1 Department of Chemistry, Graduate School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-8602, Japan
2 Core Research for Evolutional Science and Technology, Japan Science and
Technology Corporation, Japan
* Author for correspondence (e-mail: endo{at}biochem.chem.nagoya-u.ac.jp)
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Summary |
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Key words: Mitochondria, Protein import, Translocator, TOM, TIM
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Introduction |
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Information for targeting to mitochondria is contained in the mitochondrial
proteins themselves (Schatz and
Dobberstein, 1996). Typical mitochondrial targeting signals are
encoded in the N-terminal presequences, which have the potential to form
positively charged amphiphilic helices and are removed upon import of the
protein into mitochondria (Roise and
Schatz, 1988
) (Fig.
1). Some other mitochondrial proteins are synthesized without
cleavable presequences and contain targeting signals within the mature protein
(Fig. 1).
|
Import into mitochondria is mediated by translocators (also termed
translocases or translocons) in the mitochondrial membranes
(Neupert, 1997;
Pfanner and Geissler, 2001
).
The translocator is an assembly of multiple membrane-protein subunits and
performs multiple functions. First, it functions as a receptor for recognition
of the targeting and/or intramitochondrial sorting signals. Second, it
provides a protein-conducting channel through which precursor proteins cross
the membrane in an unfolded state. Third, it provides the driving force for
vectorial movement of the translocating polypeptide chain.
In Saccharomyces cerevisiae, three mitochondrial translocators
have been identified: the TOM (the translocase of the outer mitochondrial
membrane) complex in the outer membrane, and the TIM23 (TIM, the translocase
of the inner mitochondrial membrane) and TIM22 complexes in the inner membrane
(Fig. 2)
(Lill and Neupert, 1996;
Neupert, 1997
;
Koehler et al., 1999
;
Bauer et al., 2000
;
Pfanner and Geissler, 2001
;
Endo and Kohda, 2002
;
Jensen and Dunn, 2002
). These
translocators mediate protein translocation across or into the mitochondrial
membranes and guide the proteins to their destinations within
mitochondria.
|
We review recent progress in our understanding of mitochondrial protein import. In particular, we focus on the functional cooperation and separation of the translocators in the two membranes.
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The translocators |
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The TOM complex purified from Neurospora crassa is a
cation-selective high-conductance channel
(Künkele et al., 1998).
Electron microscopic analyses revealed that the holo TOM complex contains two
or three pores, each of which has a diameter of
20 Å
(Ahting et al., 1999
). Tom40 is
the main component of this channel and purified Tom40 alone can function as a
cation-sensitive channel when reconstituted into liposomes
(Hill et al., 1998
). Tom22 and
Tom5 may functionally link the receptors to the Tom40 channel
(Kiebler et al., 1993
;
Dietmeier et al., 1997
;
van Wilpe et al., 1999
).
The TIM23 complex
The TIM23 complex mediates the translocation of presequence-containing
proteins across the inner membrane. It consists of two integral membrane
proteins Tim23 and Tim17 and a peripheral membrane protein,
Tim44, and is functionally assisted by soluble matrix proteins Ssc1p and Yge1p
(Mge1p). Tim17 and Tim23 could form a protein-conducting channel. Tim23 forms
a dimer through its N-terminal domain (residues 51-101)
(Bauer et al., 1996).
Dimerization depends on the membrane potential across the inner membrane
(
), and dissociation of the dimer depends on the presence of
presequences (Bauer et al.,
1996
). Purified recombinant Tim23, when integrated into liposomes,
functions as a voltage-activated cation-selective channel that is inhibited by
presequence peptides but is activated by both presequence peptides and
(Truscott et al.,
2001
). Therefore, in the presence of
, presequences
trigger dissociation of the Tim23 dimer, probably leading to the opening of
the TIM23 channel. Although a channel-forming activity of Tim17 has not been
directly demonstrated, the sequence similarity shared by Tim17 and Tim23
suggests that Tim17 could also constitute a pore, perhaps with Tim23.
