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Translocation of mitochondrial
inner-membrane proteins:
conformation matters
Carine de Marcos-Lousa1, Dionisia P Sideris1,2 and Kostas Tokatlidis1,3
1
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology (IMBB-FORTH), PO Box 1385, Heraklion
71110, Crete, Greece
2
Department of Biology, University of Crete, PO Box 2208, Heraklion 71409, Crete, Greece
3
Department of Chemistry, University of Crete, PO Box 1470, Heraklion 71409, Crete, Greece
Most of the mitochondrial inner-membrane proteins are
generated without a presequence and their targeting
depends on inadequately defined internal segments.
Despite the numerous components of the import
machinery identified by proteomics, the properties of
hydrophobic import substrates remain poorly understood. Recent studies support several principles for
these membrane proteins: first, they become organized
into partially assembled forms within the translocon;
second, they present noncontiguous targeting signals;
and third, they induce conformational changes in
translocase subunits, thereby mediating ‘assembly on
demand’ of the import machinery. It is possible that the
energy needed for these proteins to pass across the
outer membrane, to travel through the intermembrane
space and to target the inner-membrane surface is
provided by conformational changes involving import
components that seem to have natively unfolded
structures. Such structural malleability might render
some of the translocase subunits more adept at driving
the protein import process.
Introduction
For the successful construction and function of all types of
cell, proteins must be targeted to their correct location,
both spatially and temporally, because they often function
at specific cellular sites distant from where they are made.
The magnitude of these targeting events is reflected by the
fact that up to half of the proteins in a eukaryotic cell are
translocated across or inserted into a membrane.
The landmark ‘signal hypothesis’ [1], which proposes
that proteins contain intrinsic, built-in address tags, laid
the foundations for subsequent research in this field. A
complete understanding of the targeting processes for
intracellular organelles has been greatly facilitated by the
discovery of most of the protein components that mediate
the translocation processes for many organelles. In
particular, determination of the yeast mitochondrial
proteome [2] uncovered new mitochondrial import
components, bringing the number of components
Corresponding author: Tokatlidis, K. ([email protected]).
identified to O30 and revealing two important features
of the translocation process: first, the unexpected
versatility of the mitochondrial protein import system;
and second, the crossover of pathways previously thought to
be separate (such as translocation into the outer and inner
membranes, or import into the matrix and insertion at the
inner membrane). Our understanding of the sequences of
the substrates that these import components recognize are
more limited, however, particularly for those proteins that
are imported but lack an identifiable presequence.
Such ‘presequence-less’ proteins include polypeptides
embedded in the mitochondrial inner membrane. Most of
these belong to the mitochondrial carrier family, which
transports metabolites, nucleotides and cofactors across
the mitochondrial inner membrane [3,4]. More than 35
members of this family with 28 different functions have
been identified in yeast [5]. The ADP/ATP carrier (AAC)
has been studied most extensively and has been used as a
model protein for this family. All members of the
mitochondrial carrier family are encoded by the nucleus
and therefore have to be imported into mitochondria.
Probably owing to the fundamental role of the carriers in
energy metabolism, mitochondria have developed
specialized machineries to ensure their correct targeting.
Although the function and organization of these
components remain to be understood, the signals that
mediate their translocation from the outer to the inner
mitochondrial membrane are more diverse than was
originally thought. The current idea that translocation is
achieved by a simple linear targeting signal is being
replaced by a model in which the conformation of the
precursor represents a crucial targeting determinant,
inducing a cascade of binding by specific translocase
subunits throughout the import pathway. Here, we
summarize some emerging features that might be
essential for this presequence-independent pathway:
first, conformation signals seem to be key to recognition
by the different translocase subunits; second, the
substrate itself is not a passive cargo, but might induce
downstream organization of the translocases involved
(in an assembly-on-demand mechanism); and third, many
of the components involved in this import pathway seem
to be ‘natively unfolded’ proteins.
www.sciencedirect.com 0968-0004/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2006.03.006
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Translocation intermediates reveal different stages of
import
Generating an arrested translocation intermediate is
imperative for dissecting the exact molecular mechanism
of translocation. Three main methods are usually used to
arrest import substrates at particular stages of the
translocation process (Box 1). In each approach, the
arrested translocation intermediate reflects differential
association of the precursor with both distinct translocase
components and the lipid bilayer: the main objective is to
probe the molecular environment of the imported
substrate and to ascertain how the molecules closest to
the precursor change along the import pathway.
