the al- (by 64 + 24%; P < 0.05) and u2-subunits (by 43 5 22%; P < 0.05) in the PM of red-I/IIa muscle when five independent experiments were analyzed (Fig. 1C). Surprisingly, this increase was not associated with a reduction in the content of theae subunits in the IM (Fig. 1A) or in the other fractions recovered in the sucrose gradient (results not shown), including the pellet that is nch in sarcoplasmic reticu- lum markers. Moreover, exercise did not after the content of al- and al-subunits in the CM fraction. Exercise caused a small increase in the PM content of the pl-subunit that, however, did not mach statistical significance when al1 five experiments were analyzed. Neither the IM levels of BI-subunit nor the total amount of BI- subunit in CM was affected by exercise (Fig. 1A).
Efect of exercise on the content and distribution of pump subunits in white-IIalIIb muscle. Figure 1B shows representative immunoblots from three indepen- dent experiments examining the al-, a2-, and Pa- sub- unit distribution in white-IIaIIIb muscles in the three
cantly increased the al-polypeptide (by 55 + 22%; P < 0.05) and a2-polypeptide (by 94 2 39%; P < 0.05) in the PM of white-IIa/IIb muscles in three independent experiments (Fig. 1C). As in red-I/IIa muscle, we were unable to detect a reduction in these subunits in the white-IIa/IIb muscle IM (Fig. 1B) or other fkactions h m the gradient (results not shown). Examination of the CM isolated fkom white-IIa/IIb muscles also showed no significant alteration in the total amount of al- and a2-subuaits. Xn the PM, there was a trend of the &subunit to increase with exercise, but this did not reach statistical sigdicance. There were no signifiant alterations in the Pz-levels i n either the IM or the CM (Fig. LB).
Whereas immunoblotting does not allow for the calculation of molar equivalents of pump subunits, it does diow one to compare the ratio of $- to a-subunits between different membrane fractions; i.e., one can examine whether this ratio M e r s between &actions. The ratio of Q to a was estimated in control muscles by dividing the optical density reading of the immunoblots, and this ratio was given an arbitrary value of 1.0 for the IM. The ratio of to al was found ta be 4.1 in the PM compared with 1.0 in the IM of red-I/IIa muscles. These values are in arbitrary units and do not indicate molar ratios; however, they do indicaie that there are more &-subunits per al-subunits in the PM than in the IM. In the same fkactions, the ratio of pl to a2 was found to be 6.7 in the PM compared with 1.0 in the IM. In white-IIafIIb muscles, the ratio of p2 tO u1 was 5.7 in the PM compared with 1.0 in the CM, and the ratio of p2 to a2 was 13.3 in the PM compared with 1.0 in the IM. Collectively, these resulta indicate that there is a relatively greater proportion of f3- to a-subunits in the PM than in the IM r e g d e s s of P- or a-isoform.
Effect of exercise on t h expression of pump subunit rnRNAs in red-I and white-IIb muscles. The levels of Na+-K+ pump subunit transcripts were also investi- gated &r the 1-h exercise bout by performing North- ern blots of total RNA isolated h m soleus muscle (red-1) and tensor fasciae latae or white portions of rectus femoris and vastus lateralis muscles (white-IR). Figure 2A shows representative Northern blots of the al-, az-, and pl-subunit transcripts of control and exercised red-1 muscles. In the soleus, the al-cDNA probe reacted with a single transcript of 3.7 kb. Exer- cise caused a marked increase in the abundance of the
Table 1. Protein yield in membrane fractions derived from red-I/IIa and white-IIa /IIb muscles
Pmtein Yield, pg
Muscle Mass, g Plasma membranes Internai membranes Cnide membranes
n Control Exercise C o n h l Exercise Control Exerciee Control Exercise
Red 5 3.1 5 0.15 2.72 t- 0.11 58 -+ 9.11 54 + 9.98 549 i 119 490 2 137 2,919 2 352 3,024 2 351 White 3 2.92 t 0.28 2.85 + 0.02 45.8 2 6.21 45 2 1.77 823 t 339 913 2 264 5,506 2 806 5,856 2 473
Values are means i SE; n, no. of rats. Red-I/IIa muscles of 6 control and 5 exerciaed animais were examined. Becauae of emaii yield of pure white-IIa/Iib muscles fmm each animal, muscle8 of 2 amtrol or 2 exercised rats were pooled together to f o m 3 pairs of white mntrol and exercised muscle groups starting from 6 animale in each csse. Average mass of muscles isolated h m eacb gmup and amount of protein yielded in each subcellular fraction are given. See M E ~ ~ O D S for epecific red-I/na and white-IIa/IIb muscles isolated.