Tim44, Ssc1p and Yge1p (Mge1p) form a motor that drives translocation
across the inner membrane (Stuart et al.,
1994; Rassow et al.,
1995
). Ssc1p is a mitochondrial Hsp70 (mHsp70) and cycles between
high-affinity and low-affinity states for unfolded polypeptide segments at the
expense of ATP hydrolysis (Schneider et
al., 1994
). Yge1p is a nucleotide-exchange factor for Ssc1p. Tim44
is a peripheral inner membrane protein, and Ssc1p binds to it in a
nucleotide-dependent manner. The current model proposes that two molecules of
Ssc1p, tethered to the TIM23 complex through Tim44, bind in a hand-over-hand
manner to the mitochondrial precursor protein, which emerges through the exit
of the TIM channel in an unfolded state
(Moro et al., 1999
). How Ssc1p
facilitates the movement of the precursor through the TIM channel has been a
matter of debate: two conceptually distinct models, the Brownian ratchet and
power stroke models, have been proposed
(Glick, 1995
;
Neupert and Brunner, 2002
). In
the power stroke model, Ssc1p undergoes a conformational change to generate a
pulling force on the precursor protein, which drives unfolding of a folded
domain outside the mitochondria. The Brownian ratchet model suggests the
transient local unfolding of the precursor protein, which allows translocation
of the unfolded segments through the import channel. Multiple rounds of
binding of Ssc1p to the translocated precursor segments in the matrix would
thus result in unidirectional movement and global unfolding of the precursor
protein.
Tim50: a new component of the TIM23 complex
Although several reports have described possible additional components of
the TIM23 complex, it was only in the last year that Tim50, a new component of
the TIM23 complex, was identified. Geissler et al.
(Geissler et al., 2002)
isolated the TIM23 complex from yeast cells and identified Tim50 as an
additional protein co-purifying with the complex. Yamamoto et al.
(Yamamoto et al., 2002
)
identified it by characterizing a protein previously shown to be crosslinked
to a translocation intermediate. Tim50 was also identified in Neurospora
crassa by co-isolation with the TIM23 complex
(Mokranjac et al., 2003
).
Tim50 is essential for yeast growth. When one selectively depletes it,
mitochondria lose the ability to import presequence-containing precursor
proteins but not the presequence-less inner membrane protein ADP-ATP carrier
(AAC) (Geissler et al., 2002;
Yamamoto et al., 2002
).
Anti-Tim50 antibodies block the import of presequence-containing proteins, but
not of presequence-less inner membrane proteins into mitochondria when the
outer membrane is broken open to allow the access of the antibodies to Tim50
(Yamamoto et al., 2002
).
Therefore, Tim50 is essential for the translocation of presequence-containing
mitochondrial precursor proteins across the inner membrane. Interestingly, the
effects of Tim50 depletion on import by the TIM23 complex is somehow
suppressed when the positively charged matrix-targeting signal in the
presequence is followed by a hydrophobic sorting signal/transmembrane segment
(Fig. 1) that arrests
translocation across the inner membrane
(Geissler et al., 2002
;
Mokranjac et al., 2003
).
The TIM22 complex
The TIM22 complex facilitates insertion of presequence-less polytopic
membrane proteins, including members of the metabolite carrier protein family
(e.g. AAC) and some Tim proteins
(Sirrenberg et al., 1996;
Koehler et al., 1999
;
Bauer et al., 2000
;
Pfanner and Geissler, 2001
;
Jensen and Dunn, 2002
). The
TIM22 complex functions in cooperation with a family of homologous proteins in
the IMS called small Tim proteins: Tim8, Tim9, Tim10, Tim12 and Tim13. Three
heterooligomeric complexes of small Tim proteins have been found: the
Tim9-Tim10 complex and the Tim8-Tim13 complex in the IMS, and the
Tim9-Tim10-Tim12 complex associated with the TIM22 complex in the inner
membrane. The TIM22 complex consists of at least three integral membrane
proteins: Tim18, Tim22 and Tim54. Electron microscopic analyses revealed that
the reconstituted TIM22 complex forms pores that have a diameter of
16Å (Rehling et al.,
2003
). Although precise roles of each component of the TIM22
complex in insertion of proteins into the inner membrane are not known,
purified Tim22, whose sequence shows similarity to that of Tim23, can form a
hydrophilic channel when reconstituted into liposomes
(Kovermann et al., 2002
).
Tim18 and Tim54 could stabilize the oligomeric structure of the TIM22
complex.