For the presequence-less mitochondrial inner-membrane
proteins, studies manipulating the conditions for import of
the yeast AAC have resulted in the identification of five
distinct operational import stages for this pathway [6–9],
which are now quite well characterized (Figure 1).
Transport of the carrier protein precursor from its site of
synthesis in the cytosol to the mitochondrion marks the
first stage of import and is facilitated by cytosolic
Vol.xx No.xx Monthxxxx
chaperones such as Hsp70 (stage I) [10]. Using ATP
hydrolysis as an energy source, the carrier precursor is
transferred to three Tom70 receptor dimers (stage II) [8]
and then passed through the outer membrane TOM40
complex (stage IIIa) [9]. As soon as it emerges in the
intermembrane space, the carrier precursor is bound by
the 70-kDa TIM10 complex, which comprises three Tim9
and three Tim10 molecules (stage IIIb).
Subsequent association between the TIM10 complex
and the peripherally attached Tim12 protein further
brings the carrier precursor into the vicinity of the inner
membrane [11]. At this stage, called ‘tethering’, the N- and
C-terminal domains of the carrier precursor are still in
contact with the TOM40 channel [8]. In the presence of a
low membrane potential, which is used only in vitro to
dissect the import stages operationally, the carrier
precursor is transferred to the TIM22 complex (called
‘docking’ or stage IV) [12–14]. Final insertion into the
membrane requires a high membrane potential and
lateral release from the TIM22 channel (stage V) [12].
The carrier now spans the inner mitochondrial membrane
Box 1. How to generate an arrested protein translocation intermediate
To arrest a protein in transit through a membrane translocon, three
main approaches have been used. The parameters that can be altered
each time include modification of external conditions, genetic
manipulations of the translocon, and substrate modification (Figure I).
Modification of external conditions
Modifying external conditions is the most commonly used approach
and has been instrumental in defining import stages (Figure Ia). In
most cases, changing the import conditions involves altering the
temperature of import, time or pH, depleting or dissipating energy
sources such as ATP, or changing electrochemical potential [58]. The
last option proved to be fundamental in separating the tethering and
docking phases of stage IV in the mitochondrial import of presequence-less precursors [12].
Genetic manipulations of the translocon
Genetically manipulating the translocon provides an indirect
method of arresting the substrate in transit (Figure Ib). This
approach involves the formation of a partially functional or
nonfunctional variant of a component of the translocation machinery that is known to interact with the incoming substrate. This
modified component can be created by introducing mutations in
the protein – for example, by error-prone PCR – and then by
(a) External conditions
–ATP
screening for a less active form of the protein. In this way the
incoming substrate is ‘trapped’ simply by increasing the time of
interaction and thus the time of translocation. For studies involving
a non-essential protein in the import machinery, the whole gene
can be deleted from the genome (usually in yeast). For most
components, however, the use of a thermosensitive mutant or a
galactose-inducible promoter is required.
Modification of the substrate
Modifying the import substrate involves making deletion constructs,
chimeric constructs and single or multiple point mutants (Figure Ic). In
chimeric constructs, a bulky and tightly folded domain is attached to
one end of the import substrate to ‘plug’ the import pore. Typically,
mouse dehydrofolate reductase has been used in mitochondrial
import because it can be stabilized by the addition of its specific
ligand methotrexate [59]. This method cannot be used, however, for
translocons that import folded substrates. Nevertheless, substrate
modifications are most commonly used to identify import
targeting signals.
The dissection and the precise definition of the import pathways
have been delineated by the combinatorial use of the these
approaches and have proved essential to our understanding of the
molecular mechanisms involved in this process.
(b) Genetic manipulations
of the translocon
(c) Substrate modification
DHFR
MTX
Ti BS
Figure I. Generating an arrested protein translocation intermediate. (a) Modification of external conditions. The newly synthesized protein (brown) is arrested before
insertion into the membrane (yellow) by depleting the ATP that is necessary for its release from the cytosolic chaperones into the translocon (green). (b) Genetic
manipulation of the translocon. A mutation in the translocon renders it inaccessible to the incoming substrate. (c) Substrate modification. The substrate is fused to mouse
dehydrofolate reductase (DHFR). On addition of the ligand methotrexate (MTX; blue) DHFR folds and cannot cross the translocon. The substrate is therefore ‘trapped’ in
the translocon.
