On the mechanism of preprotein import by the mitochondrial presequence translocase
Martin van der Laan
, Dana P. Hutu
, Peter Rehling
Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, Universität Freiburg, D-79104 Freiburg, Germany
Abteilung Biochemie II, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany
Received 2 October 2009
Received in revised form 5 January 2010
Accepted 11 January 2010
Available online 25 January 2010
Mitochondria are organelles of endosymbiontic origin that contain more than one thousand different
proteins. The vast majority of these proteins is synthesized in the cytosol and imported into one of four
mitochondrial subcompartments: outer membrane, intermembrane space, inner membrane and matrix.
Several import pathways exist and are committed to different classes of precursor proteins. The presequence
translocase of the inner mitochondrial membrane (TIM23 complex) mediates import of precursor proteins
with cleavable amino-terminal presequences. Presequences direct precursors across the inner membrane.
The combination of this presequence with adjacent regions determines if a precursor is fully translocated
into the matrix or laterally sorted into the inner mitochondrial membrane. The membrane-embedded
complex mediates the membrane potential-dependent membrane insertion of precursor proteins
with a stop-transfer sequence downstream of the mitochondrial targeting signal. In contrast, translocation of
precursor proteins into the matrix requires the recruitment of the presequence translocase-associated motor
(PAM) to the TIM23 complex. This ATP-driven import motor consists of mitochondrial Hsp70 and several
membrane-associated co-chaperones. These two structurally and functionally distinct forms of the TIM23
) are in a dynamic equilibrium with each other. In this review, we discuss
recent advances in our understanding of the mechanisms of matrix translocation and membrane insertion by
the TIM23 machinery.
© 2010 Elsevier B.V. All rights reserved.
Translocation of proteins across biological membranes is essential
for the survival of every living organism. Bacteria and archaea secrete
for example cell wall-associated proteins or hydrolytic enzymes for
the extracellular breakdown of polymers across the cytoplasmic mem-
brane. In the evolution of eukaryotic cells the formation or acquisition
of intracellular membrane-bound organelles allowed to distribute in-
compatible biochemical reactions into separate compartments. This
major advantage, however, confronted the eukaryotic cell with a log-
istic problem. In order to carry out complex biochemical reactions,
each organelle needs to be equipped with a distinct set of proteins.
By far the most of these proteins, however, are synthesized on cyto-
solic ribosomes. This problem is solved by a number of sophisticated
protein targeting and translocation machineries that mediate the ef-
ﬁcient and highly speciﬁc sorting of proteins to different organelles.
Importantly, to be recognized as a protein destined for a certain or-
ganelle, newly synthesized proteins must contain targeting informa-
tion in the form of speciﬁc signal sequences. These signal sequences
function like zip codes and ultimately determine the localization of a
newborn protein. Signal sequence-containing proteins are generally
termed precursor proteins, or shortly precursors. Many different types
of signal sequences have been described ranging from hydrophobic or
positively charged internal sequences or short C-terminal peptides in
the mature part of the protein to N-terminal presequences that are
typically cleaved off by processing peptidases after or during trans-
Different classes of molecular machineries for protein targeting
and transport across or integration into biological membranes exist.
The main difference between these classes is the nature of the protein-
conducting channel that allows the passage of precursors across the
lipid bilayer. Translocases that mediate the transport of large, stably
folded proteins assemble a functional protein-conducting pore only in
the presence of substrate. Examples for this are the bacterial twin-
arginine translocase  and the peroxisomal protein import machin-
ery . Other types of translocases transport proteins in a largely
unfolded conformation, thus containing no or very little tertiary struc-
ture elements . Among these are the β-barrel type translocases
located in the outer envelope membranes of organelles with endo-
symbiontic origin [5,6]. In other translocation machineries the pro-
tein-conducting channels are formed by transmembrane α-helices. In
the absence of substrate, these pores are in a closed state, which
allows maintaining the permeability barrier function of the surround-
ing membrane. Incoming precursor proteins induce the controlled
Biochimica et Biophysica Acta 1803 (2010) 732–739
⁎ Corresponding author. Tel.: +49 551 395947; fax: +49 551 395979.
E-mail address: email@example.com (P. Rehling).
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temporary openi ng of the protein-conducting c hannels (Fig. 1).