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Cooperation of the translocators |
---|
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Electron microscopic analyses have shown that mitochondria contain sites
where the outer and inner membranes are closely apposed (18-20 nm apart)
(Reichert and Neupert, 2002).
These sites are called `contact sites' or `membrane adhesion sites'.
Accumulated evidence suggests that translocation mediated by the TOM and TIM
complexes is coupled at such contact sites. For example, when
post-translational translocation of the C-terminal domain of the
presequence-containing precursor protein is inhibited in vitro, it forms a
translocation intermediate that spans the two membranes, which suggests that
the translocation takes place at contact sites
(Schleyer and Neupert, 1985
;
Rassow et al., 1990
:
Jascur et al., 1992
;
Kanamori et al., 1997
).
From TOM to TIM23
The translocation of precursor proteins through the TIM23 complex is
tightly linked to their translocation through the TOM complex, because no
soluble intermediates in the IMS can be observed. Nevertheless, the TOM and
TIM23 complexes are not permanently linked; they can interact transiently but
only in the presence of a translocating precursor protein
(Berthold et al., 1995;
Horst et al., 1995
;
Dekker et al., 1997
).
Therefore, a precursor protein translocating through the TOM complex needs to
engage with the TIM23 complex in the inner membrane to trigger formation of
the ternary supracomplex (Glick et al.,
1991
: Pfanner et al.,
1992
).
The TOM complex is distributed throughout the outer membrane. Tim23,
however, is thought to exhibit an unusual transmembrane topology in which the
C-terminal domain (residues 101-222) is integrated into the inner membrane,
whereas the N-terminal 50 residues are inserted into the outer membrane,
leaving residues 51-100 exposed to the IMS
(Donzeau et al., 2000). The
double-membrane spanning topology of Tim23 suggests its enrichment around
contact sites (Fig. 3A).
|
Studies by several groups showed that the IMS domain of Tom22
(Court et al., 1996;
Moczko et al., 1997
;
Kanamori et al., 1999
) and the
N-terminal 50-residue segment of Tim23
(Donzeau et al., 2000
) might
facilitate the transfer of precursor proteins from the TOM complex to the
TIM23 complex. The coupling of translocation across the outer and inner
membranes can be assessed by two-step import experiments in vitro. Briefly,
when incubated with mitochondria in the absence of
, which is
essential for presequence translocation through the TIM23 channel, a precursor
protein destined for the matrix stays in the TOM channel. In this
intermediate, the presequence reaches the presequence-binding site on the IMS
side of the TOM complex (the `trans site'), which involves Tom40
(Fig. 3A) (Rapaport et al., 1997
;
Kanamori et al., 1999
). This
surface-bound intermediate can be chased into the matrix by replenishment of
. Mitochondria containing a mutant Tom22 that lacks its IMS domain
exhibit no defect in the accumulation of the translocation intermediate in the
absence of
, but this impairs the passage of the intermediate into
the matrix after regeneration of
. Therefore the IMS domain of
Tom22 mediates the transfer of the presequence from the TOM complex to the
TIM23 complex.
Mitochondria containing mutant Tim23 that lacks the N-terminal 50 residues
have defects in protein import, but mitoplasts in which the outer membrane is
selectively ruptured do not (Donzeau et
al., 2000). This suggests that the N-terminal domain of Tim23 also
facilitates the transfer of precursor proteins from the TOM complex to the
TIM23 complex. Tethering of the TIM23 complex to the outer membrane by the
N-terminal domain of Tim23 may be important for efficient recruitment of the
presequence of precursor proteins associated with the TOM channel.
Tim50 also plays a role in the transfer of precursor proteins between the
TOM and TIM23 complexes. Although the presequence of the surface-bound
translocation intermediates generated in the absence of reaches
the trans site of the TOM complex, early attempts to detect a direct
interaction of this intermediate with a component of the TIM23 complex were
unsuccessful. However, Yamamoto et al.
(Yamamoto et al., 2002
) have
been able to crosslink the surface-bound intermediate and Tim50. Therefore,
the translocation intermediate lodged in the TOM channel interacts with Tim50
in the TIM23 complex, which suggests that Tim50 links translocation through
the TOM channel and that through the TIM23 channel. It is tempting to suggest
that the IMS domain of Tim50 is a receptor for the presequence. Regardless, it
interacts directly with the IMS domain of Tim23
(Geissler et al., 2002
;
Yamamoto et al., 2002
).