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3
Stage I
N
Hsp70
C
Hsp90
Stage II
N
Stage IIIa
Stage IIIb
N
Tom70
C
20
20
20
N
C
TOM40
OM
TOM40
C
TOM40
9
10
IMS
9/10
N
OM
C
TOM40
9
10
9/10
12
12
N
Low
IM
TIM22
Tethering
stage IVa
potential
C N
C
High
TIM22
potential
Docking
stage IVb
Stage V
Ti BS
Figure 1. Different stages of the import system for mitochondrial metabolite carriers. The newly synthesized protein is transported from the cytosol to the mitochondrial
membrane by the chaperones Hsp90 and Hsp70 in mammals and by Hsp70 alone in yeast (stage I) [10]. The precursor is then bound by three dimers of the receptor Tom70
(stage II) and released to Tom20 in an ATP-dependent manner [8]. The stage of association between Tom20 and the precursor is shown in parentheses because no stable
transport intermediate has been generated as yet. As the precursor moves through the Tom40 channel (stage IIIa), it is bound by the TIM10 complex (comprising Tim9 and
Tim10) in the intermembrane space (stage IIIb) [9]. The precursor is then tethered to the TIM22 complex of the inner membrane by association of the TIM10 complex with the
peripheral protein, Tim12, of the TIM22 complex (stage IVa; ‘tethering’) [11]. A membrane potential of !60 mV is adequate for a loop of the precursor to insert into the Tim22
channel (stage IVb; ‘docking’). High potential then brings the precursor carrier protein to its fully inserted dimeric functional form (stage V) [13]. Abbreviations: IM, inner
membrane; IMS, intermembrane space; OM, outer membrane.
and forms a homodimer to acquire its functional form
[15,16].
Several internal sequences are recognized as targeting
signals
What are the signals that drive a carrier precursor from
the cytosol to the inner mitochondrial membrane? In
contrast to most mitochondrial proteins, members of the
metabolite carriers family do not usually contain
N-terminal targeting signals and, in the few exceptions
that do, such extensions are dispensable for targeting and
insertion [17–19]. Owing to the highly hydrophobic nature
of the mitochondrial metabolite carriers, it has been
difficult to assess other linear signals; however, the use
of peptide scans has facilitated the identification of some
linear segments of carriers that are bound by different
components of the import machinery (Figure 2).
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All metabolite carriers share a common general topology
of three repeated modules of w100 amino acids, each
consisting of two transmembrane segments connected by a
loop (Figure 2). Whereas Tom70 seems to interact with
more hydrophobic domains of the metabolite carriers,
Tom20 – another protein import receptor of the outer
mitochondrial membrane – interacts with hydrophilic
segments of these carriers. In addition, biochemical import
assays suggest that both ionic and hydrophobic interactions are important for recognition of the precursor by
Tom70 and Tom20 [18,20].
Peptide scans have shown that transmembrane
segments TM3, TM4 and TM5 of the AAC bind more
strongly than other regions to the TIM10 chaperone
complex [21–23], arguing that hydrophobic interactions
have an important role in passage of the carrier precursor
across the intermembrane space. The C-terminal region of
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C
N
(a)
N terminus
1 2
(b)
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Module I
1
3 4 5 6
Module II
2
3
C terminus
Opinion
4
Module III
4
5
6
Tom70
Tom20
TIM10
Tim22
Ti BS
Figure 2. Linear targeting signals in the sequence of the mitochondrial metabolite carriers. (a) Topological model of a carrier in the inner mitochondrial membrane. The
transmembrane segments are numbered. (b) Linear representation of carrier regions known to bind to Tom70 [20], Tom20 [20], TIM10 [21,22] and Tim22 [12]. Each bar
represents a binding domain; stronger binding is indicated by darker shading. The regions shown are based on binding studies done on the ADP/ATP carrier (AAC) [21,22]
and the phosphate carrier (PiC) [20,12]. Yellow bar represents the P2 peptide of PiC that has been reported to induce opening of the Tim22 channel [14].
the AAC seems to be important for binding to Tom70 and
Tom20 but, surprisingly, the N-terminal part and transmembrane segment TM1 contain very little or no
targeting information. The carrier regions that bind to
other import components such as Tom40, Tim12 and
subunits of the TIM22 complex remain, however, to
be determined.
