sec22 regulates er morphology but not autophagy and is ...sec22 regulates er morphology but not...
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Sec22 regulates ER morphology but not autophagy in flies
1
Sec22 regulates ER morphology but not autophagy and is required for eye
development in Drosophila
Xiaocui Zhao1#, Huan Yang 1, 2#, Wei Liu1, Xiuying Duan1, Weina Shang1, Dajing
Xia2*, Chao Tong1 *
1 Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University,
Hangzhou 310058, China
2 School of Public Health, Zhejiang University, Hangzhou 310058, China
Running title: Sec22 regulates ER morphology but not autophagy
# Contributed equally to this work
*Correspondence to: Dr. Chao Tong, Life Sciences Institute, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China.
Tel: 86-571-88981370; E-mail: [email protected]
Dr. Dajing Xia, School of Public Health, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China.
E-mail: [email protected]
Keywords: autophagy; Drosophila; Endoplasmic reticulum; Golgi apparatus; SNARE proteins
Background: ER morphological changes are
often observed in disease conditions but the
molecular mechanisms remain unclear.
Results: Malfunction of Sec22 and its binding
partners resulted in ER expansion and defects
in fly eye development.
Conclusion: Sec22 regulates ER morphology
through regulating ER-Golgi trafficking but
not autophagy.
Significance: ER morphology changes under
the disease conditions might due to the defects
of ER-Golgi trafficking.
ABSTRACT
The endoplasmic reticulum (ER) is a highly
dynamic organelle that plays a critical role
in many cellular processes. Abnormal ER
morphology is associated with some human
diseases, although little is known regarding
how ER morphology is regulated. Using a
forward genetic screen to identify genes that
regulated ER morphology in Drosophila, we
identified a mutant of Sec22, the orthologs
of which in yeast, plants, and humans are
required for ER to Golgi trafficking.
However, the physiological function of
Sec22 has not been previously investigated
http://www.jbc.org/cgi/doi/10.1074/jbc.M115.640920The latest version is at JBC Papers in Press. Published on February 10, 2015 as Manuscript M115.640920
Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Sec22 regulates ER morphology but not autophagy in flies
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in animal development. A loss of Sec22
resulted in ER proliferation and expansion,
enlargement of late endosomes, and
abnormal Golgi morphology in mutant
larvae fat body cells. However,
starvation-induced autophagy was not
affected by a loss of Sec22. Mosaic analysis
of the eye revealed that Sec22 was required
for photoreceptor morphogenesis. In Sec22
mutant photoreceptor cells, the ER was
highly expanded, and gradually lost normal
morphology with aging. The rhabdomeres
in mutants were small and sometimes fused
with each other. The morphology of Sec22
mutant eyes resembled the eye morphology
of flies with overexpressed eyeclosed (eyc).
eyc encodes for a Drosophila p47 protein
that is required for membrane fusion. A loss
of Syntaxin5 (Syx5), encoding for a tSNARE
on Golgi, also phenocopied the Sec22
mutant. Sec22 formed complexes with Syx5
and Eyc. Thus, we propose that appropriate
trafficking between the ER and Golgi is
required for maintaining ER morphology
and for Drosophila eye morphogenesis.
The endoplasmic reticulum (ER) plays
pivotal roles in many cellular processes. In
addition to its important function in protein
synthesis, modifications, and quality control,
the ER is also critical for lipid synthesis,
autophagy, and calcium homeostasis (1-3).
The ER makes close contacts with other
organelles, including mitochondria, the Golgi
apparatus, endosomes, peroxisomes, and
lysosomes. It exchanges lipids and proteins
with these organelles to regulate their
biogenesis and function (4, 5). Thus, the ER
has diverse functions and ER malfunctions can
cause numerous human diseases, including
diabetes and neurodegeneration (6).
The ER is a membrane network that
consists of tubules and sheets that extend
throughout a cell (7, 8). The ER is highly
dynamic and constitutively undergoes
rearrangements, which are critical for ER
functions (9-11). ER morphological changes,
including fragmentation and expansion, are
often observed in cells that are subjected to
pathological insults (6). However, the
molecular mechanisms underlying these ER
morphological changes remain unclear.
Using a forward genetic screen to
identify genes that regulated ER morphology,
we obtained a complementation group with
remarkable ER proliferation and expansion.
Molecular mapping located a lesion on Sec22,
a gene encoding for a SNARE protein
enriched on the ER (12, 13). SNARE proteins
are a family of conserved proteins that mediate
membrane fusion between vesicles derived
from one sub-cellular compartment and their
targeted membranes (14, 15). In the secretory
pathway, proteins are transported by vesicle
budding, docking, and fusion. Different
SNAREs mediate the fusion step between
various vesicles and their ultimate destinies.
