7 - mitochondrial protein import in plants
TRANSCRIPT
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Plant Molecular Biology 0: 311–338, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.311
Mitochondrial protein import in plants
Signals, Sorting, Targeting, Processing and Regulation
Elzbieta Glaser1, Sara Sjöling1, Marcel Tanudji2 and James Whelan2
1 Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stock-holm, Sweden; 2 Department of Biochemistry, University of Western Australia, Nedlands 6907, Perth, Western
Australia, Australia
Key words: mitochondrial processing peptidase, molecular chaperones, plant mitochondria, protein import,
processing peptidase, protein processing, protein sorting, regulation of protein import, signal peptides
Abstract
Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes andspecific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are
synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which
is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components
and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties
of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition,
mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molec-
ular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial
processing peptidase (MPP), which in contrast to other sources, is integrated into the bc 1 complex of the respi-
ratory chain. This is the most studied component of the plant import machinery characterised to date. What are
the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are
several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage,
specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into
mitochondria.
Introduction
Plant cell constitutes an interesting biogenetic system
in which genetic information is located in three in-
tracellular compartments, the nucleus, mitochondria
and chloroplasts. Despite the fact that both mito-
chondria and chloroplasts contain their own genetic
information, most of the protein complement of these
organelles is synthesised in the cytosol. This results
in an active protein trafficking from the cytosol tothese organelles. Mitochondrial protein import process
has been studied very extensively during the past two
decades, especially in lower eukaryotes, in fungi, and
resulted in characterisation of many signal peptides,
protein translocating machineries on the outer and in-
ner membrane and involvement of both cytosolic and
organeller chaperones in the process of transport (for
reviews see [141, 167]). Although less information
is available on the mitochondrial protein import in
plants (for recent reviews [178, 211]), several new
and unique findings have been reported in the last few
years.
The mitochondrial protein import process is a
multi-step process including the following events:
(1) Synthesis in cytosol of the precursor protein con-
taining an N-terminal extension called a presequence
that functions as an organellar sorting and target-ing signal. (2) Interaction of the newly synthesised
precursor with cytosolic chaperones and other fac-
tors, to confer an import competent conformation
on the precursor, facilitate recognition and prevent
organellar mis-sorting. (3) Recognition of the pre-
cursor on the organellar membrane either by direct
interaction of the presequence with organellar recep-
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tors or by interaction of the presequence bound to
cytosolic chaperones. (4) Translocation of the precur-
sor through the organellar membranes. (5) Proteolytic
processing of the precursor inside mitochondria by
a highly specific mitochondrial processing peptidase.
(6) Assembly of the mature form of the protein into
a functional, oligomeric protein complex in a processinvolving organellar chaperones.
In this chapter we will review our present knowl-
edge on components and mechanisms involved in
mitochondrial protein import in plants with emphasis
on findings that are specific and unique for plants.
Synthesis of precursor proteins in the cytosol
The nuclear encoded mitochondrial proteins carry tar-
geting information as signal peptide located at N-
terminus. Most of the signals constitute an N-terminal
extension, the so called presequence that is cleavedoff by the mitochondrial processing peptidase (MPP)
during, or after, import into the mitochondrion, re-
sulting in the production of mature protein. Only the
outer membrane proteins and some low molecular
mass proteins of the inner membrane contain non-
cleavable signal peptides [169, 211]. We will review
the major features of the plant mitochondrial prese-
quences that lead to sorting, targeting, translocation
and processing the nuclear encoded precursor proteins
into mitochondria.
Presequence composition and structure
A collection of all available plant presequences of
nuclear encoded mitochondrial proteins from the data-
bases shown in Figure 1 contains at present over
100 sequences. Characteristic features of the prese-
quences were outlined using statistical analysis, se-
quence alignment and secondary structure predictions.
Out of a total of 737 mitochondrial precursor se-
quences from different sources, we have analysed
80 plant presequences statistically in terms of length,
amino acid composition and conserved domains [169,
178, 211]. Seventy-one sequences were predicted, or
estimated, to contain a mitochondrial processing pep-tidase (MPP) cleavage site. Out of these, 31 sequences
contained a confirmed/microsequenced cleavage site;
a final collection of 25 different presequences was
used for analysis, i.e. only one sequence representing
each protein was analysed.
The length of the known plant presequences varies
from 13 amino acid residues for the alternative oxidase
of Arabidopsis thaliana to 85 amino acid residues of
the P subunit of glycine decarboxylase from pea, with
an average length calculated to 40 amino acid residues.
Compared to mammalian, yeast and N. crassa prese-
quences, the plant presequences are in average 7–9
residues longer. The plant presequences are rich in
serine (17.1%), arginine (12,6%), alanine (12.0%),leucine (10,6%), but low in cysteine (1.0%), histidine
(1.3%), tryptophane (1.4%), tyrosine (1.4%), glutamic
acid (1.4%) and aspartic acid (1.5%). The amino acid
composition of the plant presequences is thus simi-
lar to that of other mitochondrial presequences [169,
201] i.e. they exhibit high content of basic hydroxy-
lated residues (with the exception for histidine) and
low content of acidic and aromatic residues. A unique
feature is that the plant presequences have higher con-
tent of serine (17%) as compared to that of yeast (7%),
mammals (3%) or N. crassa (10%) [178].
The existence of amino acid sequences for full-
length precursor proteins corresponding to the same
protein from different plant species enables sequence
comparison of both the presequences and mature parts
of the protein. The identity of amino acids of the
mature parts of the precursors was much higher than
within the presequences indicating a common evolu-
tionary pathway previous to the endosymbiotic event.
The acquisition of the presequence, after gene transfer
from the mitochondria to the nucleus, could have been
an independent step for different ancestors of each
species.
In order to investigate if there are any conserved
domains within the plant presequence we aligned pre-sequences from different plant species, corresponding
to the same protein [178]. Proteins for which at least
three sequences are available from different sources,
e.g. F1β, FeS, HSP 60, and superoxide dismutase
(SOD) (with an exception for alternative oxidase) have
higher identity of amino acids in the amino- and
carboxy- terminal regions, than in the central parts
of the presequences. The more conserved N- and C-
terminal regions of these presequences suggest func-
tional importance of these domains. The N-terminal
domain, or the import domain, is important for guid-
ing the precursor protein to the mitochondria. This
domain shows a rather regular alternation between ba-
sic and hydrophobic residues and has the potential to
form amphiphilic α-helix with one positively charged
and one apolar face, in contact with lipids, or other
proteins [200]. The C-terminal domain, or the process-
ing domain, most often contains a cleavage motif for
MPP and a secondary structure which is compatible
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Figure 1. Amino acid sequence of the presequences of the nuclear encoded mitochondrial proteins. The amino acid sequence of the presequence
and the first three amino acids of the mature protein are shown. The space indicates the beginning of the mature protein, if determined
directly by N-terminal sequencing the protein is indicated by an ∗. The N-terminal region of the other proteins was as indicated in the
published sequences (see Ref for details), this was judged on homology with published sequences (from plants and other organisms) for
which N-terminal information was available. 1 indicates proteins that have no cleavable presequence and the first 30 amino acids of the
mature protein are shown or the amino acids that the original authors suggested that were involved in targeting. 2 represents a naturally
occurring plant presequence that targets to the mitochondrion and chloroplast (see text for details). + = Personal communication Michael
Hodges. Abbreviations used: Plant species - At = Arabidopsisthaliana, Bn = Brassica napus, Cr = Chlamydomonas reinhardtii,
Cr u = Chenopodium rubum, Cs = Cucurbita sp, Cv = Citrullus vulgaris, Eg = Euglena gracilis, Eug = Eucalyptus gunnii,
Fp = Flaveria pringlei, Gm = Glycine max, Gv = Gracilaria verrucosa, H b = Hevea Brasiliensis, I b = Ipomea batat as,
Np = Nicotia na plubaginif olia, N t = Nicotiana tobacum, Mi = Mangoif era, Or = Oryza sativa, P a = Panicum miliaceum,
P s = P isum sativum, Sg = Sauromatum guttatum, St = Solanum tuberosum, T t = T riticum turgidum, V u = V igna unguiculata ,
Zm = Zea mays. Proteins - AA = Asparate aminotransferase, AC = Aconitase, AD = Aldehyde dehydrogenase, ANT = Adenine nucleotidetranslocator, AOX = Alternative oxidase, CcR = Ubiquinol cytochrome C oxidoreductase complex (bc1), CS = Citrate synthase, COX =
cytochrome oxidase, Cyt c1 = cytochrome c1, F1 α , β, δ, and γ = the alpha, beta, delta, epilson and gamma subunits of the ATP synthase, FAd
= d subunit of the FA portion of the ATP synthase, FC = Ferrochelatase, FH = Fumarate Hydratase, FPS = Farnesyl-diphosphate synthase, GD
= Glycine decarboxylase subunits, Gr = Gluathione reductase, HSP = Heat shock protein, HPPK DS = 6-hydroxymethyl 7, 8-dihydropterin
pyrophosphokinase 7, 8 dihydropteroate synthase, ICDH = Isocitrate dehydrogenase, MD = Malate dehydrogenase, ME = Malic enzyme, Mt
= Malate translocator, CI NADH = NADH binding subunit of complex I, PD = Pyruvate dehydrogenase, PPXI = Protoporphyrinagen, PSST
= PSST protein of complex I, RFeS = Rieske FeS protein, RSP = Ribosomal Protein, SHMT = Serine Hydroxymethyltransferase, SOD =
Superoxide dismutase, TOM = Translocase outer membrane, TufM = Translation elongation factor, UCP = Uncoupler protein.