Examples for this class of translocases are the SEC translocons of
the endoplasmic reticulum membrane and the bacterial cytoplasmic
membrane [7,8], the two TIM machineries of the mitochondrial inner
membrane and the TIC complex of the inner chloroplast membrane
. The basic principles of protein translocation by these machineries
are very similar. Three functional modules are required: i) a receptor
that recognizes distinct signal sequences and targets precursors to the
destined membrane, ii) a signal-gated protein-conducting channel
that takes over the precursor for membrane passage, and iii) a motor
device that generates a directed driving force on the precursor gen-
erally at the expense of ATP hydrolysis (Fig. 1). In addition, chaperones
on both sides of the membrane are required either to keep the pre-
cursor in a loosely folded, translocation-competent state (cis side) or to
fold the protein – in many cases after proteolytic removal of the signal
sequence – into the mature conformation (trans side) (Fig. 1). In
different translocation machineries these modules are organized in
different ways. Primary signal sequence receptors may be soluble
cytosolic proteins or membrane-integral proteins exposed to the
target membrane surface. In the bacterial SEC system, the SecA protein
serves as both, signal sequence receptor and ATP-driven cis-acting
motor . For translocases that reside in energy-transducing mem-
branes, like the bacterial cytoplasmic membrane or the mitochondrial
inner membrane, the membrane potential (Δψ) contributes an im-
portant driving force for protein transport. The carrier translocase
(TIM22 complex) of the inner mitochondrial membrane, which medi-
ates the membrane insertion of multi-spanning membrane proteins
with internal targeting signals, utilizes the Δψ as sole external driv-
ing force for protein transport . In this review, we will focus on
the presequence translocase of the inner mitochondrial membrane
(TIM23 complex), which is a very interesting example of a signal-
gated translocase. This translocase uses the Δψ as well as an ATP-
powered motor to drive protein translocation across the membrane.
Although the mechanism of protein translocation by the TIM23 com-
plex follows the basic rules described above, it shows several unique
features, which will be addressed in detail in the following sections.
2. Speciﬁc aspects of mitochondrial protein import
Protein import into mitochondria represents a particularly com-
plicated task, as these organelles are surrounded by two membrane
systems: the outer membrane and the inner membrane. These two
membranes generate two aqueous compartments, the intermem-
brane space and the matrix. Proteomic studies using highly puriﬁed
mitochondria derived from the yeast Saccharomyces cerevisiae have
shown that mitochondria contain ∼ 1000 different proteins [11,12].
Only eight of these proteins are encoded by mitochondrial DNA and
synthesized on mitochondrial ribosomes. All other proteins are im-
ported from the cytosol into the organelle. Diverse protein transloca-
tion machineries mediate the import of distinct classes of precursors
that are speciﬁcally sorted into one of the four subcompartments
[13,14]. In a recent study, Vögtle et al. have determined the N-
termini of 615 different mitochondrial proteins . This com-
prehensive analysis revealed that ∼ 70% of mitochondrial precursor
proteins are synthesized with N-terminal signal sequences that are
proteolytically processed upon import. These presequences possess a
positive net charge, most frequently between +3 and +6. Mitochon-
drial presequences exhibit a broad length distribution from 6 to more
than 90 amino acid residues. Most frequently presequences between
15 and 55 amino acids length were detected . Presequence-
containing proteins can be categorized into different classes: water-
soluble matrix proteins, membrane proteins with one or more trans-
membrane segments and intermembrane space proteins that are
initially inserted into the inner membrane and subsequently released
into the intermembrane space by proteolytic processing.
Like virtually all mitochondrial proteins, presequence-containing
precursors enter the organelle via the general translocase of the outer
mitochondrial membrane (TOM complex). The TOM complex belongs
to the class of β-barrel type translocases . The protein-conducting
channel of the TOM complex is formed by the central β-barrel protein
Tom40 . The TOM complex further contains two primary recep-
tors, Tom20 and Tom70, and the central receptor Tom22 [17,18].
Whereas Tom70 preferentially recognizes internal hydrophobic signal
sequences, Tom20 binds to N-terminal presequences and thus re-
presents the primary receptor for presequence-containing precursors
[19–22]. A number of presequence-containing precursors, which may
be particularly aggregation-prone, require both, Tom20 and Tom70,
for efﬁcient import into mitochondria . With the help of Tom22
and the small Tom protein Tom5, presequence proteins are handed
over to Tom40 and inserted into the protein-conducting pore [17,24].