Vesicle membrane localized v-SNAREs and
targeting membrane localized t-SNAREs form
helix bundles to drive membrane fusion.
Subsequently, these bundled SNAREs are
dissociated and recycled by
N-ethylmaleimide-sensitive factor (NSF) (16).
Sec22 family proteins are highly
conserved from yeast to human. In yeast, there
is only one Sec22 gene and its functions
partially overlap with those of another gene,
ykt6 (13). Sec22p is involved in transport
between the ER and Golgi compartments in
both anterograde and retrograde directions in
yeast (12). In addition, Sec22p is required for
autophagosome biogenesis by regulating the
transport of Atg9, an essential protein for
autophagosome formation, to the phagophore
assembly site (PAS) (17). In both yeast and
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plants, Sec22p/SEC22 regulates cesium
accumulation, which indicates that Sec22
proteins play a role in vacuole function (18). A
loss of SEC22 in plants also results in Golgi
fragmentation and defects in gametophyte
development (19). Studies of the rice fungi
show that Sec22 is required for conidiogenesis,
cell wall integrity, and host plant infection (20).
Several Sec22 genes, SEC22A, SEC22B, and
SEC22C, were also found in humans. In
cultured mammalian cells, SEC22 proteins are
also required for ER to Golgi trafficking (21,
22). However, knocking down SEC22B
expression in cultured mammalian cells does
not cause autophagy defects, presumably due
to redundancy (23). It was recently shown that
SEC22B also has a conserved non-fusogenic
function in plasma membrane expansion (24).
All of these studies demonstrated that
Sec22 was a key regulator of the secretory
pathway. However, surprisingly, there have
been no studies of the physiological functions
of Sec22 in animal development and
morphogenesis. In this study, we found that
Sec22 was an essential gene in flies. A loss of
Sec22 resulted in morphogenesis defects in fly
eyes. The ER was highly proliferated and its
morphology underwent very dramatic changes
in Sec22 mutant flies. In contrast to yeast
orthologs, Sec22 was not required for
starvation-induced autophagy in Drosophila.
Interestingly, a loss of Syntaxin 5 (Syx5),
which encodes for a tSNARE in Golgi, also
phenocopied Sec22 mutants, which indicated
that appropriate trafficking between the ER
and Golgi was critical for ER morphology.
EXPERIMENTAL PROCEDURES
Molecular biology
To generate a Sec22 genomic rescue
construct, a 3.3 kb genomic fragment that
contained the Sec22 gene was amplified using
the primers,
5’-TGAGATCTGTCCATGCAGAGGATGGT
CTTCG-3’ and
5’-TACTCGAGCCAGAGCTTCAGCAGAA
CCACAGC-3’, using genomic DNA that was
purified from FRT19A isogenized flies. To
generate a Sec22 expression vector, we
inserted the Sec22 cDNA into a pattB
UAS-3HA vector. A 3XHA tag was fused to
Sec22 at the N-terminus. To generate a
Flag-tagged Syntaxin 5 expression vector, we
inserted the Flag sequence and Syntaxin 5
cDNA into the pattB UAS expression vector.
The Flag tag was fused to the N-terminus of
the Syx5 protein. A Myc-Eyc expression
vector was generated by inserting full length
eyc cDNA into the pattB UAS-Myc expression
vector. Myc was fused to the N-terminus of the
Eyc protein.
Fly strains
A Sec22 mutant was isolated from an
ethane methylsulfonate (EMS) screen as
previously reported (25). The fly strains
purchased from the Bloomington Drosophila
Stock Center (Indiana University,
Bloomington) were: Df(1)ED6443,
Df(1)BSC534, Dp(1;3)DC012, Dp(1;3)DC013,
P[ey-FLP.N]2; P[EP] Syx5 EP2313P [neoFRT]
40A, P[UASp-GFP.KDEL], and
P[UASp-RFP.KDEL]10. Sec22-genomic-
rescue transgenic flies, UAS-3XHA-Sec22,
UAS-Myc-Eyc, and UAS-Fiag-Syx5
transgenic flies were generated by the Core
Facility of Drosophila Resource and
Technique (Institutes of Biochemistry and Cell
Biology, Shanghai) using standard injection
procedures. Syx5 RNAi fly stains were from
the Tsinghua Fly Stock Center (Beijing,
China).
cDNA analysis of the Sec22 mutant
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Total RNA was isolated from 25 wild
type larvae or larvae with homozygous
mutation in Sec22 using Tirzol reagent
(Invitrogen) according to the manufacturer’s
instructions. cDNAs were synthesized using a
First Strand cDNA Synthesis kit (Invitrogen)
with Oligo dT primers and used in PCR
reactions with the Sec22 -specific primers P1F:
5’-ATGGCGCTGCTGACCATGATAG-3’ and
P1R: 5’
-TTACAGAACCCAGAAATACATG-3’, or
primer P2F:
5’-GTGCTGGCGGGTAACTACTAC-3’ and
P2R:
5’-TTACAGAACCCAGAAATACATG-3’.