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Figure 1. Continued.
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Figure 1. Continued.
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Figure 1. Continued.
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Figure 1. Continued.
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for processing [178]. Both domains may overlap. Be-
low, structural properties of the presequences with
relevance for their functions will be discussed.
Targeting properties of presequences
Despite the fact that over 100 plant sequences areavailable, the mitochondrial targeting ability of only
about 10 of these sequences has been demonstrated
experimentally, and detailed analysis has only been
carried out on a handful of sequences. The mitochon-
drial targeting ability of a presequence can be deter-
mined by (1) the use of chimeric constructs containing
the presequence and a marker gene and targeting ex-
amined in vitro and in vivo, (2) the use of synthetic
peptides to inhibit targeting of authentic or chimeric
constructs in vitro and (3) the use of site-directed mu-
tagenesis to test the role of specific residues with in
vitro studies. All approaches have advantages and dis-
advantages. The use of marker constructs is useful
to detect import but in vivo they suffer from the fact
that the nature of the passenger protein may greatly
effect targeting ability. Chloramphenicol acetyl trans-
ferase (CAT) as a passenger protein is targeted more
efficiently than β-glucuronidase (GUS) to plant mito-
chondria [174]. Lack of targeting observed in some
cases may be explained by structural features of dif-
ferent constructs ([207]). Also the proteolytic accessi-
bility in the cytosol of a chimeric construct may vary
greatly with the addition of ‘linker sequences’ [230].
The presequence of the Nicotiana plumbaginifolia
F1β subunit can target passenger proteins to mitochon-dria [30, 32], and although deletions in the C-terminal
region don’t abolish targeting they reduced the ef-
ficiency quite dramatically [32]. This implies that
although the N-terminal portion is sufficient for tar-
geting, the C-terminal region plays a significant role.
Deletion of the N-terminus abolish targeting, and this
is supported with in vitro studies with synthetic pep-
tides derived from the presequences [91]. A similar
functional role for the maize superoxide dismutase
presequence comes from deletion studies [219].
A number of other studies with the adenine nu-
cleotide translocator (ANT) and F1δ presequence re-port that additional residues from the mature region
are required for mitochondrial targeting [100, 136,
223]. Although Mozo et al (1995 [136]) who used
the GUS reporter gene showed that the presequence
of ANT is not sufficient for targeting in vivo, Winning
et al. (1992 [223]) showed that an additional 20 amino
acids from the mature protein did support mitochondr-
ial targeting with dihydrofolate reductase (DHFR) as
a marker. It cannot be concluded that the N-terminal
region of the adenine nucleotide translocator does not
play a role in mitochondrial targeting. Even though
mature ANT can be imported to the mitochondrion
[223], a study with chimeric precursors containing
both mitochondrial and chloroplast targeting peptidesshowed that the first sequence (most N-terminal) ex-
erted the targeting information [174]. Therefore the
N-terminal presequence of the ANT may play a role
in targeting in vivo.
Site directed mutagenesis studies of the prese-
quence of the soybean alternative oxidase have been
carried out to elucidate the targeting requirement of
a plant mitochondrial precursor protein (Tanudji et
al., unpublished). Approximately 30 different mu-
tants were constructed to elucidate the role of indi-
vidual residues. It was found that arginine residues
throughout the presequence were important for target-
ing ability. However, not just arginine (or positive)
residues in the region of the predicted amphiphilic α-
helix were important, but arginine residues near the
processing site also played an important role in tar-
geting. Changing two arginine residues at the −2 and
−10 region of the presequence (outside the predicted
helical region) inhibited import as much (80%) as
changing two arginines (−20, −30) predicted to be in
the helix forming region. Additionally a region of the
presequence that contained the putative amphiphilic
α-helix was not sufficient to support efficient import
into isolated soybean mitochondria (Tanudji et al., un-
published). This study would indicate that the entirepresequence is important for targeting, which is in
general agreement with the deletion studies for the
F1β if the amount of import is examined. It does also
indicate that an amphiphilic α-helix alone is not the
only requirement for an efficient targeting to plant mi-
tochondria. It will be of interest to investigate if the
in vitro results are consistent with the in vivo studies
using chimeric constructs.
Sorting of precursors between mitochondria and
chloroplasts
Mitochondrial and chloroplastic precursor proteins
were shown to interact in the cytosol with molecular
chaperones, heat-shock proteins of 70 kDa (HSP70s)
(for review see Hartl, 1996), mitochondrial import
stimulation factor (MSF) [72] and also a presequence
binding factor (PBF) [137]. These proteins seem to
co-operate in binding to newly synthesised precursor
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proteins, assist their folding, prevent aggregation [67],
keep them in a transport-competent form (unfolded
or loosely folded) and convey them to import recep-
tor complexes on the surface of the organelle [190].
Despite the fact that there exist distinct structural dif-
ferences in mitochondrial and chloroplastic targeting
peptides [201] it is possible that cytosolic factors thatinteract with the targeting peptides contribute to the
specificity of protein targeting and/or to prevention of
miss-sorting [121].
A number of studies demonstrated that mitochon-
drial protein import in plants was highly specific, i.
e. mitochondria appear to import precursors of mi-
tochondrial proteins only, while chloroplasts appear
to import precursors of chloroplast proteins only [18,
214]. Using a homologous in vitro organelle import
system, i.e., isolated spinach mitochondria and chloro-
plasts, we have shown that import was specific for both
organelles [214]. Also, recently, specific targeting into
isolated mitochondria and chloroplasts has been re-
ported for proteins from C. reinhardtii [144]. The
C. reinhardtii precursors of the F1α subunit and the
Rieske FeS protein were imported into C. reinhardtii
mitochondria with high efficiency in a membrane po-
tential dependent manner. The C. reinhardtii F1β sub-
unit, containing a C-terminal extension in addition to
the N-terminal presequence, was imported with much
lower efficiency. Also, the import of mitochondrial
heterologous precursor proteins from higher plants,
soybean alternative oxidase and the N. plumbaginifo-
lia F1β subunit, was much less efficient. A number of
studies using transgenic approaches also showed highspecificity of targeting into plant mitochondria. The
chimeric constructs consisting of the presequence of
N. plumbaginifolia F1β coupled to CAT or glutamine
synthase were specifically targeted into mitochondria
[18, 83].
Mis-sorting of proteins has also been reported in
a few cases. Hurt et al. (1986 [194]) reported that
the RuBisCO transit peptide from C. reinhardtii could
direct proteins, mouse DHFR and yeast cytochrome
oxidase subunit IV, into yeast mitochondria. However,
the system was non-homologous and the chloroplast
transit peptides from C. reinhardtii were later reported
to contain an amphiphilic α-helix, characteristic of mi-
tochondrial targeting peptides [63]. Huang et al. (1990
[89]) showed that the mitochondrial presequence of
the yeast cytochrome oxidase subunit Va can function
both as a mitochondrial and chloroplastic targeting
peptide in transgenic tobacco. However, it was con-
cluded that this ‘mis-targeting’ did not represent a
physiological pathway, as in vitro studies showed that
the import of this chimeric construct occurred at zero
degrees and was not receptor mediated. Both these
cases of mis-sorting are examples of mis-sorting in
heterologous systems. However, Criessen et al. (1995
[37]) have reported that the pea glutathione reduc-
tase precursor protein is directed both to chloroplastsand to mitochondria in transgenic tobacco. The PsaF
protein from C. reinhardtii but not the PsaK protein
has been shown to be imported in vitro into spinach
mitochondria and also recently into C. reinhardtii mi-
tochondria, in vitro [92, 144]. However, characteristics
of the import process were peculiar. This process was
shown to be independent of the presequence, as a
mutant protein devoid of presequence was protease-
protected upon incubation with spinach mitochondria.