Fig. 1. Effect of exemise on Na+-K+ pump subunit content of plasma Control t membranes (PM), intemal membranes (IM), and crude membranes
a Exercise (CM) h m red-I/IIa and white-lla/IIb muacles. Ten microgaxm of PM, IM, and CM from red-I/IIa (A) and white-ïia/IIb ( B ) muscle8 of control (C) and exercised CE) animals were subjected to sodium dodecyl d a t e -
i * O polyacrylamide gel ele~trophoresi~ and immunoblotted to detect ai-, az-,
pl-, and BTsubunits of the pump. Five micmgranu of either kidney CKi) or braixi (Br) crude membranes were used as positive standards. Monoclonal antiai- CMck-Il and antisz- Wcù-2) antibodiea were diluted l:M)O, ,md polyclonal anti+ and anti-j3z-antibodies were diluted 1:1,000. Monoclonal antibodies were detemed with shep mI-labeled anti-moue immunoglobulin G (0.1 pCi/ml, ICN Radiochemicals) and polyclonal antibodies with 1251-labeled protein A (0.1 pCi/ml, ICN Radiochernicab). Immunoreactive bands in al1 fractions were quantitated by scanning densitometry, and r d t a were normalued relative to protein content on polyvinylidene difluoride filters as described in METBODS. Rernilta of independent immunoblotting experimenta quentitating plasma mem- brane content of al- and un-suhni& of red-I/IIa (n = 5 ) and white-Da/
or! -
a2 . ai Ilb (n = 3) muscles were normalized relative to resting control values and are summarized in C. Values are means 2 SE. *P < 0.05 (paired Red-Mla muscles White-IlaAlb muscles ,-,,,).
al-mRNA levels relative to the controls (Fig. 2A). The a2-cDNA probe recognized a major transcript of 3.4 kb and a very faint one of 5.3 kb (Fig. 2A). The existence of transcripts of different sizes is documented and may be due ta differences in polyadenylation, splicing, or pro- cessing of mRNA (Ref. 13 and references therein). The pl-cDNAprobe reacted with a major transcript of 2.7 kb and two very minor ones of 2.3 kb and 1.9 kb (Fig. 2A). In contrast to al, exercise elicited only a faint increase in the cc2- and Pl-transcript levels of soleus muscle without reaching statistical significance. As expected (17), there was no detectable mRNA of the @,-isoform in the soleus muscle. Figure 2C summarizes the results of three ta six independent experiments examining the effect of exercise on the abundance of al-, a2-, and Pl-transcripts in soleus muscle. Exercise increased significantly the mRNA of the al-subunit of pump by 50 rt: 7% (P < 0.05) but not of the as- or @ I - ~ ~ b u n i t s .
Figure 2B illustrates representative Northern blots showing the mRNA levels of al- and &-subunits of the pump in white-IIb muscles from resting and 1-h exer-
cised rats. Both the al- and the P2-cDNA probes recog- nized a single transcript in the RNA isolated fiom the white-1% muscles. In agreement with earlier observa- tions (17), transcripts of the Pl-subunit were not de- tected in the RNA isolated fkom the white-IIb muscles. Surprisingly, we were unable ta detect the a2-subunit transcript in these muscles (see Fig. 2). Figure 2C summarizes the results of three to four independent experirnents examining the effect of exercise on the transcripts of these subunits in white-1% muscles. In contrast to red-I muscle, exercise did not alter signifi- cantly the al-mFWA in white-1% muscles. However, a significant increase (by 64 -r: 19%; P < 0.01) in the Pz-transcript was detected in the exercised white-Ilb muscles relative to the controls (Fig. 2, B and C).