The PCR products were sequenced.
Immunostaining for fly fat bodies
Dissected fly fat bodies were fixed
fixed with 3.7% formaldehyde in PBS for 20
min, and then washed 3 times with PBST
(PBS + 0.1% Triton X-100). Tissues were
incubated with a primary antibody (rabbit
anti-HA used at 1:1000; CST) overnight,
extensively washed, and then incubated with a
secondary antibody (Alexa-488 conjugated
used at 1:500; Invitrogen) at room temperature
for 1 h. After extensive washing, samples were
mounted in vector shield and observed by a
confocal microscope.
Phalloidin and immunostaining of the
photoreceptor cells
For the late pupae stage flies, the
dissected brain-eye complexes ware fixed in
PBS with 4% formaldehyde for 20 min. For
the adult flies, heads were fixed in PBS with 4%
formaldehyde for 1 hour upon removal of the
proboscis. Then the photoreceptor cells were
dissected and fixed for 15 min. After the
fixation, the tissues were washed 3 times with
PBST (PBS + 0.4% Triton X-100) and stained
with primary antibody mouse anti-Rh1 (1:50,
Developmental Studies Hybridoma Bank)
overnight at 4 ºC. After extensive washing
with PBST, secondary antibody (1:500,
Invitrogen) and Alexa-488 labeled phalloidin
(1:1000, Invitrogen) were incubated with the
tissues for 2 hours at room temperature.
Samples were washed with PBST for 4 times
and mounted in vector shield and observed by
a confocal microscope.
Cell culture and immunoprecipitation (IP)
S2 cells were cultured at room
temperature and transfected using Opti-PEI.
For Co-IP, cells were harvested at 48 h after
transfection and lysed with lysis buffer. Cell
lysates were incubated with beads (anti-HA,
anti-Myc, or anti-Flag) at 4 ºC for 4 hrs in
lysis buffer. After extensive washing, the IP
products were analyzed by Western blot using
anti-HA (1:1,000), anti-Myc (1:1,000), or
anti-FLAG (1:1,000), followed by an
HRP-conjugated secondary antibody
(1:5,000).
Transmission Electron Microscopy
For electron microscopic observations
of photoreceptors, fly heads were dissected
and fixed in 4% paraformaldehyde and 1%
glutaraldehyde at 4⁰C overnight. Then, the
samples were post-fixed in 2% OsO4 for 2 hrs,
followed by dehydration in ethanol and
propylene oxide. Samples were embedded in
Embed-812 resin (Electron Microscopy
Sciences). TEM images were acquired using a
transmission electron microscope equipped
with a digital camera.
Confocal Microscopy
All mounted samples were examined
and imaged with a confocal microscope
LSM710 (Carl Zeiss) outfitted with a
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Plan-Apochromat 63X 1.4NA oil immersion
objective lens (Carl Zeiss). Data were acquired
using Zen 2010 Software and processed with
Photoshop CS (Adobe).
RESULTS
Identifyinga complementation group, ERM1,
with ER morphology defects.
To identify mutants that had ER
morphology defects, we screened about 700
chemically-induced lethal mutations on X
chromosome (25). We labeled ER structures
using Cg-Gal4 driven UAS-KDEL-GFP
expression in the Drosophila fat body cells and
made mutant clone negatively marked with
RFP (Fig. 1A). By comparing the patterns of
the KDEL-GFP in the RFP negative mutant
cells with those in the neighboring RFP
positive wild type cells, we identified one
complementation group, ERM1. KDEL-GFP
punctate structures became larger and
accumulated inside the ERM1 mutant clones
(Fig. 1B: A’, a’, B’, and b’), which indicated
that the ER was over-proliferated and had
expanded. To determine whether other
organelles were also affected in these mutant
clones, we also examined the patterns for
Rab7-GFP, ManII-GFP (26), and Mito-GFP,
which label late endosomes, the Golgi
apparatus, and mitochondria, respectively.