Furthermore, the protease-protection also appeared
to be independent of mitochondrial import receptors,
as it was not inhibited by a synthetic peptide corre-
sponding to a mitochondrial presequence [92]. The
transit peptides of two chloroplast envelope proteins,
the triose-3-phosphoglycerate phosphate translocator
(TPT) and a 37 kDa protein of unknown function, have
the ability to form an amphiphilic α-helix, a feature
considered to be essential for mitochondrial but not
for chloroplastic targeting sequences [28, 201]. Import
studies of these proteins showed that they interacted
with yeast mitochondria in a receptor dependent man-
ner [28]. Chimeric constructs containing TPT transit
peptide and 5 or 23 amino-terminal residues of the
mature TPT coupled to CAT were also imported into
plant mitochondria in vitro [175]. However, in vivo intransgenic tobacco, the construct containing the transit
peptide and 5 residues of the mature TPT was found
in the cytosol, whereas, the construct containing 23
residues was specifically imported into chloroplasts.
These studies clearly show that the import process has
more stringent specificity in vivo [175].
In summary, the above described results indi-
cate that some chloroplast transit peptides contain
sufficient information for specific interaction with
mitochondrial import receptors, however, they also
show that import specificity between mitochondria
and chloroplasts is maintained in vivo. Interestingly, it
has been reported that transit sequences of chloroplast
precursor proteins but not mitochondrial or peroxi-
somal precursors are phosphorylated by a plant spe-
cific cytosolic protein kinase and that phosphorylated
precursors bind to chloroplasts [205]. Dephosphory-
lation seems to be required to complete the precur-
sor translocation process across the membranes. This
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phosphorylation-dephosphorylation cycle of chloro-
plast destined precursor proteins might represent one
of the events involved in a specific sorting process
between mitochondria and chloroplasts [205]. If an
amphiphilic α-helix is not the only requirement for
plant mitochondrial import, the few chloroplast pre-
cursor proteins that do contain such a motif in thetransit peptide may only be targeted to plant mitochon-
dria quite inefficiently. Along with other factors that
may contribute to targeting specificity such as chaper-
ones [121] it seems that targeting specificity is quite
high. What the studies on specificity do indicate is
the possible artefacts of using heterologous systems
with chimeric constructs as the nature of the passen-
ger protein and it’s susceptibility to various proteases
may differ between the authentic construct and the
chimeric construct used in import studies ([174, 207,
230]) Tanudji et al., unpublished).
Protein import machinery
Components of the outer membrane translocase
Several components of the mitochondrial import ma-
chinery (translocase) of the outer membrane (Tom)
have been identified in plants, potato (p) Tom 20
[81], Vicia faba (Vf ) Tom 40 [152] and outer mem-
brane HSP 70 [133]. Additionally, mtHSP 70 [209]
has been cloned and expressed sequence tags exist for
Tim 17 from Arabidopsis and rice, and Tim 23 from
Arabidopsis.Tom 20 from potato has been cloned, it has a pre-
dicted molecular mass of 23 kDa. It displays only
20% identity with other Tom 20 but similarity values
rise to 50% and it is also proposed to contain a tetra-
tricopeptide repeat (TPR), a characteristic of many
components of the mitochondrial import machinery
[12, 13, 81]. Preincubation of mitochondria with an-
tibodies to pTom 20 inhibited the import of a variety
of precursors by 30% to 40%. The lack of complete
inhibition is not unexpected as similar studies in yeast
suggest several receptors with overlapping specifici-
ties [161]. pTom 20 is imported into mitochondriawithout the need for a protease (trypsin) sensitive
receptor on the outer membrane [81]. The character-
isation of Tom 20 from plants represents an important
step in understanding the import process as further
studies with this components may help to understand
how specificity of targeting is maintained. For in-
stance when h Tom 20 was cloned and overexpressed
in yeast (y) it was shown that it could complement
a y Tom 20 mutant. However, in contrast to the y
Tom 20 it did not support import of artificial precur-
sors under the influence of a ‘cryptic’ mitochondrial
targeting sequence [128]. This indicates that similar
components within different phylogenetic groups may
display substantially different properties.A Tom 40 component has been identified from Vi-
cia faba, with an apparent molecular mass of 42 kDa.
This protein was identified with antibodies raised
against Neurospora crassa ( Nc) Tom 38 and y Tom
40 (formally ISP 42) [152]. These antibodies inhib-
ited import of several precursor proteins into isolated
mitochondria. This component is proposed to be one
of the central proteins of the general insertion pore in
fungi. However, no further information of its identity
or function is currently available in plants. The other
component of the outer membrane that has been listed
is the outer membrane (OM) HSP 70 [133]. No role
in the import process has been shown for this com-
ponent. It is not present in N. crassa or yeast but a
similar component has been reported from mammals
[122]. As different HSP 70 isoforms play several roles
in the import process it is interesting to speculate that
the OM HSP 70 may also be involved in the import
process in higher organisms. It is possible that pre-
cursors proteins are passed from cytosolic HSP 70 to
the OM HSP 70 before binding to the outer membrane
receptors.
Import pathways
Three import pathway that have been elucidated for
plant mitochondria are outlined in Figure 2. The sim-
plest pathway exists for p Tom 20 (pathway 1) which
does not require a trypsin sensitive components on the
outer membrane [81]. The import conditions used in
this study employed both ATP and substrates to gener-
ate a membrane potential so the involvement of these
components cannot be ruled out. However, in yeast, a
membrane potential is not required for the import of
outer membrane proteins [118].
The third import pathway outlined in Figure 2 may
be described as the general import pathway [141].The import of proteins in this pathway requires both
extra- and intramitochondrial ATP, cytosolic factors,
outer membrane proteinaceous receptor(s); precursor
proteins that use this pathway are usually processed
by MPP (non-cleaved precursors may also use this
pathway). It is likely that this pathway uses other com-
ponents such as Vf Tom 42, Arabidopsis thaliana ( At )
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Figure 2. Diagrammatic representation of three pathways of protein import into plant mitochondria. Pathway 1 shows the import of an outer
membrane protein (Tom 20) which inserts spontaneously into the outer membrane [81]. Pathway 2 shows the import of the ATPsynthase
FAd subunit precursor protein from soybean. This precursor protein is synthesised with an N-terminal presequence which interacts with the
outer and inner membrane translocation machinery components. This precursor protein does not use ATP-dependent cytosolic factors for
targeting through the cytosol [44]. Pathway 3 shows the import of the soybean alternative oxidase precursor protein. This precursor protein is
also synthesised with an N-terminal presequence and requires ATP-dependent cytosolic factors. Import via pathways 2 and 3 converges with
the requirement for a membrane potential across the inner membrane, and ATP in the matrix for driving the inner membrane translocation
machinery [44]. Components of the inner and outer translocation machinery which have been identified in plants are labelled in larger lettering.
HSP70, Tom 20 and Tom 40 [81, 133, 152] on the outer membrane, Tim 23, Tim 17 ( Arabidopsis thalianaEST database), and mtHSP 70 [202],
in the inner membrane and matrix. Other components labelled in smaller lettering which have been identified in fungi are shown. Components
involved in processing and assembly have not been shown.
Tim 17 and Tim 23 but since the specific involve-
ment of these components has not been experimentally
demonstrated in plants they will not be further dis-
cussed. The requirement for external ATP in this
pathway may come from two sources, cytosolic HSP
70 and/or an additional factor. Although it was previ-
ously thought that cytosolic HSP 70 had a requirement
for ATP, characterisation of the mitochondrial import
stimulating factor (MSF) from rat using yeast mito-
chondria indicates that it is MSF that has the obligate
ATP requirement and that precursors bound to cytoso-
lic HSP 70 may be imported without ATP [73, 74,104]. However, this has only been shown with the
adrenodoxin precursor and it has not be applicable
to all precursors using the general import pathway.
Additionally, it should be remembered that rat mito-
chondria contain an outer membrane HSP 70 that is
absent in yeast and the use of a heterologous system
may not give a clear picture. In vivo, assuming that
cytosolic HSP 70 would bind to precursor proteins and
that ATP would be present, it is likely that ATP is used
by cytosolic HSP 70 in the import process.
There is evidence that another cytosolic factor in
addition to cytosolic HSP 70 is required in the plant
mitochondrial import pathway. This comes from stud-
ies using wheat germ as a translation lysate rather
than rabbit reticulocyte lysate, the assay used to iden-
tify and purify MSF [131]. Mitochondrial precursors
translated in the wheat germ translation system were
imported into mitochondria at a very low efficiency.Addition of rabbit reticulocyte lysate stimulated im-
port. This factor was shown to be N-ethylmaleimide
(NEM) sensitive and required ATP, similar to purified
MSF from rat liver. It has been shown that import
of the soybean FAd precursor into soybean mitochon-
dria could be supported by the wheat germ alone [44].