DISCUSSION
Electrical stimulation of short duration increases the ouabain-sensitive Rb+ uptake in red and white skeletal muscles (5, 10, I l ) , indicating increased activity of the
Contml Exercise
Fig. 2. Effed of exercise on Na+-K+ pump subunit transcript levels in red-1 and white-IIb mudes. FiReen mimgrame. of total RNA h m red-1 (A) and white-Llb (BI muscles were subjected to ~ o r t h e i blotting by us- ing isoform cDNA probes specific for
8 2 the al, ar, BI-, and &-subunits (see METHODS). Reactivity of major band of
1 each isoform wss quantitated, and C E resulîa of 3-6 independent experi-
ments were normalized relative to resting control and are ehown in C. Values are meana i SE. *P < 0.05; **P < 0.01 (unpaireci t-test). Note: we
2.8 kb + could not detect atmRNA in white- IIb mudes andyzed. Previously we have detected lower a2-transcript lev- els in extensor digitorum longus and white gastrocnemius muscles (am- tainhg both type IIa and type Iib fibers) compared with soleun mucle (17). Because white-iXb muscles used for mRNA measurements in preaent study are almost exclizeively com- posed of IIb fibers (89-99961, the pre- sent observation euggeats that pure type LTb fibers may express very low or ni1 levels of a*-mRNk
1 I t I
Red4 muscles White-llb muscles
Na+-K+ pump. This function is likely vital for the continued performance of the muscle during exercise. However, the exact mechanism whereby pump a c t i m is elevated during acute exercise is large'ly iInknown. In the present study, we began to examine the basis for the increase in pump activity during exercise by analyzing whether there are changes in pump subunits at the plasma membrane and whether these changes are associated with variations in the mRNA levels in red-1 and white-IIb skeletal muscles after a single bout of exercise. This became possible because of the recent availability of isoform-specific antibodies and cDNA probes to the pump subunits and the development in our laboratory of strategies to isolate purified plasma membranes from srnall amounts of skeletal muscle (7, 9, 15). These procedures allowed us to anaiyze muscles composed rnainly of type I plus IIa fibers (red) type IIa plus 1% fibers (white). We observed that a single bout of exercise can increase the al- and a2-protein abundance in isolated plasma membranes in both red-I/IIa and
white-IIa/IIb muscles (Fig. l), indicating that the plas- ma membrane Na+-K+ pump content can be regulated by exercise of short duration. The results suggest that a rise in the plasma membrane al- and a*-subunits can be part of the mechanism of elevation of pump activity in response to exercise, particularly because these are the cataiytic subunits of the enzyme. These results are compatible with earlier findings that ion-pump activity is increased in both red (soleus) and white (extensor digitonun longus) rat skeletal muscles after electrically stimulated contraction (12), although quantitative dif- ferences were noted between the muscles. Our observa- tions are also in agreement with observations of in- creased binding of cardiac glycosides to skeletai muscle of humans undergoing 1 h of bicycle exercise (18). In contrast, there was an increase in the rate of [3H]oua- bain binding to electrically stimulated muscle during the first minutes of stimulation, ascribed to a change in pump activity rather than to pump number (11). Be- cause of the differences in the duration and means of
one, the molecular events involved -
each case rnay differ.
I n principle, the increase in al- and a2-subunits at the plasma membrane could be brought about by synthesis of new pump subunits, recruitment of preex- isting enzymes from intracellular sites, or stabilization of the plasma membrane-associated complexes in this compartment. Because there was no statistically signifi- cant change in pump subunit content in crude (i.e., total) membranes (Fig. 1, A and B), it seems unlikely that new subunit synthesis contributes to the increase in plasma membrane subunit content. On the other hand, our results did not provide information regarding the existence of a donor pool of preexisting pumps because there was no change in subunit content in the intracellular fiactions isolated. Yet, i t is possible that small membrane pools are not detected by the fraction- ation procedure. Indeed, there is precedence that there is a pool of intracellular membranes endowed w i t h GLUT-4 glucose transporters that provides tramport- ers to the plasma membrane in response to exercise and is not purified by the isolation procedure used (9).
The observation that a-subunits increase in the PM without an apparent concomitant increase in $-sub- units can arise from several hypothetical scenarios. 1) It is possible that aaubunits are recrultd to the plasma membrane where they associate with tkee B-subunits. Indeed, this phenomenon was shown to occur in insect sf-9 cells (a), and there are suggestions for independent intracellular tratFic of the a- and f3-subunits in response to insulin in mammalian muscle (16). We estirnabd the relative proportions of the pump subunits in PM and 1 . . The ratio of f3- to a-subunits was severalfold higher in the PM than in the IM regardless of isoform or muscie fiber type. These estimates suggest that the PM compartment may maintain a pool of @-subunits that could potentially associate with newly recruited a-sub- units, thereby allowing the formation of functional pump complexes. 2) Our results cannot d e out, how- ever, that the larger proportion of P-subunits in the PM may obscure detedion of a M h e r increase in the PM in response to exercise. Thus we cannot formally dis- card the possibility that there is translocation of pre- formed a-f3 dimmers in response to exercise. 3) Fïnally, the amount of any protein at the ce11 surface depends on its rate of cycling through this compartment, deter- mined by the rates of arriva1 and retrieval. The results obtained are also compatible with an increase in the permanence of a-subunits at the plasma membrane in response to exercise, which could be due to decreased rate of retrievd from the plasma membrane. Such a mechanism of regulation of the distribution of pump subunits would also indicate that a) the or- and f3-sub- units of the pump have different rates of arrival to and retrievd from t.he plasma membrane and b ) the rate of retrieval of a-subunits from this compartment is faster under resting conditions than during exercise.