There was a dramatic enlargement and
accumulation of Rab7-GFP punctate structures
inside ERM1 mutant clones (Fig. 1B: C’, c’,
D’, and d’). The Golgi marker ManII-GFP
became diffusely distributed as compared to its
distribution in a control (Fig. 1B: E’, e’, F’,
and f’). The mitochondria pattern appeared to
be intact inside these mutant clones (Fig. 1B:
G’, g’, H’, and h’). These data suggested that
the ER, late endosomes and Golgi were all
affected in ERM1 mutant cells.
ERM1 mutants correspond to Sec22
mutants
Because the ERM1 phenotypes were
interesting, we performed duplication and
deficiency mapping. ERM1 was rescued by an
X chromosome duplication that covered a
region from 1A1 to 2C1. Several overlapping
deficiencies that covered this region were used
to further narrow down the mutant lesion.
Df(1)BSC534 did but the overlapping
Df(1)ED6433 did not rescue the lethality of
ERM1, which indicated that the mutation was
localized in the region deleted in Df(1)ED6433
but not in Df(1)BSC534. To further pinpoint
the mutant region, small duplications that
covered the deleted region in the deficiencies
were used for a complementation test. ERM1
was rescued by two overlapping small
duplications, Dp(1;3) DC012 and Dp(1;3)
DC013, which narrowed the putative gene
down to nine genes (Fig. 2A). Sequencing
revealed point mutations in Sec22 (Fig. 2B). A
splicing acceptor G to A mutation at the end of
the third intron was detected in ERM1. To
analyze the transcripts in this mutant, we
isolated mRNA from the mutant larvae, made
cDNAs, and sequenced the cDNAs with two
pair of primers. Two different cDNAs were
identified: Sec22 mu1 and Sec22 mu2 (Fig.
2C). An alternative splicing acceptor site was
adopted by Sec22 mu1, which resulted in a
deletion of part of the 4th exon. Wild type
Sec22 encodes for a SNARE protein that
contains a longin domain and a synaptobrevin
domain (Fig. 2D). The frame shift caused by
the deletion in Sec22 mu1 resulted in the
mis-translation of the most part of the
synaptobrevin domain. Sec22 mu2 had an
un-spliced intron and that led to a premature
stop codon in the synaptobrevin domain.
To confirm that ERM1 mutants indeed
corresponded to Sec22 mutants, a genomic
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fragment that contained only the Sec22 gene
was introduced into these mutants (Fig. 2A).
The genomic rescue construct not only fully
rescued the lethality of ERM1 to adulthood but
also rescued the ER morphology defects in the
mutant clones (Fig. 2E). It indicated that Sec22
was the gene affected in this mutant.
Sec22 is not required for autophagy in
Drosophila
A recent study with yeast showed that
Sec22 is required for autophagosome
formation. Because the ER is degraded in the
autophagosome through “ER-phagy” (27), the
accumulated morphologically abnormal ER in
Sec22 mutants could be due to an autophagy
malfunction.
To test if Sec22 was required for
autophagy in flies, we examined different
autophagy markers in Sec22 mutant cells after
their starvation. We labeled autophagy
vacuoles in fat bodies by expressing different
markers driven by a fat body Gal4 driver,
Cg-Gal4 (28). We negatively labeled the Sec22
mutant clones with RFP in third instar larvae
fat bodies, and then examined the different
autophagy markers after starvation for 4 hours.
Autophagy initiation requires type III
PI3K activity, which can be monitored based
on the intracellular distribution of FYVE-GFP.
Without PI3K activity, FYVE-GFP signals are
diffusely distributed inside cells. If PI3K is
activated, then FYVE-GFP exhibits punctate
staining (29). In Sec22 mutant cells,
FYVE-GFP exhibited a punctate distribution
that was similar to that in control cells (Fig. 3
A, A’), which indicated that PI3K activity was
intact.
Atg8 is an ubiquitin-like protein that is
required for autophagosome formation and
autophagy progression (30). Without
starvation, Atg8 signals are diffusely
distributed inside cells. Upon starvation, Atg8
forms punctate structures and labels
autophagosomes. Once autophagosomes fuse
with lysosomes, Atg8 is degraded inside
autolysosomes. In Sec22 mutant clones,
Atg8-GFP punctate structures were formed
and these patterns were comparable to those in
controls’ (Fig. 3B, B’), which indicated that
Sec22 was not required for autophagosome
formation.
To monitor autophagosome and
lysosome fusion, Atg8-GFPRFP was used as a
marker. GFP signals but not RFP signals are
quenched inside acidic autolysosomes; thus,
autophagosomes will appear yellow and
autolysosomes will appear red (31). If there
are fusion defects between autophagosomes
and lysosomes, then most Atg8 punctate
structures will appear as yellow; otherwise, the
majority of Atg8 punctate structures will
appear as red. The yellow and red signals of
Atg8-GFPRFP punctate structures in Sec22
mutant clones were same as those in control
cells (Fig. 3C, C’), which indicated that Sec22
was not required for autophagosome
maturation.