Because the N and C extremities of the carriers are
sensitive to externally added protease at stage III during
import into isolated mitochondria [8], it is tempting to
postulate that these regions signal that the carrier is
anchored in the outer membrane TOM40 channel. Release
from the TOM40 machinery is then mediated by the
TIM10 complex [24], presumably through its recognition
of the internal transmembrane segments TM3, TM4 and
TM5. Finally, insertion into the inner membrane mediated
by interaction with the inner-membrane TIM22 complex
causes release of the N and C termini from the TOM40
complex. Module III of both the Neurospora crassa AAC
and the yeast dicarboxylate carrier has been reported to
contain the information essential for insertion into the
inner membrane [6,7,25]. In agreement with this hypothesis, a synthetic peptide called P2, corresponding to the
N-terminal portion of module III (the C-terminal third) of
the phosphate carrier (PiC), can stimulate the activity of
reconstituted TIM22 channels [14].
Taken together, the data available thus suggest that
targeting information is spread throughout the whole
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amino acid sequence of the metabolite carriers. It remains
open how these fragmentary targeting signals are decoded
and recognized along the import pathway in vivo. A trivial
possibility is that the various translocase components bind
to different segments of the metabolite carrier, which is
maintained in an unfolded state throughout the import
pathway. A more likely mechanism in our view is that the
discontinuous targeting segments cooperate to form
conformational states of the carriers that elicit preferential binding of the translocase components. Although
this latter mechanism is more intriguing and provocative,
several recent studies have provided experimental evidence in support of it.
Cooperation of noncontiguous segments as conformational signals
Proteins with a presequence insert their N-terminal part
into the intermembrane space first and thus are translocated ‘linearly’ through the outer membrane TOM40
channel [26,27]. By contrast, the AAC does not have a
presequence and it adopts a loop conformation when it
emerges from the TOM40 channel [7,8]. This channel is
made up of two pores, each with a diameter of 20–22 Å
that is wide enough to accommodate up to two polypeptide
chains [26,28,29]. Translocation of the whole carrier is
then mediated by structural alterations of Tom40, which
probably occur through interaction with the other
components of the TOM40 complex [27]. Adopting a loop
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(a)
(b)
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(c)
(d)
5
(e)
(f)
C
6
N
5
1
2
3
4
C
N
6
1
C
N
5
2
OM
1
3 4
TOM40
56
2
9/10
3 4
N
IMS
TIM10
C
9/10
9/10
N C
2 3 1 46 5
IM
TIM22
Figure 3. Conformations adopted by the mitochondrial metabolite carriers during import. The import of carrier proteins is proposed to be conformation dependent.
Represented here, on the basis of carrier regions known to bind to different import components (see Figure 2), are the probable conformations that a carrier adopts
throughout its import. Because the TOM40 channel contains two pores that can each accommodate only two transmembrane segments (i.e. one module), translocation of all
three modules requires a sequential mechanism. Module III shows strong binding to Tom20 and thus would be retained more efficiently. By contrast, module I is bound
weakly to Tom20 and is probably translocated into the TOM40 channel first, followed by module II (a). Module III is inserted after probable movement of the TM3 and TM4
segments further inside the TOM40 channel (b). At this point, the carrier is emerging from the TOM40 channel and can bind to the TIM10 complex (c). This conformation is
compatible with reports showing that (i) the ADP/ATP carrier (AAC) is inserted as a loop into the TOM40 channel; (ii) the N- and C-terminal regions remain accessible from the
outside whereas the carrier can be crosslinked to TIM10; and (iii) module II has a strong affinity for the TIM10 complex, whereas the TM1 and TM6 segments have the weakest
affinity. Because these two transmembrane segments seem to be the last regions to be released from the TOM40 channel, they probably contain signals for retention of the
carrier in the TOM40 channel. Insertion into the TIM22 channel has been reported to involve initially module III, the membrane potential response element of the carrier [7,25].