At the trans side of the outer membrane the presequence binds to the
intermembrane space domain of Tom22 (Tom22
) [25–28]. In the
absence of a Δψ across the inner mitochondrial membrane, prese-
quence-containing precursors remain arrested at this stage of import
(TOM intermediate) . Only in the presence of a Δψ these precur-
sors are further transferred to the TIM23 complex, the presequence
translocase of the inner mitochondrial membrane (Fig. 2). The TIM23
complex catalyzes both, full translocation into the matrix and lateral
membrane insertion of presequence-containing precursors depend-
ing on the type of signal information.
Fig. 1. Modular organization of signal-gated protein translocation machineries. A precursor protein is synthesized on the cis side of the membrane (usually the cytosol) with a
speciﬁc signal sequence (red box) and is kept in a loosely folded conformation by molecular chaperones. The signal sequence is recognized by a receptor on the surface of the target
compartment. Docking of the signal sequence to the receptor module of the translocation machinery triggers opening of the protein-conducting channel (PCC) and the precursor
protein is inserted into the PCC module. A directed driving force is generated by an import motor at the expense of ATP hydrolysis. Within the target compartment (trans side of the
membrane) the signal sequence is cleaved off and the mature protein folds into its native conformation.
733M. van der Laan et al. / Biochimica et Biophysica Acta 1803 (2010) 732–739
3. Direct transfer of precursors from the TOM to the
It is well known that the presequence-containing precursors can
be arrested at a late stage of import into the matrix at which they span
both outer and inner membranes [30–33]. This indicated that pre-
cursors are directly handed over from the TOM complex to a translo-
case in the inner mitochondrial membrane, giving rise to the formation
of such two-membrane-spanning intermediates. Hence, the import
of presequence-containing precursors across outer and inner mem-
branes is a tightly coupled process. Indeed, supercomplexes containing
the TOM complex of the outer membrane and the TIM23 complex
of the inner membrane connected by a precursor in transit could be
The membrane-embedded core of the TIM23 complex consists
of three proteins: Tim23, Tim17 and Tim50 (Fig. 2). Tim23 forms the
protein-conducting channel of the TIM23 complex. The exact mole-
cular nature of this channel is unclear. It has been speculated that it
consists of more than one copy of Tim23. A presequence as well as a
Δψ across the inner membrane are required to open the Tim23-
channel [39–41], which is reﬂected by reversible conformational
changes in the quaternary structure of the TIM23 complex upon
manipulation of Δψ and addition of presequence-containing pre-
cursors . The Tim23 protein consists of an N-terminal intermem-
brane space domain (Tim23
) and four transmembrane segments.
Alder et al.  have shown by a site-speciﬁc crosslinking approach
that transmembrane segment 2 of Tim23 lines an aqueous channel
across the membrane. The spectral properties of a ﬂuorescent probe
attached to residues on the water-accessible face of this transmem-
brane segment are signiﬁcantly changed in the presence of a precursor
arrested during translocation suggesting that this aqueous channel
may represent the protein-conducting pore. Tim23
is in close
proximity to Tom22
[27,44] and involved in the initial binding of
presequence-containing precursors to the TIM23 complex [39,42,45].
thus constitutes a part of the TIM23 complex receptor
domain. Tim17 is homologous to Tim23  and was suggested to
modulate the gating of the transmembrane channel formed by Tim23
[47,48]. Tim17 is required for both matrix translocation and lateral
membrane sorting. These two functions can be genetically separated,
as mutants of TIM17 have been isolated that are speciﬁcally affected in
the import of matrix-targeted or inner membrane-sorted precursors,
respectively . The Tim50 protein consists of a single N-terminal
transmembrane segment and a large intermembrane space domain
). This intermembrane space domain keeps the Tim23-
channel in a closed state in the absence of a precursor [40,42].