To examine autophagy flux in Sec22
mutant cells, we checked the patterns of p62
(32), an adapter protein that is digested via
autophagy. There was no difference in the p62
protein distribution between Sec22 mutants
and wild type controls’ (Fig. 3D, D’).
In summary, Sec22 was not required
for autophagy in fly fat body cells. Thus, the
accumulated ER and morphological changes
were not due to autophagy failure.
Syx5 interacts with Sec22 to regulate ER
morphology
Because autophagy was not affected
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by a loss of Sec22, the accumulation of
morphologically abnormal ER could have
resulted from other conserved functions of
Sec22. In yeast, Sec22 forms a complex with
the Golgi SNARE protein Sed5p to mediate
the fusion of ER-derived vesicles with the
Golgi compartment (33). In Sec22 mutant fat
body cells, the ER was expanded and Golgi
morphology had changed, which indicated that
Sec22 indeed was required for membrane
trafficking between the ER and Golgi in flies.
Thus, we hypothesized that trafficking defects
between the ER and Golgi had resulted in the
ER morphological changes.
To test this, we first examined
whether Syntaxin 5 (Syx5), encoded by the fly
ortholog of sed5, could bind to Sec22. We
performed immunoprecipitation (IP) using
cultured S2 cells and found that Sec22 indeed
formed a complex with Syx5 (Fig. 4A). When
we knocked down Syx5 in fly fat bodies using
RNAi, we found that KDEL-RFP punctate
structures became larger and had accumulated
inside cells (Fig. 4B, C). This indicated that
the ER had expanded in Syx5 RNAi tissues, a
phenotype that was very similar to the Sec22
mutants. Sec22 is a v-SNARE on the ER
membrane and Syx5 is a t-SNARE on the
Golgi compartment. The ER morphology
change in those cells that had lost either
SNARE protein suggested that the trafficking
failure between the ER and Golgi had
triggered ER expansion.
Interestingly, Syx5 overexpression in fat body
cells also led to ER expansion similar to the
ER morphology in the Syx5 RNAi tissues,
suggesting Syx5 overexpression has dominant
negative effects (Fig. 4E). In yeast, ER-Golgi
transport requires four SNARE proteins,
Sec22p, Bos1p, Bet1p, and Sed5p (encoded by
a yeast ortholog of Syx5), to form complexes.
The dominant negative effects of Syx5
overexpression probably is due to the extra
Syx5 competing with the endogenous Syx5 to
form ineffective complexes with other
endogenous SNARE proteins, such as Sec22
or the proteins encoded by the orthologs of
Bos1p or Bet1p. Strikingly, when we
co-expressed Sec22 and Syx5, the ER
punctuate structures in the fat body cells were
much larger than those in the cells with Syx5
overexpression alone (Fig. 4D-F). Since Sec22
overexpression did not have ER morphology
defects, the enhancement of the Syx5
overexpression phenotypes suggested that
Sec22 might form a complex with Syx5 and
together they were a better competitor than
Syx5 alone to titrate out of other SNARE
proteins. These data implied that Sec22 and
Syx5 might function in a same pathway to
regulate ER morphology.
Sec22 is required for eye morphogenesis
The dramatic ER morphological
changes in the Sec22 mutant cells suggested
that Sec22 played a critical role in regulating
ER function. However, the physiological
function of Sec22 has not been previously
investigated in animals. We found that a Sec22
hemizygous mutant cannot survive to the
adulthood. To investigate whether Sec22 was
required for nerve system development and
neuron homeostasis, we generated Sec22
mutant clones in the eye using the eyFLP
system. In wild type control eyes, round,
tightly packed and oval shaped rhabdomeres
were in a trapezoid arrangement. In Sec22
mutant eyes, irregularly shaped rhabdomeres
were small and sometimes fused with each
other (Fig. 5A, a, C and c). Inside
photoreceptor cells, the ER was highly
expanded with numerous stacks and gradually
lost its normal morphology with aging (Fig.
5A-d). Occasionally, lipid droplets could be
detected in the cytoplasm, another hallmark of
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ER malfunction (Fig. 5A-d).
It has been reported that mis-trafficking of
Rhodopsin 1 (Rh1) leads to retinal
developmental defects and degeneration (34,
35). Rh1 is a light sensor that presents in the
R1-R6 photoreceptor cells. During fly
photoreceptor cell development, Rh1 is
synthesized and matured in the ER and
transport to the rhabdomeres through the Golgi.