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Significantly this import did not require external ATP
and was not sensitive to NEM treatment of the precur-
sor. This implies that it did not require this additional
factor, that is either inactivated or absent in wheat
germ. Additionally, protease treatment of mitochon-
dria showed that import of the FAd precursor used a
less protease sensitive protein component on the outermembrane, indicating that a different receptor may be
involved [44]. Therefore, an additional import path-
way (pathway 2, Figure 2) has been proposed to exist
in plant mitochondria (Figure 2).
Evidence suggests that import pathways 2 and 3
converge at a common point for translocation into mi-
tochondria. This is supported by the fact that both
pathways require a membrane potential and that pre-
cursors are processed by MPP ([44], unpublished
data). The translocation force for import is postulated
to be derived from Tim 44 in combination with mtHSP
70, although it is still unclear whether a Brownian
ratchet or translocation motor model is the mecha-
nism of import [72, 86]. In plants mtHSP 70 has been
cloned from a number of sources (Figure 1) but no
direct experimental evidence exists for its role in im-
port. In spinach, we have shown that mtHSP 70 is
partially associated with the mitochondrial inner mem-
brane [202]. It is likely that it fulfills a similar function
as in yeast and the proposal that mtHSP 70 may regu-
late import in plants is discussed in section on control
of protein import.
Mechanism of translocation
Import (translocation) of proteins into mitochondria is
inhibited by components that abolish the membrane
potential such as the ionophore valinomycin, and un-
couplers CCCP and FCCP [30, 77]. Using a potassium
driven membrane potential it has been concluded from
studies in fungal systems that it is the ψ compo-
nent of the membrane potential that is required [125,
153]. No specific studies in plants have addressed this
question.
A variety of respiratory substrates as malate, succi-
nate, glycine and NADH have been shown to support
precursor import into plant mitochondria in varioussystems [31, 49, 102, 203, 209, 212]. We have com-
pared the efficiency of each of these substrates to
support import with soybean mitochondria. We have
also investigated if respiration via the alternative path-
way alone can support import. We have found that
succinate and then NADH support import best of two
different precursors. Added NADH is only oxidised
via the external NAD(P)H dehydrogenase [48, 132,
134, 198], and can support import via the alternative
pathway alone. It indicates that a sufficient membrane
potential can be generated in the absence of any proton
translocation via the well characterised complexes of
the cytochrome chain [43, 206].
It is puzzling why import should differ with dif-ferent substrates if only a low membrane potential (40
to 60 mV) is required as suggested from the studies
in fungal systems [125, 153]. Using succinate as a
substrate to support import, a competitive inhibitor
to complex II, malonate, we could clearly demon-
strate that the import of the FAd was inhibited with
10 mM malonate, whereas the alternative oxidase was
not fully inhibited with 50 mM inhibitor. This strongly
suggests a difference in the magnitude of the mem-
brane potential required for the import of these two
precursor proteins [43]. A different threshold of mem-
brane potential has been reported to be required for
different precursors in fungi [125]. This study showed
that the requirement for different magnitude of the
membrane potential corresponded to a property of the
presequence, and was not a consequence of the length
of the mature protein. It was proposed that the greater
number of positive residues in the presequence the
lower the requirement for a membrane potential. As
both the alternative oxidase and FAd precursor have
five positive residues this cannot be the only factor
that causes the difference seen with these two pre-
cursors. An explanation for the difference may be the
nature of the mature protein. The insertion of the pre-
sequence into and across the inner membrane seemsto be a reversible event [141]. However, the presence
of a hydrophobic segment may stabilise the translo-
cation intermediate [71]. As the FAd protein is very
hydrophilic [181], the difference in the requirement
for a membrane potential may lie in the fact that a
higher threshold is required to maintain the prese-
quence of the FAd precursor on the inside of the inner
membrane compared to the alternative oxidase. The
latter is a transmembrane protein and once its pre-
sequence has inserted across the innermembrane the
hydrophobic segments may directly insert into the in-
ner membrane or stabilise this precursor. Considering
the branched nature of the respiratory chain in plant
mitochondria, close examination of the membrane po-
tential requirement with a variety of precursors and
chimeric constructs is warranted, measuring both im-
port and membrane potential under similar conditions,
to understand the translocation of proteins to various
locations within the mitochondrion.
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The ψ component of the membrane potential is
proposed to exert its effect via an electrophoretic ef-
fect on the positively charged presequence [77]. This
is supported by a study which elegantly showed that
import of a fusion protein containing the targeting do-
main of Tom 70 (and DHFR) can be inserted into the
inner membrane of ruptured rat liver mitochondria in aψ dependant manner, but removal of the three posi-
tive signals in the targeting domain, results in insertion
into the inner membrane that becomes independent of
ψ [129].
A second role for the membrane potential has
emerged with the characterization of the import ma-
chinery. It has been demonstrated that Tim 23 acts
as a voltage sensor, undergoing dimerization in the
presence of a membrane potential, the extent of dimer-
ization being dependant of the extent of the membrane
potential. Binding of the matrix targeting domain of a
presequence dissociates the dimer opening the import
channel [6]. The two different roles for the membrane
potential are not contradictory as it would appear
from the studies showing that the threshold of mem-
brane potential for dimerization [6] is lower than that
required for the electrophoretic effect on precursors
[125]. This issue, however, has not been addressed in
the same study.
Another factor that may affect protein import
is redox status of SH-groups of the inner mem-
brane translocase. We have investigated the effect of
sulfhydryl group reagents on import of the in vitro
transcribed/translated precursor of the F1β subunit
of the ATP synthase (pF1β) into Solanum tubero-sum mitochondria [203]. We have used a reducing
agent, dithiothreitol (DTT), an alkylating membrane-
permeant, N-ethylmaleimide (NEM), a non-permeant,
3-(N-maleimidopropionyl)-biocytin (MPB), an SH-
group specific agent 5,5-dithiobis-(2-nitro-benzoic
acid) (DTNB) as well as an oxidising cross-linker,
copper. DTT slightly stimulated the mitochondrial
protein import, whereas NEM and Cu2+ were in-
hibitory. Inhibition by Cu2+ could be reversed by
addition of DTT. We have dissected the effect of the
SH-group reagents on binding, unfolding and trans-
port of the precursor into mitochondria. The efficiency
of receptor-mediated binding of pF1β to mitochon-
dria increased in the presence of DTT, NEM or Cu2+.
This demonstrated that the inhibitory effect of NEM
and Cu2+ on the efficiency of import was not due to
the interaction of the SH-group reagents with import
receptors. Modification of pF1β with NEM prior to
the import resulted in stimulation of import, whereas
no effect was seen with DTT and Cu2+. It h as
been reported that treatment of the alternative oxi-
dase precursor with 5 mM NEM inhibits its import
into mitochondria. This was proposed to be due to
inactivation of an NEM sensitive factor [44]. Import
of pF1β through a receptor-independent bypass-route
and into mitoplasts was sensitive to DTT, NEM andCu2+ in a similar manner as import into mitochondria.
Both DTNB and a membrane-impermeant, MPB were
shown to inhibit protein import into mitoplasts. These
results taken together indicate that protein import into
plant mitochondria is inhibited by modification of the
SH-groups of the protein import machinery located on
the outer surface of the inner mitochondrial membrane
[203].
A question that arises with regard to the translo-
cation process in plant mitochondria is the timing of
the processing event with respect to the location of
the precursor. Considering the membrane location of
MPP (next section), the question arises if precursors
are processed during or after translocation. The latter
would require that matrix located proteins would have
to come into close proximity with the inner membrane
after import whereas the former would require that the
cytochrome bc1 complex would be physically close to
the import site. It has been reported that characteris-
tic inhibitors of MPP, EDTA and orthophanenthroline,
inhibit import into plant mitochondria, in contrast to
the situation in fungal systems [216]. Metal chelators
which could not pass the inner membrane did not in-
hibit import indicating existence of a metal dependant
translocation step on the inside of the inner membrane.Further characterisation of the Tim components of the
import machinery may help to answer this question
by using immunolocalisation or immunoprecipitation
techniques.
It can be concluded that the current understanding
of the translocation process in plants is at an early
stage and heavily relies on studies from yeast sys-
tem to interpret the available experimental data from
plant systems. Although some of the components have
been identified, functional characterisation still needs
to be carried out with a variety of precursor proteins
as it cannot be presumed that these components have
the same properties in plants as they have in simpler
fungi. The case of h Tom 20 compared to y Tom 20
exemplifies the need for caution in trying to fit exper-
imental data from one system with the characterised
components of another system [128]. The other area
that needs to be further investigated with reference to
the translocation mechanism is the involvement and
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consequences, if any, of the integrated MPP into the
cytochrome bc1 complex. Although studies with puri-
fied MPP (see below) indicate no link between elec-
tron transport and processing it would be of interest to
test this out with intact mitochondria.