Biochemical studies have indicated that Tim50
exerts its effect on
the protein-conducting channel by modulating the oligomeric state of
Tim23 rather than by forming a physical plug . Moreover, Tim50
plays an important role in precursor protein recognition at the TIM23
interacts with Tim23
to form the receptor
domain of the TIM23 complex (Fig. 2) [37,44,50,51]. Based on a
mutational analysis in yeast it has been proposed that this interaction,
which is crucial for protein translocation by the TIM23 complex, is
mediated by coiled-coil domains in Tim23
(amino acids 68–85) and
(amino acids 274–291) . Several lines of evidence show
that Tim50 is intimately involved in the transfer of presequence-
containing precursors from the TOM to the TIM23 complex. Tim50
can be crosslinked to Tom22
as well as to the presequence of
translocation-arrested precursors indicative of a close spatial proxim-
ity [44,52]. Whether Tim50
directly binds presequences or just
facilitates their interaction with Tim23
remains to be clariﬁed.
can be crosslinked to the mature part of
precursor proteins in the absence of a Δψ across the inner membrane,
when the presequence is still bound to Tom22
(Fig. 2) . It has
further been shown that functional Tim50 is required for the formation
of a stable TOM intermediate of the presequence-containing polytopic
inner membrane protein Oxa1 .
The essential TIM23 core complex (TIM23
) dynamically asso-
ciates with an additional inner membrane protein, Tim21 (Fig. 2 )
. Like Tim50, Tim21 is evolutionary conserved and consists of an
N-terminal transmembrane segment and an intermembrane space
). The positively charged Tim21
associates to the negatively charged Tom22
domain via electro-
static interactions [49,53]. Moreover, Tim21
competes with posi-
tively charged presequences for binding to Tom22
the binding sites on Tom22
and presequences at least
partially overlap (Fig. 2) [49,53]. These data suggest that Tim21 faci-
litates the handover of precursor proteins from the TOM to the TIM23
complex by displacing the presequences from Tom22
. Based on the
experimental evidence described above we propose the following
Fig. 2. Transfer of a precursor protein from the TOM complex to the TIM23 complex. Upon passage of a precursor protein with a positively charged N-terminal presequence through
the protein-conducting pore of the TOM complex in the outer mitochondrial membrane (OM) the presequence binds to the intermembrane space (IMS) domain of Tom22 (left
panel). The mature part of the protein contacts the IMS domain of Tim50, which together with the IMS domain of the Tim23 protein forms the TIM23 receptor domain at the surface
of the inner membrane (IM). Transfer of the presequence from Tom22 to the TIM23 receptor domain and subsequent insertion into the Tim23 protein-conducting channel is
facilitated by Tim21 and requires the membrane potential (Δψ) across the inner membrane (right panel).
734 M. van der Laan et al. / Biochimica et Biophysica Acta 1803 (2010) 732–739
model for precursor transfer from the TOM to the TIM23 machinery
(Fig. 2): i) when a presequence-containing precursor emerges from
the Tom40 channel at the intermembrane space site of the outer
membrane, the presequence binds to Tom22
, while most of the
mature part of the precursor is still located within the Tom40 channel
or in the cytosol. This TOM intermediate is stabilized by Tim50, which
contacts the region of the precursor adjacent to the presequence.
relieves the presequence from its in teraction with
through a competitive mechanism. iii) In the presence of a
Δψ across the inner membrane the presequence is handed over to the
TIM23 complex, where it binds to the TIM23 receptor module formed
. iv) Presequence binding to the Tim23/
Tim50 receptor domain leads to the Δψ-dependent opening of the
Tim23-channel and to the insertion of the precursor into the protein-
conducting pore. The Δψ exerts an inward-directed electrophoretic
force on the presequence that moves it towards the matrix side .