The ER defects are likely to cause
mis-trafficking of Rh1 (34, 35). We therefore
tested whether there is Rh1 trafficking defects
in the Sec22 mutants. We made Sec22 mutant
clones in the fly eyes and stained the tissue
with anti-Rh1 antibody and Phalloidin to
examine the distribution Rh1 in the
photoreceptor cells at the late pupae (84h APF)
and adult stages. There was a massive
accumulation of Rh1 in the cytoplasm (Fig.
5E), which might explain the degeneration
phenotypes we observed in Sec22 mutant eyes.
Syx5 mutants, Sec22 mutants, and Eyc
overexpression animals all have similar ER
phenotypes
Studies of Syx5 functions in the fat
body cells suggested that it might form a
complex with Sec22 to regulate ER
morphology. We then tested whether Syx5
mutants also had phenotypes similar to those
of Sec22 mutants in the eyes. Indeed, the
rhabdomeres were small and irregularly
shaped in the Syx5 mutants. The photoreceptor
cells were also packed with over-proliferated
ER and lipid droplets (Fig. 6A, a).
The photoreceptor morphogenesis
defects and ER accumulation in Sec22 mutants
were reminiscent of a mutant called eyes
closed (eyc) (36). eyc encodes for a fly p47
protein that forms a complex with p97 ATPase
to regulate membrane fusion (37). Eyc
overexpression during the early pupal stage
resulted in fused rhabdomeres. Eyc
mis-expression at a later stage resulted in ER
proliferation in the photoreceptor cells (Fig.
6B, b) (36). The trafficking of Rh1 is defective
in the fly eyes with Eyc overexpressed (36).
All of these phenotypes were very similar to
what we observed in Sec22 mutant eyes. To
test whether Sec22 and Eyc interacts with each
other, we performed IP experiments with
cultured fly S2 cells. Indeed, Sec22 bound to
Eyc (Fig. 6C, D). We wondered whether Sec22,
Syx5, and Eyc function in a same pathway to
regulate ER morphology and photoreceptor
cell morphogenesis. We knocked Syx5 known
or overexpressed Eyc in the Sec22 mutant
background using MARCM technology and
analyzed the ER morphology in the fat body
cells. Both Syx5 RNAi and Eyc overexpression
in the Sec22 mutant cells led to cell growth
defects. The cells became very small, which
hindered the ER morphology analysis (Fig. 6
E-J). The synergistic effects of regulating cell
size suggested these proteins may have
paralleled functions in cell growth. But
whether they work together in regulating ER
morphology is still an open question.
Since both Eyc and Syx5 interact with
Sec22, we hypothesized that Eyc might
compete with Syx5 to bind Sec22. However,
we did not observe any decrease of the
interaction between Sec22 and Syx5 when Eyc
was overexpressed (Fig. 6K). We also did not
observe any alleviation of ER morphology
defects in Eyc overexpression cells when
Sec22 was overexpressed. Therefore, Eyc
overexpression phenotypes were not due to
competing Sec22 with Syx5 (Fig. 6L-O).
DISCUSSION
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Losses of Sec22 and Syx5 result in ER
expansion
The ER is highly dynamic and its
morphology is tightly regulated (10, 11).
Abnormal ER morphology is associated with
hereditary spastic paraplegias (HSP) and
related disorders (38). In this study, we found
that both Sec22 and Syx5 mutants had an
over-proliferated ER and expanded ER lumens.
Sec22 is primarily localized on the ER and
Syx5 is found on the Golgi. The interaction
between Sec22 and Syx5 mediates the fusion
of ER-derived vesicles to Golgi cysts. The
similar ER phenotypes between Sec22 and
Syx5 mutants suggested that defective
ER-Golgi trafficking resulted in ER
proliferation and morphology defects.
ER proliferation has been found in
cells that were exposed to multiple
pathological insults; however, the molecular
mechanisms involved are less well understood
(6). It would be interesting to test whether the
trafficking route between the ER and Golgi is
blocked in these disease conditions. It is still
not clear how these trafficking defects result in
ER proliferation and morphology defects. ER
stress could result in ER proliferation. Thus, it
needs to be determined whether these Sec22
mutants have ER stress responses.
Eyc, p97 and ER dynamics
SNARE proteins can assemble into
unproductive cis-SNARE complexes that need
to be dissociated by two ATPases: NSF
(N-ethylmaleimide-sensitive factor) (39) and
p97 (40-42). After their dissociation, SNAREs
are available to form trans-complexes and
promote another round of fusion. p47 is an
adaptor protein that mediates the interaction
between p97 and a SNARE complex (43, 44).