Mitochondrial protein processing
The mitochondrial processing peptidase (MPP) re-
moves the amino-terminal targeting signal, the prese-
quence, from nuclear-encoded mitochondrial precur-
sor proteins, during or shortly after protein import into
mitochondria [77, 166]. A striking feature of MPP
is that it is a general peptidase, as it acts on several
hundred mitochondrial precursor proteins, yet MPP is
specific as it recognises a distinct cleavage site and
specifically cleaves off the presequences, that show
low sequence similarity, in one single cut. Proteolytic
removal of the targeting peptide is not essential for im-
port [94, 228, 232], Sjöling, unpublished), but might
be necessary for proper folding of the protein [162].
The subunits of the MPP were the first geneti-
cally identified components of the import machinery
[228]. It consists of two structurally related subunits,
α-MPP and β-MPP, which co-operate in processing.
Both subunits are essential for processing. In plants,
the MPP has been purified from potato tubers [21]
spinach leaves [56, 60] spinach roots (Sjöling, unpub-
lished) and wheat [26]. The plant MPP was shown to
reside in the inner mitochondrial [59] integrated into
the cytochrome bc1 complex of the respiratory chain[21, 54, 60]). Thegenes encoding the potato MPP have
been cloned [21, 53, 55]) and shown to be homologous
to each other.
In mammals and yeast both subunits are soluble
in the matrix as a heterodimer, whereas in N. crassa,
the matrix subunits are isolated as monomers [79, 148,
229] and 70% of β-MPP can be found associated to
the mitochondrial inner membrane and it is identical to
the core 1 protein of the bc1 complex of the respiratory
chain [171].
Early studies of plant mitochondrial protein
processing showed that detergent extracts of lysedcauliflower and Vicia faba mitochondria could cleave
N. crassa precursors to mature proteins [214] and that
mitochondrial extracts were unable to cleave chloro-
plast precursors [213]. The processing activity of plant
mitochondria was found in the membrane fraction [59]
and could not be dissociated from the membrane by
high pH, high ionic strength or chaotropic reagents.
Addition of the soluble fraction, the matrix, did not
affect the activity of the membrane fraction.
The plant cytochrome bc1 complex has a simi-
lar subunit composition as the corresponding enzymes
of mammals and fungi. It comprises 10 polypeptides
ranging in molecular mass from about 60 to 8 kDa [21,
56], including the Rieske-FeS protein, cytochromesc1 and b. The exception is the apparent existence, on
SDS-PAGE, of three to four core proteins, instead of
two, as found in the bc1 complex of mammals and
N. crassa [165, 210]. Three core proteins have been
observed in the bc1 complex of spinach leaves, beet
[11, 21] and yeast [123,191]. There is even the appear-
ance of four core proteins in the bc1 complex of potato
wheat [26] and spinach roots (Sjöling, unpublished).
It was suggested that these extra polypeptides, which
could be seen on SDS-PAGE, were due to the exis-
tence of incompletely processed core precursors [123,
191]. The polypeptides, however, were always present
in equimolar ratio. Therefore, the existence of more
than two core proteins is rather the result of occurrence
of isoenzymes. Blue native gels show a composi-
tion of potato bc1 complex with the same molecular
mass as bovine bc1 complex, indicating that the potato
bc1 complex consists of only two core proteins per
monomer [96]. Immunoprecipitation using antibody
against one of the isoforms in potato reveal an enzyme
complex containing only two core proteins and gene
specific oligonucleotides reveal that the genes encod-
ing α-MPP of potato are differently expressed in all
tissues analysed but transcript levels do not vary be-
tween tissues [96]. The core proteins of the spinachbc1 complex were immunological [56, 60] and by se-
quence analysis [21] identified as MPP subunits [21].
The genes for the isoproteins have probably arisen
by gene duplication followed by sequence divergence
[47]. The biological significance of these isoforms re-
mains to be determined. Possibly they act as a buffer
against detrimental mutations effecting the highly ex-
pressed genes or it is the result of the polyploidy of
cultured plants [55, 96].
Characteristics of the MPP/bc1 complex
The molecular mass of the MPP/bc1 complex cor-
responds to a dimer of the bc1 complex [57]. The
dimeric form of the bc1 complex has been observed in
other species by chromatography, electron microscopy
and X-ray crystallography [150, 168, 210, 227]. It is
not known if the monomeric forms in the dimer co-
operate in the respiratory electron transfer neither if
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the dimeric form is essential for peptidase activity in
plants.
The plant MPP is active over a broad pH range,
pH 6–11 (with optimum at pH 8) and over a broad
temperature range, 10–50 ◦C (with optimum at 35 ◦C).
The plant MPP is a metallopeptidase. This is in ac-
cordance with MPP from other organisms [14, 127].There is no effect on the processing activity by in-
hibitors of the serine-, cystein-, amino- aspartic- or
thiol-type protease. On the other hand, the metal
chelators, ortho-phenantroline and EDTA, totally in-
hibit the processing activity. The processing activity is
not dependent on exogenous metal ions [56, 57] but it
is slightly stimulated by the addition of Ca2+, Mn2+,
or Mg2+. Co2+, Fe2+, Zn2+, Cu2+ or Ni2+ inhibit
the peptidase activity [58, 66, 177]. It has been shown
that inhibition by heavy metals, or transition metals,
is a common feature amongst many zinc containing
metallo-peptidases [35, 115]. In reconstitution exper-
iments with spinach apo-MPP, prepared by dialysis
against ortho-phenantroline and then buffer to remove
the chelator, the activity could be restored by the addi-
tion of Mn2+, Zn2+ and partially by Mg2+, Co2+ and
Cu2+ [58]. Mn2+, Mg2+, Co2+, and Cu2+ are divalent
cations frequently found to substitute for zinc in met-
allopeptidases [4]. Particle Induced X-ray Emission
(PIXE), Inductively Coupled Plasma-Atomic Emis-
sion Spectroscopy (ICP-AES) and Total Reflexion
X-ray Fluorescence (TRXE) show that the spinach
MPP/bc1 complex contains iron, copper, calcium and
zinc [58]. High ionic strength inhibits the spinach
leaf MPP activity [56] at higher concentrations (>0.1 m), however, it does not dissociate the spinach core
proteins from the bc1 complex. Surprisingly, KCl con-
centrations above 1M stimulate the processing activity
of potato MPP [53].
As the plant cytochrome bc1 complex is involved
in both protein processing and respiration, one may
wonder whether protein processing is dependent on,
or affected by, the redox state of the bc 1 complex or
on respiration. Complete oxidation or reduction of the
redox centra of the bc1 complex in sub-mitochondrial
particles (SMP) in the presence of respiratory sub-
strates and respiratory chain inhibitors has only slight
or non effect on the processing activity. Reduction of
the bc1 complex with DTT or dithionite diminishes
the processing activity to about 50%, probably by
effecting metal/ligand interaction. In summary, even
though there is a bifunctionality of the oligomeric
MPP/bc1 complex, there is no correlation between
electron transfer and protein processing in vitro [56].
Soluble plant MPP
Preliminary characterisation of the processing prop-
erties of plant mitochondria has shown that plants
contain a processing peptidase with similar proper-
ties to fungal MPP [213, 214]. Despite the fact that a
processing activity can be clearly located and isolatedfrom the mitochondrial inner membrane, a process-
ing activity located in the matrix fraction was also
reported from Vicia and spinach as earlyas 1992 and in
subsequent studies, although it was not characterised
in detail [59, 80, 103]. We showed that the matrix
located activity was not due to an ATP dependent
protease as it was not dependent on ATP. Likewise,
there was no evidence for non-specific breakdown
such as smearing or appearance of other breakdown
products on SDS-PAGE [103]. Additional evidence
for the location of a processing peptidase in the matrix
of plant mitochondria came from the studies in which
the purified N. crassa β-MPP subunit restored the
processing activity in solubilized plant mitochondria
which were immuno-depleted with antibodies against
N. crassa β-MPP and unable to catalyse the process-
ing activity [214]. This is in contrast to studies of the
now extensively characterised membrane located MPP
from plants which have shown that the processing ac-
tivity cannot be separated from the cytochrome bc1
complex.
In a combined effort from four laboratories, we re-
investigated the possible presence of a matrix-located
processing activity in plant mitochondria, in terms
of its ability to generate mature size products fromprecursor proteins, its specificity, and its inhibitor
sensitivity [186]. We have used three mitochondrial
precursor proteins and two plant species. We inves-
tigated occurrence of an additional, matrix located
processing activity, by incubation of the precursors
of the soybean mitochondrial proteins, alternative ox-
idase, the FAd subunit of the ATP synthase and the
tobacco F1β subunit of the ATP synthase, with the
membrane and soluble components of mitochondria
isolated from soybean cotyledons and spinach leaves.