4. Protein import into the matrix requires an ATP-driven
Whereas Δψ is sufﬁcient for the initial translocation of the pre-
sequence across the inner membrane, full translocation of a precursor
into the matrix requires the activity of the ATP-driven presequence
translocase-associated motor (PAM) (Fig. 3). The core component of
PAM is the mitochondrial Hsp70 (mtHsp70), which is tethered to the
protein-conducting channel via the adaptor protein Tim44 [55–58].In
the ATP-bound form, mtHsp70 loosely associates with the incom-
ing precursor via its peptide-binding domain. Precursor binding to
mtHsp70 triggers ATP hydrolysis and induces a conformational change
leading to closure of the peptide-binding domain around the pre-
cursor. A recent study by Becker et al.  has highlighted the impor-
tance of interdomain communication within mtHsp70 for protein
translocation. The precise nature of the following step has been a
matter of long debates in the ﬁeld. The Brownian ratchet model
proposes that ADP-bound mtHsp70 immediately dissociates from
Tim44 and thus from the protein-conducting channel. By random
diffusion short segments of the precursor become temporarily ex-
posed to the matrix. Backsliding of the precursor is prevented by
the association of a second mtHsp70 molecule to these segments
(“trapping”) [57,60,61]. The power-stroke model suggests that ATP
hydrolysis ﬁrst induces an inward movement of the peptide-bind-
ing domain relative to the nucleotide-binding domain, which is still
attached to Tim44. This results in an active pulling of the precursor
towards the matrix side [62–65]. Most likely, both mechanisms con-
tribute to the import of precursors into the matrix. Whereas thermal
diffusion and trapping is sufﬁcient for the import of unfolded or loosely
Fig. 3. Two different forms of the TIM23 complex. Distinct forms of the TIM23 complex mediate full translocation of matrix-targeted precursor proteins or lateral sorting of inner
membrane-targeted precursor proteins, respectively. The TIM23
complex receives precursor proteins from the TOM complex. Tim21 facilitates the transfer of precursors from
the TOM complex to the TIM23
complex by displacing the positively charged N-terminal presequences from Tom22. Inner membrane-sorted precursors contain hydrophobic
stop-transfer signals adjacent to the N-terminal presequences. These sorting signals trigger the lateral release of precursors from TIM23
into the lipid bilayer. Δψ-dependent
inner membrane sorting of precursor proteins is facilitated by the association of TIM23
with respiratory chain supercomplexes consisting of the cytochrome bc
(complex III, bc
) and cytochrome c oxidase (complex IV, COX). For matrix translocation the TIM23 core complex recruits the presequence translocase-associated motor (PAM). This
process is accompanied by the release of Tim21. The central component of PAM is mitochondrial Hsp70 (mtHsp70), which is tightly regulated by ﬁve co-chaperones: Tim44, Mge1,
Pam18 (Tim14), Pam16 (Tim16) and Pam17. Matrix translocation requires both the Δψ and ATP hydrolysis by mtHsp70. For both types of precursor proteins matrix processing
peptidase (MPP) removes N-terminal presequences on the matrix side of the membrane.
735M. van der Laan et al. / Biochimica et Biophysica Acta 1803 (2010) 732–739
folded precursor proteins, the translocation of more stably folded
domains was shown to require active pulling by mtHsp70 [63,64].
Additionally, it has been suggested that unfolding of precursor pro-
teins is supported by Δψ .
For a long time is was thought that Tim44 and mtHsp70 together
with the matrix-localized nucleotide exchange factor Mge1 [67,68]
constitute the import motor for translocation of precursors through
the TIM23 complex. Today, however, we know that PAM consists of
at least three additional components that regulate the ATPase activity
of mtHsp70 in space and time: Pam18, Pam16 and Pam17 (Fig. 3). The
Pam18 protein consists of a small, N-terminal intermembrane space
domain, a single transmembrane segment and a conserved J-domain
on the matrix side of the membrane [69–71]. J-proteins (named after
the prototypical member of this family, DnaJ ) transiently inter-
act with Hsp70 chaperones and strongly increase their ATPase acti-
vity. Indeed, it was shown that the J-domain of Pam18 stimulates the
ATPase activity of mtHsp70 [69,70]. Pam18 is found in a stable com-
plex with the J-like protein Pam16, which is peripherally associated
with the inner membrane and required for the recruitment of Pam18
to the TIM23 complex [73–78]. J-like proteins exhibit overall homo-
logy to J-proteins, but lack the canonical HPD-motif that is essential
for the ATPase-stimulating activity of J-proteins . Accordingly,
Pam16 does not increase the mtHsp70 ATPase activity. Pam16 in
fact inhibits the ATPase-stimulating activity of Pam18 indicating
that Pam16 and Pam18 together ﬁne-tune ATP hydrolysis rates of
mtHsp70 . The crystal structure of the Pam16/Pam18 complex
revealed multiple intimate contacts between both proteins .