Interestingly, overexpression of Eyc (fly
ortholog of p47) mimicked the loss of Sec22
and Syx5, which suggested that Eyc functioned
within a paralleled or a same pathway as
Sec22 and Syx5. We found that Sec22 could
bind to Eyc. However, Eyc overexpression
phenotypes are not due to Eyc competing
Sec22 with Syx5. It is possible that the excess
Eyc interferes the organization of the whole
SNARE complex required for ER-Golgi
trafficking. It is also possible that Eyc works
in a paralleled pathway independent of Sec22
to regulate ER morphology.
Sec22 and autophagy
Autophagy is a critical degradative
pathway that is required for cellular
homeostasis (45). SNAREs are important for
membrane homophilic fusion during
autophagosome formation (17, 46). Sec22
facilitates Atg9 recruitment to the PAS and,
therefore, is required for autophagosome
formation in yeast (17). However, in our study,
Sec22 was dispensable for starvation-induced
autophagy in flies. In yeast, autophagosomes
are generated from the PAS, which has not yet
been identified in fly and mammalian cells
(31). It is possible that different autophagic
initiation structures determine whether Sec22
is required for autophagy. Interestingly, no
autophagy defects are observed when SEC22B
is knocked down in mammalian cells (46).
In yeast, ykt6 function overlaps with
that of Sec22 (13). Ykt6p is required for
autophagosome formation in yeast (17), and
Ykt6p is highly conserved. It is possible that
Ykt6 and Sec22 have redundant roles during
autophagy.
The ER is one of the membrane
sources of the autophagosome (47). In Sec22
mutants, the ER was highly proliferated. The
lumens of the ER were expanded and ER
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Sec22 regulates ER morphology but not autophagy in flies
10
morphology changed dramatically. However,
autophagosome formation was not affected in
Sec22 mutants, which indicated that
appropriate ER morphology was not
absolutely required for autophagosome
formation.
In summary, we found that trafficking
between the ER and Golgi mediated by Sec22
and Syx5 was critical for maintaining ER
morphology. Sec22 was also required for
development and eye morphogenesis in flies.
ACKNOWLEGEMENT
We thank Dr. Hugo Bellen, Bloomington
Drosophila Stock Center, and the
Developmental Studies Hybridoma Bank for
providing reagents. C. Tong was supported by
the National Basic Research Program of China
(2012CB966600, 2014CB943100), the
National Natural Science Foundation of China
(31271432), and Specialized Research Fund
for the Doctoral Program of Higher Education
(20130101110116)
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FIGURE LEGENDS
Figure 1. Identifying a complementation group with ER morphology defects
(A) . Screening strategy used to identify mutants with ER morphology defects. (B). Complementation
group ERM1 had an expanded ER, enlargements of late endosomes, and abnormal Golgi
morphology. However, mitochondria were intact. CTL and mutant clones are marked by a loss of
RFP (red) and outlined by the dashed lines. (a’-h’) Green channels of (A’-H’). KDEL-GFP was
used to label the ER, Rab7-GFP was used to label late endosomes, ManII-GFP was used to label
the Golgi apparatus, and Mito-GFP was used to label mitochondria.
Figure 2. ERM1 mutants correspond to Sec22 mutants.
(A) Deficiency and duplication mapping located the ERM1 lesion to a small genomic fragment that
contains 9 genes, including Sec22. (B). Sanger sequencing reviewed a G to A mutation at the
splicing acceptor site on Sec22. (C). Diagram indicating the genomic organization and the
resulting mRNA of the Sec22 locus. The exons were shown as bars with different colors and the
introns were shown as broken lines. The positions of the primers used for RT-PCR were indicated.
The point mutation on ERM1 resulted in two transcripts (Sec22 mu1 and Sec22 mu2) different
from the wild type (Sec22 WT). The alignment of the Sec22 mu1 with Sec22 WT showed that a
deletion in Sec22 mu1 resulted from an alternative splicing site used by Sec22 mu1. The
alignment of Sec22 mu2 with Sec22 WT showed that an intron was failed to be spliced and an
insertion was produced in Sec22 mu2. (D). Diagram indicating the domain organization of Sec22
protein. The proteins encoded by Sec22 WT, mu1, and mu2 were shown. (E). Sec22 genomic
rescue construct rescued the ER morphology defects of Sec22 mutant clones. The ER was labeled
with KDEL-GFP. The clones were negatively labeled with RFP.
Figure 3. Sec22 is not required for starvation-induced autophagy.