A matrix-located peptidase specifically processed the
precursors to the predicted mature form in a reactionwhich was sensitive to o-phenanthroline, a characteris-
tic inhibitor of MPP. The activity was also inhibited by
NEM, an inhibitor of fungi MPP. The specificity of the
matrix peptidase was illustrated by the inhibition of
processing of the alternative oxidase precursor in both
soybean and spinach matrix extracts upon altering a
single amino acid residue in the targeting presequence
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(−2Arg to Gly). Additionally, there was no evidence
for general proteolysis of precursor proteins incubated
with the matrix. The purity of the matrix fractions was
ascertained by spectrophotometric and immunologi-
cal analyses. The results demonstrate that there is a
specific processing activity in the matrix of soybean
and spinach corresponding to 50% and 20%, respec-tively, of the total mitochondrial processing activity.
The identity of the matrix peptidase is not known.
In a lower eukaryotic photosynthetic organism C.
reinhardtii, in vitro processing studies revealed that
in contrast to the situation in higher plants, and in
accordance with studies in yeast and mammals, the
processing of the precursors was catalysed by a matrix
located peptidase and not by a peptidase integrated
into the cytochrome bc1 complex of the respiratory
chain [144]. Interestingly, maturation of the homolo-
gous precursors inside mitochondria required addition
of ATP during import. In the absence of ATP, pre-
cursors were protease-protected inside mitochondria,
but most of the imported proteins existed in a non-
processed form, indicating requirement for ATP for
import of the precursor into the matrix.
The different location of MPP in various organisms
is proposed to represent divergence from a single orig-
inal evolutionary event [27]. This is in agreement with
a monophyletic origin for mitochondria [68].
A new family of metallo-endopeptidases
Sequence alignment shows that the MPP subunits of potato, rat, yeast, N. crassa and the core proteins
of the bc1 complexes, share sequence similarity with
the pitrilysin family [156–158]. The pitrilysin family
includes the pitrilysins from E. coli, the insulin de-
grading enzymes from mammals and Drosophila, the
N-arginine dibasic convertase from rat and a couple
of additional enzymes. Pitrilysins are highly specific
metallo-endopeptidases, and like the MPP, recognise
their substrates without defined amino acid residues
around the scissile bond, showing that recognition is
on the basis of higher order structure rather than of
the amino acid sequence [1, 8]. By site directed muta-genesis, an inverted zinc-binding site was discovered
that was used to define a new family of metallo-
endopeptidases, the pitrilysin family [5, 7, 9, 151].
A glutamate in this zinc binding motif possibly has
the function as electron donor for a hydrogen bond
to a water molecule which attacks the carbonyl at the
peptide bond to be cleaved [29]. The HXXEH74−76E
signature is conserved in all β-MPPs but degenerate in
α-MPPs and core proteins.
Substrate specificity of processing
How does the MPP recognise such a diversity of mito-
chondrial precursor proteins? What features determinethe MPP cleavage? What are the structural features
of the precursor protein that are recognised by the
MPP? Is the sequence of a few amino acids around
the scissile bond sufficient or do other structural el-
ements contribute to the recognition of the cleavage
site? These questions have been addressed using sev-
eral approaches, including statistical analysis of plant
presequences for common features, in vitro studies of
affinity of chemically synthesised targeting peptides
for MPP, structural analysis of presequence peptides
and site directed mutagenesis of precursor proteins.
Except for the common cleavage motifs R-3 and
R-2, [64, 169, 178, 201] no consensus sequence has
been identified within the presequences. Since there is
a significant amount of variability in the presequence
among different precursors, we and others have pro-
posed that MPP recognises higher order structural
features [93, 109, 176, 178, 179, 208], possibly in
conjunction with basic residues [84, 88] rather than a
specific amino acid sequence. Although the local argi-
nine motifs, R-3, R-2, represent parts of the features
enhancing precursor processing, the motifs are highly
degenerate and can even be found elsewhere in the
precursor protein since arginine residues are present
at multiple positions in all proteins. MPP, however,does not cleave at the other sites, making it obvious
that the R-3 or R-2 motifs are not sufficient for spe-
cific cleavage, but that additional common features are
required.
Studies have shown that synthetic peptides corre-
sponding the C-terminal portion the presequence of
the F1β ATPase subunit of N. plumbaginifolia inhibit
processing of pF1β [176] to a higher extent com-
pared with an N-terminal peptide. These results show
that the C-terminal peptide has higher affinity for the
spinach MPP and that MPP recognises the C-terminal
domain rather than the N-terminal domain of the pF1βpresequence. This is consistent with results of studies
of alignment of plant presequences. In those studies
several presequences were found to contain conserved
N-terminal and C-terminal domains. This may have
functional importance, the N-terminal domain being
important for targeting and import and the C-terminal
domain for interaction with MPP [178]. Shorter pep-
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tides corresponding to 4, 6 and 11 amino acid residues
relative to the cleavage site of the pF1β presequence
had no significant inhibitory effect on processing. This
could indicate either that a peptide longer than 11
amino acid residues is required for interaction with
MPP, or that these shorter peptides lack a feature im-
portant for interaction with MPP, which is presentin the longer C-terminal peptide, or both. Interest-
ingly, by secondary structure prediction [176] and
circular dichroism analysis (Sjöling, unpublished) the
longer pF1β C-terminal peptide was shown to contain
a helical element which is not present in the shorter
C-terminal peptides. The helical structure is proposed
to facilitate interaction of the precursor with MPP.
Similar results were shown with the soluble rat liver
MPP [179], indicating that both the membrane bound
plant MPP and the soluble mammalian MPP recognise
similar higher order structure
In addition, a mutant peptide corresponding to the
22 C-terminal residues of the pF1β presequence with
a reduced helix (through the substitution of a serine
for a proline residue which brakes the helix) does
not have the same affinity for spinach MPP as a 22
residues long C-terminal wild-type peptide containing
the intact helix (Sjöling, unpublished). Disrupting the
same C-terminal helix of pF1β by site-directed muta-
genesis of the presequence of the complete precursor
protein inhibits processing (Sjöling, unpublished). It is
possible that a C-terminal helix of the presequence fa-
cilitates the interaction with MPP through helix/helix
packing with the peptidase, or that the helical segment,
followed by a rather loose structural part of the ma-ture chain, constitutes a transition point functioning
as recognition element for MPP as suggested by von
Heijne [201].
The MPP of differentorganismsresides in different
mitochondrial suborganellar compartments. Results
show that despite the fact that the plant MPP is in-
tegrated into a membrane-bound oligomeric complex
in vivo, whereas the mammalian MPP is soluble, the
recognition of the substrate is conserved, and the same
structural features upstream of the cleavage site are
recognised by both the spinach and the rat MPP [179,
208]. In addition, results also show that the presence of
a typical mitochondrial cleavage site is not always suf-
ficient for processing by either soluble or membrane
bound MPP.
The necessity of basic amino acid residues within
the presequence for the targeting of mitochondrial pre-
cursors to the mitochondria has been accepted [2, 76,
77] however, the role of basic residues for MPP cleav-
age is not understood. Mutagenesis studies of both
mammalian [2, 142, 146, 182] and plant precursor
proteins (Tanudji et al., unpublished, Sjöling, unpub-
lished) suggest that basic residues at proximal as well
as distant positions upstream relative to the cleavage
site are important for specifying the cleavage. An argi-
nine residue is suggested to be required for the peptidebond cleavage and possibly for docking the precursor
at the correct site for MPP cleavage [149]. According
to the cleavage site classification by a self organising
network [169] the role of arginine residues seems to be
similar in different organisms, although location and
topology of MPP varies in different organisms.
Mutational analysis of plant presequences
The specificity of protein processing has been
analysed by site directed mutagenesis of prese-
quences. Possibly the recognition site for MPP is
physically separate from the actual cleavage site in
the precursor protein, as has been suggested by Bed-
well and co-workers [10]. The role of both proximal
and distal arginine residues and secondary structure
for processing has been investigated using the soybean
alternative oxidase and the tobacco F1β precursor.
The arginine residue at position −2 from the cleav-
age site of the alternative oxidase presequence, which
is suggested to be part of the cleavage motif [64,
84, 169, 178, 201] could not be substituted by a
glycine, leucine, alanine, glutamine, threonine, or
phenylalanine residue without inhibition of process-
ing or processing at an incorrect cleavage site (Tanudjiet al., unpublished). −2Arg could not be exchanged by
other basic residues such as lysine or histidine without
partial or total inhibition of processing.