This complex does not signiﬁcantly stimulate the ATPase activity
of mtHsp70. Thus, in order to allow Pam18 to activate the mtHsp70
ATPase, the Pam16/Pam18 complex must undergo large conforma-
tional rearrangements, which may be facilitated by the TIM23 com-
plex or other components of PAM. How this is brought about is
In addition to the association with Pam16, several other protein–
protein interactions are required to stably recruit Pam18 to the TIM23
complex and to bring Pam18 into close proximity to mtHsp70. Both,
the small intermembrane space domain, which binds to Tim17, and
the transmembrane segment of Pam18 contribute to its association
with the TIM23 core [49,77,78]. Tim44 has been crosslinked to Pam18
and Pam16 and a tim44 yeast mutant has been isolated, in which
Pam18/Pam16 fail to bind to the protein-conducting channel [74,77,
80]. Similarly, in a yeast strain lacking Pam17, an association of Pam16/
Pam18 with the TIM23 complex is impaired . Pam17 is an integral
membrane protein that can be crosslinked to Tim23 [80,82]. A genetic
interaction of PAM17 and TIM44 has been demonstrated . Inter-
estingly, inactivation of Tim44 leads to an accumulation on Pam17
at the protein-conducting channel pointing to an intricate dynamic
interplay between Pam17, Pam16, Pam18 and Tim44 in the reaction
cycle of PAM . Thus, the import-driving activity of PAM is the result
of a tightly regulated cooperation between distinct modules. Taken
together, PAM represents one of the most complex Hsp70 systems,
which we are only beginning to understand in its molecular details.
5. Matrix translocation versus inner membrane sorting: two
distinct forms of the TIM23 complex
A particularly fascinating aspect of the TIM23 complex is its re-
markably dynamic behavior in response to the type of incoming pre-
cursor. Although this issue is controversially discussed in the ﬁeld,
several lines of evidence indicate that the TIM23 complex is not a
static entity, but rather switches between different structural and
functional states (Fig. 3). Transport of proteins into the inner mem-
brane is thought to occur by lateral diffusion of the precursor out of
the Tim23 channel [82,84]. Proteins that undergo this sorting process
contain hydrophobic sorting signals downstream of the presequence.
These sorting signals stall precursor translocation across the inner
membrane and are then laterally released from the Tim23 channel by
a yet undeﬁned mechanism that involves Tim17 [49,85]. The presence
of proline residues in the hydrophobic core of the sorting signal has
been found to strongly disfavor translocation arrest and instead to
promote precursor transport into the matrix . Moreover, muta-
tions that inactivate components of the PAM machinery do not affect
inner membrane sorting of precursors [69–71,73,74,80,81,86]. This
clearly indicates that lateral sorting – in contrast to matrix trans-
location – is independent of the activity of PAM and solely requires
the Δψ as energy source. In accordance with this notion, a PAM-free
form of the TIM23 complex has been isolated from mitochondria,
which consists of TIM23
and Tim21 [41,49,87,88]. This complex
form has been termed TIM23
, as it was shown to catalyze the Δψ-
dependent insertion of laterally sorted precursor proteins into the
lipid bilayer upon reconstitution into cardiolipin-rich proteolipo-
somes (Fig. 3) . In agreement with this ﬁnding TIM23
found in association with a sorted precursor protein in transit into
mitochondria . On the other hand a motor-containing form of
TIM23 has been found that lacks the Tim21 protein but consists of
in association with PAM for translocation of precursor
proteins into the matrix (TIM23
) [49,81]. Genetic and biochem-
ical data suggest that TIM23
are in a dynamic
equilibrium with each other. For example, overexpression of Tim21
leads to dissociation of Pam17 from TIM23
 and a relative
increase of the amount of PAM-free TIM23 complexes , whereas
overexpression of Pam17 leads to a loss of Tim21 from TIM23
The idea that Pam17 triggers the release of Tim21 is in line with other
studies suggesting that Pam17 is involved in early steps of the PAM
reaction cycle [80,83]. Additionally, mutations that induce an in-
creased dissociation of PAM subunits from TIM23
lead to a shift of
the TIM23 pool into Tim21-containing complex forms . However,
it is still under debate, if a complete displacement of PAM indeed
occurs in vivo . It has been reported that small substoichiometric
amounts of Tim21 can be co-isolated with components of PAM, which
has been taken as supporting evidence for a model that proposes
a single-entity TIM23-PAM machinery [44,82,90]. Alternatively, this
small pool may represent yet another snapshot of the dynamic rear-
rangements within the TIM23 complex. Some laterally sorted pre-
cursor proteins possess folded domains downstream of the sorting
signal and require PAM activity for translocation of these folded
domains across the outer membrane [86,91]. Intermediate forms of
the TIM23 complex may play an important role in the import of such
precursors. It has been reported that deletion of PAM17 causes syn-
thetic growth defects with mutations in TIM23 and TIM50 that affect
the intermembrane space receptor domains of Tim23 and Tim50 .