The regulation of pump subunit content at the plasma membrane by exercise is reminiscent of the stimulation caused by insulin. A 30-min exposure to the hormone
Pl-subunits in- mixed ( rd and white) rat skeletal muscles (16, 21). However, in the case of insulin, the donor pools that supply pump subunits to the plasma membrane were identifid (16). Similady, insulin causes recruilment of the GLUT-4 glucose transporter h m an intracellula. donor pool, which is recovered with the IM. Thus insulin stimulates the translocation of both glucose transportera and pump subunits from intracel- lular membrane organelles isolated with t h e IM, whereas exercise rnay re&t transporters and pump subunits to the plasma membrane fiom an intracellu- lar compartment that is not isolated by the present procedure. F'uture investigations will examine this possibility. Further differences exist between the ac- tions of insulin and exercise on the Na+-K+ pump because insulin recruits to the plasma membrane the u2- and pl-subunits (16), whereas exercise increases both al- and az- but not P-subhts (Fig. 1).
In addition to c h g i n g the content of a-subunits in the PM, the single bout of exercise increased the amount of a,-mRNA in soleus muscle and P2-mRNA amount in white-IIb muscles, whereas the az- and f31-transcripts in soleus and the al-transcripts in white- IIb muscles were unaltered (Fig. 2). The rise i n al- mR,NA with exercise in soleus is compatible with earlier studies showing increase in the mRNA levels of this subunit in response to elevated intracellular Na+ in other tissues (6, 22). I t is conceivable that elevated intracellular Na+ during exercise rnay signal the tran- sctiption of al-gene in red-1 and the pz-gene in white- IIb muscles. The muscle f32-isoform increases selec- tively also in response to thyroid hormone treatment (2). %ken together, our results suggest that biosynthe- tic mechanisms can be triggered by signals initiated by contraction during a single exercise bout. The lack of concordance between the increase in protein at the plasma membrane and mRNA content suggests that there is regulation of the abundance of subunit tran- script during a short exercise bout, which probably does not contribute to change the cellular levels of pumps during this period. This is not without precedence because the abundance of mRNAof the pump (2,13,23) and of other gene products such as the GLZTT-4 glucose transporter (20) ofkn do not correlate with the levels of the polypeptides they encode. Our results suggest that the pump subunits rnay be ini t idy regulated posttran- scriptionally and that the increase in mRNA rnay serve as a subsequent mechanism to sustain pump activity through prolonged exercise or during the postexercise period. It is conceivable that elevation in al- and P2-mRNA rnay in fact lead to increase of the protein content at later stages of exercise, but this was not addressed by the exercise protocol used. Nevertheless, this possibility is compatible with the obse~a t ion that exercise training augments the number of ouabain binding sites in both rat and human skeletal muscles (Ref. 5 and references therein). The exact signals involved in the increase of the
catalytic subunits of the pump at the plasma mem- brane in response to exercise need to be investigated.
aciruulurx: uit: u~uv~ lry UL ult: p u p AU sneleuu uua~;it:a (5). These hormones may mediate the effect of exercise on the pump. It h s been postulated that the rise in intracellular Ca2+ that takes place during exercise may be a signal involved in the exercise-induced glucose transporter recruilment to the ce11 surface (24). This possibility should also be considered for the elevation in a-subunits in this cornpartment detected in this study.
In conclusion, 1 h of treadmill running increases the plasma membrane content of the catalytic subunits al and a 2 of the Na+-K+ pump in both red-I/IIa and white-IIa/IIb skeletal muscles. This is the k t evi- dence that a single exercise bout can regulate the amounts of specific pump subunit isofonns at the plasma membrane. De novo pro& synthesis of pump subunits does not seem t o be the mechanism for the increase of the al- and a*-polypeptides in the plasma membrane. Although not directly exadnecl, the results are compatible with either the recruitment of aubunits from a yet-unldentified intracellular membrane pool or with increased retention of pumps at the cell surface. The exercise bout also elevated the a & R N A in red-1 muscles and pz-mRNA in white-IIb muscles, perhaps for protein synthesis at a later stage.
We thank Anna Engel, Dimitrios Dimitralcoudia, and Simon Fisher for technid support. We are grateful to Dr. Kathieen Swead- ner for the aupply of the monoclonal anti-q- and antiu2-antibodies, Dr. P. Martin-Vasdo for the polyclonal anti-fJ1- and anti-Pa- antibodies, and Dr. J. Ljngrell for the cDNA probes.
This work was supported by grants from the Medical Research Council of Canada to k Klip (MT-12601) and h m the Canadian Diabetea Association to M. Vranic. T. 'Psakiridis was eupported by a University of "hronto Open Studentship and an Ontario Graduate Scholarship.
Address for reprint requests: A. Klip, The Hospital for Sick Children, Toronto, Ontario M5G UL8, Canada.
Received 22 August 1995; accepted in final form 9 November 1995.
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