Through this interaction, Tim50 stabilizes the form of Tim23 that has the
N-terminal segment inserted into the outer membrane, and probably thereby
increases the efficiency of the transfer of a precursor protein from the TOM
complex to the TIM23 complex (Fig.
3A) (Yamamoto et al.,
2002
).
From TOM to TIM22
Substrate proteins for TIM22-mediated translocation contain multiple
hydrophobic segments to be inserted into the inner membrane. How can such
highly hydrophobic proteins cross the aqueous IMS to reach the TIM22
complex?
Import of AAC comprises several distinct steps, and the intermediates at
each have been characterized. AAC travels from the cytosol to the inner
membrane through a series of Tom and Tim protein complexes: first the TOM
complex, then the Tim9-Tim10 complex, the Tim9-Tim10-Tim12 complex and finally
the TIM22 complex (Fig. 3B) (Koehler et al., 1998;
Sirrenberg et al., 1998
;
Ryan et al., 1999
;
Truscott et al., 2002
). In the
absence of
, AAC accumulates at the point at which it interacts
with both Tom40 and Tim10 of the Tim9-Tim10 complex. Replenishment of
allows the translocation intermediate to interacts with Tim12 of
the Tim9-Tim10-Tim12 complex, which is associated with the TIM22 complex. This
requires functional Tim10. As the purified Tim9-Tim10 complex binds
specifically to the transmembrane segments of AAC, Curran et al.
(Curran et al., 2002
) have
suggested that the Tim9-Tim10 complex, which is partially soluble in the IMS,
has a chaperone-like action on unfolded hydrophobic AAC in the aqueous IMS.
However, there is no direct evidence for a soluble AAC intermediate chaperoned
by Tim9-Tim10. Instead, AAC can be directly pass from the TOM complex to the
TIM22 complex with the aid of the Tim9-Tim10 and Tim9-Tim10-Tim12 complexes,
minimizing its exposure to the soluble compartment
(Endres et al., 1999
). As in
the case of the TIM23 complex, there is no stable interaction between the TOM
complex and the Tim9-Tim10, Tim9-Tim10-Tim12 or the TIM22 complex in the
absence of translocating AAC.
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Functional separation of the translocators |
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The TOM complex, however, cannot use or ATP because there is
no
across the outer membrane and no ATP-dependent chaperone in
the IMS. When translocation by the TOM complex is coupled with that by the
TIM23 or TIM22 complex, the TOM channel can operate as a passive pore to allow
passage of the polypeptide segment, which is `pulled' by the TIM complex with
the aid of
and/or ATP. However, when translocation is uncoupled
from the inner-membrane translocators, the TOM complex has to use different
energy sources to ensure vectorial movement of the polypeptide chain to the
IMS.
TOM-mediated translocation unplugged
There are several cases in which translocation across the outer membrane is
uncoupled from that across the inner membrane. Indeed, under normal
circumstances, the TOM complex has to take up precursor proteins destined for
the matrix or inner membrane and drives their transmembrane movement until
they can engage with the translocator systems in the inner membrane.
Reflecting this ability, TOM complexes in outer membrane vesicles or purified
and reconstituted into liposomes can transfer the N-terminal presequence of a
precursor protein to the trans side of the membrane, although the rest of the
polypeptide remains outside the vesicles
(Mayer et al., 1995a;
Stan et al., 2000
). This
transfer of the presequence across the membrane is mediated by an array of
presequence-binding sites on the TOM complex that are spatially arranged in
order of affinity for the presequence
(Schatz, 1997
;
Komiya et al., 1998
). Indeed,
the TOM complex contains presequence-binding sites on both sides of the
membrane, and presequences can spontaneously go through the TOM channel to
reach the trans site on the IMS side
(Rapaport et al., 1997
;
Rapaport et al., 1998
;
Kanamori et al., 1999
).