How important is the position of the proximal
arginine residue? There are plant precursors which
are processed although they lack a proximal arginine
residue and the presequence of tobacco pF1β does not
contain an arginine residue at position −2 but at posi-
tion −5. The −5Arg, in tobacco F1β could however be
substituted by a leucine or alanine residue without in-
hibition of processing. Results show that the proximal
arginine of alternative oxidase does not have to be atposition −2 but can also be at position −4 of the cleav-
age site in order for the precursor to interact correctly
with the active site of MPP. The optimal efficiency of
processing though, is achieved with a −2Arg.
Basic residues, distal to the cleavage site, have
been suggested to be important for processing in some
studies of mammalian precursors [87, 88, 142, 146,
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149, 182] but not in others [76]. Studies of plant pre-
cursor proteins show that distal arginine residues to the
cleavage site of the tobacco F1β are not important for
the affinity of the presequence to MPP. The alternative
oxidase contains distal arginine residues at positions
−10, −30 and −35 and a lysine residue at position
−20. However, results indicate that −10 Arg is notimportant for processing whereas the other distal ba-
sic residues as distant of 30 and 35 residues from the
actual cleavage site, somewhat surprisingly, affected
processing of the soybean alternative oxidase with pu-
rified spinach MPP. However as a triple mutant of the
three distal positive residues was processed efficiently,
and a deletion mutant that had two of the distal positive
residues removed, this suggests that the distal residues
play a structural role essential for processing with the
entire presequence. Changing some of the residues
in single or double mutants can cause inhibition of
processing with purified MPP, further changes or dele-
tions relieves this inhibition. This indicates that these
residues do not have a role in the catalytic mechanism.
(Tanudji et al., unpublished).
It may be difficult to determine whether certain
amino acid positions within a given presequence are
particularly critical for cleavage function, or con-
versely, whether large segments of the presequence
contribute collectively to the functionality of the speci-
ficity. However, we can conclude from the muta-
tional analysis of the plant presequence that the most
proximal arginine is important, but not essential, for
processing of the soybean alternative oxidase (Tanudji
et al., unpublished) but not the tobacco F1β precur-sor (Sjöling et al., unpublished). In addition, basic
residues distant to the cleavage site, even those located
at the N-terminusof the presequence, are important for
processing of alternative oxidase. Surprisingly, dele-
tion of the amino acids flanking the scissile bond
of alternative oxidase, −1 serine and +1 glutamate,
inhibit but do not abolish processing.
Together with several other mutational analysis of
the alternative oxidase and the F1β precursor it can
be concluded that the secondary structure of the pre-
sequence is important, for processing, in some cases
together with distal and proximal arginine residues.
However, the conclusions are based on results of in
vitro studies. It is possible that additional factors may
influence the efficiency of processing in vivo.
Model of interaction of precursors with the MPP/bc1
complex
From our observations, and others, and with the
known structure of the bovine bc1 complex, we can
model the interaction and recognition mechanism of
a precursor protein with the plant MPP/bc1 complex.If the structure of the bovine core proteins, correlates
with the plant MPP/core protein structure, we can an-
ticipate that the presequence has to be flexible enough
to reach into the cavity of the core/MPP subunits to
the active site. Indeed, it has been shown that the pre-
sequence has to be flexible in order to be processed
[208]. We have also shown that the secondary structure
of the presequence is important for affinity to MPP
and thereby also for cleavage. A helix, proximal to
the cleavage site, would facilitate binding of the prese-
quence to MPP by helix/helix interaction. The amino
acids lining the wall of the cavity enclosed by the core
proteins in the bovine bc1 complex, are mostly hy-
drophilic and laced with negatively charged residues
[227], creating an environment which would be prone
to interact with positively charged presequences.
Two sets of basic residues enhance proteolysis in
several presequences, the proximal arginine within the
degenerate cleavage motif, and distant basic residues
relative to the cleavage site. The distance between the
proximal and distal residues varies between precur-
sors. Within this stretch a proline or glycine residue
may serve as a flexible linker and a helix breaker.
It would be possible for the basic charges to inter-
act with negative charges on MPP, and to present thescissile bond to the water on the metal in the active
site. A degenerate version of the potentially active
site of MPP, the inverted zinc binding motif, can be
found in the core 1 protein of the bovine bc1 complex,
Y91XXE94H95X 76E171. The residues Y91E94 and H95
are located on a helix facing the cavity and the core
protein 2 protein, not the matrix. Another helix, con-
taining the distal E, spans right above and across the
YEXXH helix [227]. This site, if we can correlate the
bovine structure to the spinach bc1 /MPP, may well be
the active site of MPP.
Models of evolution of the plant MPP
Why is plant mitochondrial MPP attached to the bc1
complex? The bifunctionality of this protein complex
has been suggested to reflect the co-evolution of two
enzymatic activities [26]. Bacteria have a bc1 com-
plex which lacks core proteins. The evolution of the
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MPP and core subunits could have started with an an-
cestral protease which was hydrophilic and located in
the cytosol of bacteria, in correspondence to pitrilysin.
During endosymbiosis the processing peptidase might
have become attached to the membrane as it was ad-
vantageous for the function of the early MPP to be
located close to the protein import sites. Alternativelythe bc1 complex was dependent on new subunits ex-
posed to the matrix side for protection from proteases
that evolved in the matrix [15].
After gene duplication, a two component process-
ing peptidase evolved, corresponding to the present
situation in plant mitochondria. The detachment of
MPP from the bc1 complex in yeast, mammals and
partly N. crassa could reflect the necessity of inde-
pendent regulation of respiration and mitochondrial
import. The extra subunits of the bc1 complex could
have become indispensable for protection against pro-
teolytic degradation and assembly of the complex
[170] and therefore another gene duplication resulted
in the core proteins without catalytic activity and solu-
ble MPP in the matrix. The core proteins in yeast and
mammals would in this situation be evolutionary relics
of the processing peptidase [26].
What does not fit in this model is that the process-
ing activity of the mitochondrial processing peptidase
is independent of respiration [57] and therefore there
was no necessity for the MPP subunits to become de-
tached from the bc1 complex in yeast and mammals.
The evolution of the MPP subunits could have started
with a prokaryotic ancestor where a proteolytic ac-
tivity was integrated into the primitive bc1 complex,showing that the proteolytic activity could have been
present in the bc1 complex before the acquisition of
the core proteins.
Control of protein import into plant mitochondria
The control of protein import into mitochondria is an
area where studies with plants lead rather than are
derived from other organisms. This is a desirable sit-
uation as investigations in this area will not have to
overcome established paradigms that have been es-tablished in simpler organisms. Controlling the level
of protein within an organelle by controlling import
into that organelle would add another regulatory step
to the many characterised post-transcriptional control
steps known in plants [61]. It is generally believed
that the protein import into mitochondria is a consti-
tutive process. This impression arises from the fact
that the half-life of precursors in the cytoplasm is
generally very short [75, 189], deletion of an import
component in yeast is usually compensated by other
components or is lethal [141], and in vitro import stud-
ies usually have to use non-physiological inhibitors
to prevent import. That mitochondrial protein import
is constitutive is confounded by the fact that thesestudies are carried out with simple fungi grown under
ideal conditions, import under these circumstances is
probably constitutive and apparently rapid [141]. One
exception to the apparent universal constitutive import
ability of mitochondria in non-plant systems was seen
with two P450 precursor proteins from bovine. It was
clearly demonstrated in a number of reports that only
steroidogenic tissues could support the import of these
precursors, despite the fact that mitochondria from
other tissue were shown to be import competent for
other precursor proteins [126, 147]
In an attempt to develop an in vitro import sys-
tem from tobacco we have uncovered two possible
modes of regulation [45]. It has been puzzling, why
isolated respiratory competent tobacco mitochondria
only supported very low levels of import. Our stud-
ies demonstrated that it appeared that mitochondria
isolated from the dark phase of growth clearly im-
ported precursor proteins at much higher (∼4-fold)
levels than mitochondria isolated from the light period.
The failure of mitochondria isolated from the light
phase to import protein was apparently at the translo-
cation stage of import, as binding and processing of
the precursor protein was not effected by the time of
isolation. This indicated that some component of thetranslocation machinery was either absent or inacti-
vated during the light phase. Extending these studies
clearly showed that the inhibition was not directly due
to light as plants that were clonally propagated did
not excert this rhythmic variation in import. Clon-
ally propagated plants are continually wounded (or
stressed) and this appears to over rule any regula-
tion seen with the diurnal growth conditions. [43].
Likewise wounding of plants, by cutting off half the
leaves on the plant, allowing recovery for 1 to 2 weeks
showed not rhythmic pattern in import [43]. These
studies indicate that mitochondrial import in plants is a
regulated process and that this regulation is responsive
to outside signals.