Because module II is firmly bound to TIM10, module I is more likely to be inserted before module II into the second pore of the TIM22 channel (d). The lateral opening of TIM22
will then release one of the two modules (I or III) into the membrane, enabling module II to be translocated into the pore. The same mechanism subsequently releases the
whole carrier from the TIM22 complex, facilitating its assembly in the inner mitochondrial membrane and acquisition of its final conformation (e,f). The 3D X-ray structure of
the imported substrate in the IM is reproduced, with permission, from Ref. [60] (www.nature.com).
conformation would also enable exposure of the central
TM3 and TM4 domains of the AAC to the intermembane
space, rendering them available for binding to the TIM10
complex [22]. At stage IV, the AAC has been shown to
insert in a conformation-dependent manner into the two
pores of the TIM22 channel [13]. At this stage, the
C-terminal module III inserts into the first pore, inducing
gating of the other pore, which can in turn accept another
module. Taken together, these data show that conformational signals consist of noncontiguous sequences that
interact with each other to form a structural targeting
element (Figure 3).
Consequently, alteration of this import-competent
conformation of the carrier would cause mistargeting.
Indeed, two recent independent studies using either the
AAC or the PiC have reported that deletion variants of
these two carriers fused to mouse dehydrofolate reductase
(DHFR) do not insert into the inner membrane but are
instead mistargeted to a different location [30,31]. For
example, constructs containing any two modules were
targeted to the intermembrane space after import, and
one module alone was targeted to the matrix via the
TIM23 complex in a TIM10-independent manner. In
similar experiments, Adam et al. [11] found that deletion
variants of the Neurospora crassa AAC imported into
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Saccharomyces cerevisiae mitochondria were also targeted
to an aqueous compartment but could be still crosslinked
to the TIM10 complex [7]. They used a heterologous
carrier and slightly different deletion constructs, however,
which makes comparison between the studies difficult.
Moreover, although the DHFR moiety that was fused to
the N-terminal, C-terminal or both ends of the wild-type
carrier does not affect carrier import or localization [8], the
possibility cannot be excluded that it might affect the
structure of the deleted proteins and thus the accessibility
of some signals, as has been suggested by Vergnolle et al.
[30]. In this way, a single module fused to DHFR would not
acquire the ‘loop’ conformation but rather a linear one and
thus would be committed to the ‘default’ TIM23 pathway.
By contrast, two modules could ensure a loop conformation and would be recognized by the TIM10 complex on
emerging from the outer membrane pore. It follows that
the presence of the third module, which represents the
membrane potential response element, is not sufficient for
efficient insertion in the inner membrane [7,30,31].
The dependence of the import process on conformational
signals made up of noncontiguous segments of the
polypeptide argues for a modification of the commonly
held view – which has served well for most of the soluble
precursors studied so far – that extensive unfolding is
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necessary for the import process. In fact, recognition of
conformational signals rather than specific amino acid
sequences might explain the fact that all carrier proteins
tested (which share a similar topology and similar
structure despite a sequence homology of only 20–50%)
follow the same import pathway. Such a mechanism could
hold for other membrane proteins that are unrelated to
the carrier family, but share some redundant conformational features. Such conformational features would
enable the import machinery to import efficiently a large
variety of substrates, but how could they ensure recognition in every case?
Recognition of conformational signals by natively
unfolded translocases
For some time, the lack of atomic resolution structures for
components of the mitochondrial protein import machinery
hampered our understanding of the structural basis of the
protein import pathways. Good progress has now been
made with determination of the solution structure of the
cytosolic domain of Tom20 [32] and more recently the
crystal structure of the human TIM10 complex [33].
Accumulating evidence, obtained mainly by NMR and
small angle X-ray scattering, suggests, however, that
several of the protein import components seem to be
intrinsically unstructured (Box 2). Such proteins are
abundant in eukaryotic genomes and are commonly
involved in molecular interactions. They are characterized
by at least a partial lack of folded structure and an
extended conformation with high intramolecular flexibility and little secondary structure [34–36]. For the
carrier import pathway, which depends on recognition of
conformational signals, this lack of structure is
particularly relevant.