No obvious autophagy defects could be detected in Sec22 mutant clones (absence of RFP: red in
A, A’, C, C’, D, D’). (A, A’). FYVE-GFP indicated that PI3K activity, whose activity is
required for autophagy initiation, was intact in Sec22 mutant cells. (B, B’). Upon starvation,
Atg8-GFP formed punctate structures inside fat body cells. Both control (CTL) and Sec22 clones
had similar responses to starvation. (C, C’). MARCM clones of CTL and Sec22 mutant cells
showed the Atg8-GFPRFP signals for monitoring autophagosome and lysosome fusion. There
was no difference between CTL and Sec22 cells. (D, D’). Distribution of p62, an autophagy
substrate, in Sec22 mutant cells was similar to that in CTL cells.
Figure 4. Syx5 interacts with Sec22 to regulate ER morphology
(A) Flag-tagged Syx5 pulled down HA-tagged Sec22 (top two panels). HA-tagged Sec22 also pulled
down Flag-tagged Syx5 (bottom two panels). (B-F). Syx5 RNAi (C) and overexpression (E)
resulted in KDEL-RFP labeled ER proliferation and expansion. Sec22 overexpression alone had
no effect on the ER morphology (D), but enhanced the ER morphology defects when
co-expressed with Syx5 (F). Cg-Gal4 was used to drive the expression of KDEL-RFP,
Syx5shRNA, UAS-Flag-Syx5, and UAS-3XHA Sec22.
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Sec22 regulates ER morphology but not autophagy in flies
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Figure 5. Sec22 is required for eye morphogenesis
(A, a, C, and c). In one-day-old wild type control (CTL) eyes, round, tightly packed, oval shaped
rhabdomeres were in a trapezoid arrangement (A, a). In age-matched Sec22 mutant eyes (C, c),
the rhabdomeres were small and sometimes fused with each other (blue arrows). The ER was
highly proliferated (red arrows) and lipid droplets (yellow arrows) were accumulated. (B, b, D, d).
The ER gradually lost its shape and became highly accumulated inside photoreceptor (PR) cells
with aging. (E).The Rh1 trafficking was defective in Sec22 mutant PR cells. The Sec22 mutant
clones were generated by eyFLP/FRT system. The mutant area was outlined and the typical
mutant PR cells were pointed with yellow arrows. Pha (Phalloidin, red), Rh1 (blue).
Figure 6. Syx5 mutants, Sec22 mutants, and Eyc overexpression animals have similar ER
phenotypes
(A, a). One-day-old Syx5 mutants had phenotypes similar to those of Sec22 mutants. Rhabdomeres
were small and fused (blue arrows), ER was proliferated (red arrows) and lipid droplets were
accumulated (yellow arrows). (B, b). GMR-Gal4 driven Eyc overexpression resulted in ER
proliferation (red arrows) and lipid droplets accumulation (yellow arrows). (C). HA-tagged
Sec22 pulled down Myc-tagged Eyc. (D). Myc-tagged Eyc also pulled down HA-tagged Sec22.
(E-J). Syx5 RNAi and Eyc overexpression in the Sec22 mutant cells led to cell growth defects.
ER was labeled with KDEL-RFP (red). (E). Control clone (CTL). (F). Sec22 mutant clone. (G).
Syx5 RNAi clone. (H).Syx5 shRNA expressed in Sec22 mutant clone. (I). Eyc overexpressed
clone. (J). Eyc overexpressed in Sec22 mutant clone. (K). Eyc overexpression cannot interfere
with complex formation between Syx5 and Sec22. S2 cells with indicated protein expressed.
HA-tagged Sec22 was immunoprecipated with anti-HA anibody and the amount of Flag-tagged
Syx5 was detected with anti-Flag antibody. Eyc expression did not reduce the amount of Syx5
interacted with Sec22. (L-O). Sec22 overexpression cannot rescue the ER morphology defects
caused by Eyc overexpression. ER was labeled with KDEL-RFP (red). (L). Control. (M).
Cg-Gal4 driven UAS-3HA-Sec22 overexpression. (N). Cg-Gal4 driven UAS-Myc-Eyc
overexpression. (O). Cg-Gal4 driven both UAS-3HA-Sec22 and UAS-Myc-Eyc expression.
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TongXiaocui Zhao, Huan Yang, Wei Liu, Xiuying Duan, Weina Shang, Dajing Xia and Chao
development in DrosophilaSec22 regulates ER morphology but not autophagy and is required for eye
published online February 10, 2015J. Biol. Chem.
10.1074/jbc.M115.640920Access the most updated version of this article at doi:
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