Another level of control that has been uncovered
with plant mitochondria is developmental level. In
tobacco mitochondria it was found that the FAd sub-
unit of the ATP synthase could not be imported into
younger tobacco leaves (2 cm in diameter – 6–8 weeks
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old) but could be imported into older tobacco leaves
(6 cm wide – 20 weeks old), in contrast the alternative
oxidase couldbe imported into both sets of leaves [45].
Developmental regulation of import has also been re-
ported with pea leaf mitochondria [49]. In this study
a dramatic decrease in import was observed between
day 6 and day 30 with pea leaves. This decline inimport was proposed to be due to a decline in the
level of mtHSP 70 as the amount of this protein also
decreased over a similar time period. However, the
decline in import was not strictly correlated with the
decline in mtHSP 70 levels, examination of the data
presented showed that even though import displayed
a five fold decrease from day 16 to day 30, that the
decrease in mtHSP 70 was less than two fold over the
same period. This indicates that other components or
factors effecting the activity of mtHSP 70 may also be
involved. A similar decrease in import ability has also
been reported for pea plastid import [42]. Therefore,
the decrease in mitochondrial import with pea mito-
chondria may represent an integrated cellular decrease
in organelle biogenesis with increasing maturity.
In a study of import during soybean cotyledon de-
velopment a 5-fold decline in import was observed
(Huang and Whelan, unpublished). Soybean cotyle-
dons are useful tissues for developmental studies as
they undergo a complete life cycle from germination
to senescence in approximately 20 days. Similar to the
study with pea mitochondria, a rise the amount of the
alternative oxidase was observed, but in contrast, we
detected a decline in components of the cytochrome
chain, in particular the Rieske FeS and cytochromeoxidase subunit II. The decline in these components
appeared to precede the decline in import. We propose
from these studies that the decrease in mitochondrial
import represents a general decline in mitochondrial
biogenesis. The fact that induction of the alternative
oxidase appears to be opposite to this general decline
can be explained by the fact that mRNA expression for
this subunit rises during this senescence period (Mc-
Cabe et al., unpublished) and this compensates for the
low import ability of mitochondria.
We believe that the recently uncovered mecha-
nisms of control of the protein import process repre-
sent the first studies and that the area is very fruitful for
further studies. Tissue, developmental and rhythmic
control of import are all possible avenues of regulation
of mitochondrial import. Additionally, it will be of in-
terest to uncover how the distribution of dual targeted
proteins is regulated in each organelle. One possibility
is that mitochondria (and plastids) are not constantly
importing proteins and that there exist factors that reg-
ulate the ability of organelles to import proteins; these
factors would determine the amount of proteins in an
organelle.
Concluding remarks
In the past ten years, impressive progress has been
made in characterisation the mitochondrial protein
transport machinery in plants. Although it appears
that the basic features of mitochondrial protein import
are conserved between all mitochondria [31], sev-
eral unique features have been unravelled in plants
in comparison to yeast and mammals. These features
include the following events and require further stud-
ies (i) structural characteristics of plant presequences;
(ii) the sorting phenomenon between mitochondria
and plastids, (iii) the involvement of cytosolic and
mitochondrial molecular chaperones, (iv) recognition
of the presequence by the import machinery and by
the processing peptidase, and (v) the rhythmic and
developmental regulation of the import process.
Statistical analysis of all available plant prese-
quences from the databases showed that plant prese-
quences are in average 7–9 amino acid residues longer
than in other species. They are enriched in especially
Ser, the basic residue Arg, and also Ala and Leu but
contain few Cys, His, Trp, Glu, Asp. There are typ-
ical MPP cleavage motifs with Arg in position −2
from the cleavage site R-X-A/S-T/S, or −3, R-X-X-
A/S-T/S, but the R-10 motif was not found in plantpresequences, indicating that MIP may not exist in
plant mitochondria.
In plants, the occurrence of the two endosymbiotic
organelles, mitochondria and chloroplasts requires
higher organellar specificity for protein import than in
non-plant sources. It is not known how this specificity
is maintained, but high specificity of import can be
achieved using specific targeting signals in combina-
tion with cytosolic factors. It has been also suggested
that specific cytosolic factors may direct mRNAs of
nuclear encoded organellar proteins into the vicinity
of a target organelle and increase specificity of import[120]. It is also possible that the receptor complexes
in plants contain additional components which would
contribute to a high specificity for organellar import in
plants. Next steps of investigations may thus involve
identification of novel or plant specific chaperones and
their role in the guidance of the newly synthesised
proteins to different intracellular biogenetic pathways
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and also detailed characterisation of import receptor
complexes.
The cloning of several nuclear encoded mitochon-
drial proteins has allowed more detailed studies of
protein import into plants. However to date only the
import of one outer membrane protein (pTom 20) has
been studied. A few inner membrane proteins havebeen studied, but none in detail and little has been
done on assembly. Further studies to analyse different
import pathways will be of great interest. Of particu-
lar interest will be proteins that are nuclear encoded
in some plants but organelle encoded in others. An
example of this is seen with cytochrome oxidase II,
where it is nuclear encoded in some leguminous plants
[143]. Will this protein have novel features in import
or will it use the old import machinery. High order of
complexity has been observed in protein import into
thylakoids (Robinson et al., this issue).
Mitochondrial processing peptidase catalyses cleav-
age of several hundred mitochondrial precursor pro-
teins that are nuclear encoded, synthesised on cytoso-
lic polyribosomes and imported into mitochondria. In
contrast to non-plant sources where MPP is a matrix
enzyme, the plant mitochondrial MPP is localised in
the inner membrane and constitutes an integral part
of the bc1 complex of the respiratory chain. The bc1
complex in plants is thus bifunctional, being involved
both in respiration and in protein processing. How-
ever, despite the integration, the processing activity
is not dependent on the electron transport. Recent
studies unravelled also existence of a matrix located
processing activity in addition to the activity integratedinto the bc1 complex. It will be of interest to iso-
late and clone the gene of the protein catalysing this
activity and to understand basis for co-existence of
the membrane-bound and matrix-located processing
activities.
Although the length of the presequences ranges
from 8 to 121 residues (in all sources) and there
are no sequence similarities between the mitochon-
drial presequences and no consensus for the cleav-
age site, the presequence is cleaved off in a single
proteolytic process. Both the membrane-bound inte-
grated MPP/bc1
complex of plants and the soluble
mammalian MPP recognise similar higher-order struc-
tural elements upstream of the cleavage site that are
important for processing. The secondary structure
with flexibility and stabilising elements, hydrophobic-
ity and charge of the presequence seem to influence
the interaction with MPP. Prediction analysis of sec-
ondary structure shows that a helix structure followed
by an extended conformation is common in most
plant presequences, that could facilitate recognition
for processing by the unique plant MPP which is in-
tegrated into the bc1 complex of the respiratory chain.
As the structure for the mammalian bc1 complex is be-
coming unravelled, it will be interesting to model the
plant MPP/precursor interaction to understand recog-nition event and specificity of processing.
The regulation of protein import into mitochon-
dria in plants represents a new and potentially one
of the most far reaching aspect to emerge in recent
studies. The lower eukaryotes that have been exten-
sively studied are undifferentiated and in studies with
mammalian tissues, mature organs have been used.
In both these systems mitochondria are basically in a
constant environment, maintained by the growth con-
ditions with simple fungi and by integrated metabolic
control with higher animals. However, plants must be
able to respond rapidly to changing circumstances and
therefore it is not so surprising that they may have ad-
ditional levels of control not seen in other organisms.
To date both developmental and rhythmic control have
been uncovered. It is likely that tissue effects also exist
in plants. However if the mitochondrion can control
when it imports proteins it provides another means
to determine specificity. It also adds an extra level
of complexity to the communication that takes place
between the mitochondrion and the nucleus of which
there is little known in plants [185]. It will be of inter-
est to uncover as to how the mitochondrion ‘decides’
or is ‘told’ to import proteins. Will the different im-
port pathway be regulated differently and if so whatdistinguishes these pathway for such regulation. A
combination of different approaches will be necessary
to gain insights into this area.
In conclusion, over the last ten years, there has
been significant progress in the understanding of the
basic properties of the plant mitochondrial import
process. Several unique aspects of the process have
been discovered. Future studies using both in vitro
and in vivo techniques, combined with the informa-
tion from genome sequencing projects, will lead not
only to a greater understanding of this process in
plants but will also contribute to the understanding
of mitochondrial biogenesis in higher eukaryotes in
general.
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Acknowledgements
This work was supported by grants from The Swedish
Natural Science Research Council (NFR) and The
Swedish Foundation for Strategic Research (SSF) to
EG and from The Australian Research Council to JW.
We are grateful to P. Dessi for Figure 2.
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