The cytosolic domain of the Tom70 receptor, which
contains the TPR motifs responsible for binding the
carrier in the cytosol, is unstable and prone to exist in a
state of native disorder compared with the fully folded,
globular protein [37]. Likewise, the C-terminal domain of
Tom22, which is located in the intermembrane space and
responsible for binding presequences as they emerge from
the Tom40 channel, has an amino acid composition
consistent with being natively disordered [38]. Intrinsically disordered proteins often show signs of local and
limited residual structure, which can compact down into a
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folded state when substrates or partner proteins bind [34].
Thus, these more flexible domains in proteins will be more
efficient for binding a broad range of substrates, such as
members of the carrier family and other polytopic proteins
of the mitochondrial inner membrane.
This feature is probably not restricted to these two
receptors and could be probably extended to other proteins
of the import machinery – for example, Tim50 or the
TIM10 complex. In fact, Tim9 and Tim10, the two
components of the TIM10 complex, have been reported
to be partially folded molten globules [39]. The recently
published crystal structure of the human TIM10 complex
[33] has confirmed the presence of disordered segments in
the N and C termini of both subunits [40] that form
‘tentacles’ radiating out from a central core [33,39].
Substrate sensing is mediated at least partly by the
N-terminal flexible segment of the Tim10 subunit [21].
The detailed mechanism of recognition by the TIM10
complex is still not understood; however, structural
rearrangements of the TIM10 complex that occur on
binding of the substrate seem to be important [33].
How would such proteins release their substrate, which
is bound mainly through hydrophobic interactions? The
bacterial chaperone protein GroEL illustrates a system of
structural changes that take place to release bound
substrate proteins from hydrophobic binding sites
[41–43]. We can speculate that similar mechanisms
apply to the mitochondrial protein import machinery.
Thus, the TIM10 complex would release bound substrate
after changing conformation on binding to another
partner – for example, Tim12 in yeast mitochondria.
Such a mechanism would imply that a cascade of
conformational changes provides the energy required for
passage of the substrate through the various stages from
the outer membrane, via the intermembrane space, to the
inner membrane. Similar to the TAT system in prokaryotes and thylakoids [44,45], the import machinery will
associate on demand, depending on the nature of the
incoming precursor.
Thus, the mitochondrial import system for the innermembrane hydrophobic proteins seems to be much more
dynamic than we used to think. Chacinska and collaborators [46] have recently reported that the TIM23 complex,
which recognizes presequence-containing precursors, also
shows such dynamic behavior: after the presequence is
Box 2. Structurally disordered chaperones
Chaperones are sophisticated machines that assist in the folding of
other proteins. Because no chaperone is specialized in the folding of
one specific substrate, by definition they need to be able to recognize
numerous targets that often have unrelated sequences. This feature
suggests that these proteins are likely to contain intrinsically
disordered regions, because such domains offer unique versatility in
the recognition process of target molecules. This feature is particularly
important for hydrophobic protein chaperones because disordered
segments can interact rapidly with the target, thereby preventing
aggregation of proteins such as the mitochondrial metabolite carriers.
Proteins containing disordered regions are mainly involved in cell
signaling or function as regulatory proteins [36]. The presence of these
regions in chaperones is a relatively new concept. Although some
chaperones have been reported to be totally disordered (a-synuclein
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and a-casein) [34], most have well-structured domains with small
regions comprising disordered stretches of amino acids [34–36]. The
latter type includes small heat-shock proteins (Hsp25 and Hsp31) in
addition to GroEL, DnaK and DnaJ [41–43].
Tim9 and Tim10, the chaperones specific for mitochondrial
membrane proteins, have been shown to be molten globule proteins
characterized by a high level of secondary structure, no defined
tertiary structure and a high affinity for hydrophobic substrates [39].
Recently, Tom70 and Tom22, two receptors of the mitochondrial
import machinery, have been reported to be intrinsically disordered
[37,38]. Binding to the target substrate induces folding of the
disordered regions or a conformational modification of the chaperone
that will trigger subsequent release of the substrate to the downstream component of the pathway.
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bound, linkage of the TOM and TIM channels is mediated
by components that are predicted to have natively
disordered segments (Tom22, Tim50 and Tim21).