Does the TOM complex possess a trans site for presequence-less inner
membrane proteins? AAC goes through the TOM channel not as a linear chain but
in a loop conformation (Endres et al.,
1999; Wiedemann et al.,
2001
). The AAC segments exposed to the IMS can bind to the
Tim9-Tim10 complex in the IMS at the exit of the TOM channel, which suggests
that small Tim proteins provide the high-affinity trans binding site for
presequence-less proteins (Endres et al.,
1999
; Curran et al.,
2002
). In both presequence-containing and presequence-less
proteins, the precursor proteins should be removed from the high-affinity
binding site at the end of the sequential binding array by an exergonic
reaction. The bound segments are indeed cleared from the trans site of the TOM
complex or small Tim proteins by the TIM23 or TIM22 complex, respectively, and
this requires
.
The TOM complex has at least two other mechanisms to drive translocation of
proteins across the outer membrane. These mechanisms are exemplified by the
proteins targeted to the inner membrane or IMS, including cytochrome
b2, cytochrome c1 and cytochrome
c. Precursors of cytochrome b2 and cytochrome
c1 have IMS sorting signals near the N termini
(Fig. 1) and follow the
stop-transfer pathway. This process consists of two steps, the first of which
requires and an ATP-dependent chaperone, mHsp70, but the second
of which is independent of
and mHsp70 (Glick et al., 2993;
Gärtner et al., 1995
).
The TOM complex drives the second step to transfer polypeptide domains
downstream of the sorting signals to the IMS without the aid of the TIM
systems. In the `anchor diffusion' mechanism
(Glick et al., 1991
;
Glick et al., 1993
;
Esaki et al., 1999
)
(Fig. 4A), the N-terminal
segment in the presequence that precedes the sorting signal of a precursor
protein crosses both the outer and inner membranes with the aid of
and an ATP-dependent chaperone, mHsp70 in the matrix (the first
step). However, when its sorting signal contacts the TIM23 complex,
translocation through the TIM23 channel is arrested and the hydrophobic part
of the sorting signal is anchored to the inner membrane. The rest of the
polypeptide, which is no longer exposed to the matrix, becomes disconnected
from mHsp70. The sorting signal anchored in the inner membrane then laterally
diffuses away from the site of close contact with the TOM complex, thereby
pulling the rest of the polypeptide through the TOM channel into the IMS (the
second step). Cytochrome b2 and cytochrome
c1 probably use this mechanism to transfer their mature
domains to the IMS when processing of the sorting signals in the presequences
is retarded (Wachter et al.,
1992
; Glick et al.,
1993
; Arnold et al.,
1998
; Esaki et al.,
1999
). The inner membrane proteins, including D-lactate
dehydrogenase, that are N-terminally anchored to the inner membrane and face
the IMS (Fig. 1) may well use
this mechanism to move across the outer membrane as well
(Rojo et al., 1998
).
|
A second mechanism involves folding of an N-terminal domain that has
already crossed the outer membrane and can function as a trap in the IMS to
drive translocation of the C-terminal part of the protein by a Brownian
ratchet mechanism (Fig. 4B). In
the case of cytochrome b2 fusion proteins, the mature part
of cytochrome b2 contains a heme-binding domain (HBD) in
the N-terminal region, which can fold independently of the rest of the
molecule. As the HBD is just downstream of the sorting signal in the
presequence, it moves across the outer membrane with the aid of coupled
translocation of the presequence through the TIM23 complex and
and mHsp70 (Glick et al.,
1993
) (the first step). When the processing of the sorting signal
of the presequence takes place faster than translocation of the rest of the
molecule across the outer membrane, the anchor diffusion mechanism cannot
complete translocation, because the N terminus is free in the IMS. Instead,
the tightly folded HBD in the IMS prevents Brownian backsliding, but not
forward movement, of the reminder of the molecule in the TOM channel, which
results in the forward displacement of the C-terminal part of the protein
across the outer membrane (Esaki et al.,
1999
) (the second step). Cytochrome c, a soluble protein
in the IMS, also uses folding in the IMS induced by the attachment of heme as
a driving force for translocation across the outer membrane
(Mayer et al., 1995b
). Many
small and single-domain proteins, including small Tim proteins in the IMS, may
also employ this mechanism to cross the outer membrane without engagement with
the TIM23 or TIM22 complex, because they often bind to co-factors or metals in
the IMS, which promote tight folding of the proteins
(Segui-Real et al., 1993
;
Steiner et al., 1995
;
Sirrenberg et al., 1998
).
![]() |
Conclusions and perspective |
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Acknowledgments |
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References |
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