Versatility of the presequence-less inner-membrane
proteins
The mitochondrial metabolite carriers are not the only
inner-membrane polytopic proteins that lack a cleavable
presequence. For the TIM22 pathway, for example, three
more substrates – Tim23, Tim17 and Tim22 itself [47,48] –
have been identified that do not carry an N-terminal
presequence but instead contain the targeting information
within the mature protein. Although little is known about
the import of Tim22, the targeting signals of Tim23 and
Tim17 have been studied more extensively. These two
proteins have an internal positively charged region in
their C-terminal domain that could mimic an N-terminal
presequence. When fused to DHFR, however, this region is
directed to the matrix via the TIM23 pathway, presumably
because it is recognized by an acidic segment in the
N-terminal portion of Tim23 [48]. Thus, this positive
region could function as a targeting signal to the TIM23
pathway but might be masked during acquisition of its
conformational state during import.
In fact, a mutant of Tim23 in which the positively
charged residues of the matrix-facing loops were replaced
by alanine residues, crossed the outer membrane and
could be efficiently crosslinked to the TIM10 complex, but
did not integrate into the inner membrane [49,50]. These
observations demonstrate that a specific conformation is
adopted by Tim23 that drives it to the TIM22 pathway
independent of the presence of the charged C-terminal
region, whereas proper insertion into the inner membrane
seems to require charged amino acids present in the
matrix loops. Studies using truncated versions of Tim23
have clearly shown that, similar to import of the AAC, all
modules are required to cooperate for insertion into the
inner membrane [49,51,21]. Thus, even though these
three proteins have a sequence and function that are
completely different from those of the metabolite carriers,
they seem to share some structural similarity. It is thus
apparent that the substrates of the TIM22 pathway
possess segments that enable them to acquire a specific
conformation that is essential for correct targeting and
integration in the inner membrane.
Concluding remarks
Protein sorting in organelles is usually driven by a signal
peptide that is sequence-specific. Examples include the
KDEL sequence for retention in the endoplasmic reticulum
(ER) lumen, the PTS1 and PTS2 signals for targeting of
soluble peroxisomal proteins, the twin arginine motif for
targeting to the TAT system in thylakoids and prokaryotes,
and positively charged amphipathic helices for targeting to
mitochondria. The presence of a signal peptide is not a
prerequisite, however, because proteins without such
regions can be targeted to mitochondria, chloroplasts or
peroxisomes [52–55]. In fact, peroxisomes and chloroplasts
can import completely folded proteins. For mitochondria,
the current view is more complicated because a precursor
has to be unfolded and translocated linearly. This concept is
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7
also applicable to precursors directed to the ER that are
released from the ribosome directly into the Sec machinery;
however, cooperation between the transmembrane segments during translocation through the pore and insertion
into the ER membrane has been reported [56]. Similarly, in
mitochondria the polytopic presequence-less proteins of
the inner membrane must be partially folded to be
efficiently imported.
We have discussed the main requirements, in terms of
sequence and conformation of the substrate, for efficient
translocation into mitochondria. During its translocation
from the TOM40 to the TIM22 complex, the substrate
protein acquires a precise, import-competent conformation. Specific internal sequences have a synergistic
role in creating this conformation. The acid chain
hypothesis for the import of presequence-containing
proteins into mitochondria suggests that increasingly
stronger ionic interactions with acidic domains of translocase components draw the presequence inside
mitochondria [57].
To explain conceptually the translocation of the innermembrane proteins discussed here, we propose an
extension of this idea that takes into account the fact
that mainly hydrophobic interactions must be involved.
A key difference, which would also explain the existence
of conformation-specific signals, is that hydrophobic
interactions become thermodynamically and physiologically important only when considerable structural
complementarity exists among the interacting partners.
This characteristic contrasts with ionic interactions,
which are directional and central to recognition of the
presequence. Thus, the presence of natively disordered
regions in the import machinery can optimize
recognition by enabling a rapid and efficient adaptation
of their structure to the various incoming precursors.
This new concept is emerging as an important feature of
the mitochondrial import machinery, which might
assemble on demand for challenging incoming precursors such as the inner-membrane proteins. To find the
exit of the import labyrinth, flexibility is the mitos
(meaning thread in ancient Greek) that needs to
be unraveled.
Acknowledgements
Supported by IMBB-FORTH funds, the University of Crete, the UK
Medical Research Council, and the European Social Fund and National
resources. C. dM-L. is a FEBS postdoctoral fellow, and D.P.S. was
supported by a PENED grant from the Greek General Secretariat of
Research and Technology.
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