This raises the possibility that Pam17 is involved in the transmission
of a signal from the receptor domain of the TIM23 complex to the sites
of PAM recruitment.
Interestingly, the PAM-free TIM23
complex was found to asso-
ciate with respiratory chain supercomplexes in mitochondria in a
Tim21-dependent manner (Fig. 3) [88,92,93]. These supercomplexes
consist of the cytochrome bc
complex (complex III) and cytochrome
c oxidase (COX, complex IV). Tim21 can be chemically crosslinked
to Qcr6, a subunit of complex III, indicating a close proximity of these
proteins in organello . The interaction of TIM23
ratory chain complexes contributes to the process of inner membrane
sorting of precursor proteins. It was shown that TIM23
tory chain association renders the sorting process less sensitive to
a reduction of Δψ, but has no inﬂuence on matrix transport .
However, the exact mechanism, by which TIM23
chain coupling affects the energetics of precursor insertion into the
inner membrane, is still unknown. One study has raised the possibility
that respiratory chain complexes may also be involved in the re-
cruitment of the Pam18/Pam16 module of PAM to TIM23
A pool of Pam18 and Pam16 was found associated with respiratory
chain complexes. This interaction appears to be direct, as in contrast
736 M. van der Laan et al. / Biochimica et Biophysica Acta 1803 (2010) 732–739
co-isolation of Pam18 and Pam16 with tagged complex
III is independent of Tim21 . It is therefore possible that upon
initiation of PAM recruitment, Pam18 and Pam16 are transferred from
respiratory chain complexes to TIM23
. Finally, an interaction of
the ADP/ATP carrier Aac2 with both, the TIM23-PAM machinery and
the respiratory chain has recently been demonstrated [94,95].
6. Concluding remarks
In summary, the TIM23 complex of the inner mitochondrial mem-
brane shares basic features with other protein translocation systems.
It is composed of a receptor module and a signal-gated protein-con-
ducting channel and associates with a trans-acting import motor.
The distinguishing property of the TIM23 machinery is the dynamic
coupling of the translocase core complex to different partner protein
complexes in the outer membrane, inner membrane and matrix: i) at
the initial stages of the import reaction precursor proteins are directly
handed over from the TOM to the TIM23 complex. A recent study by
Chacinska et al.  shows that TIM23
associates with the TOM
complex and a precursor in transit early during the translocation pro-
cess to form a supercomplex. ii) For matrix translocation PAM is
recruited to TIM23
. For matrix-targeted precursors this PAM-
bound form still remains associated in a supercomplex with TOM .
iii) For lateral sorting into the membrane TIM23
respiratory chain complexes to facilitate Δψ-dependent steps of the
reaction. Moreover, recent studies have clearly demonstrated that the
negatively charged phospholipid cardiolipin is important for proper
functioning of the TIM23 machinery [41,97,98]. The molecular details
of these coupling events and how they facilitate the movements of
precursors across or into the membrane remain to be elucidated.
Work in the authors' laboratories was funded by the Deutsche
Forschungsgemeinschaft, Sonderforschungsbereich 746, and the
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[Show abstract] [Hide abstract] ABSTRACT: Mitochondria are intracellular power plants that feed most eukaryotic cells with the ATP produced by the oxidative phosphorylation (OXPHOS). Mitochondrial energy production is controlled by many regulatory mechanisms. The control of mitochondrial mass through both mitochondrial biogenesis and degradation has been proposed to be one of the most important regulatory mechanisms. Recently, autophagic degradation of mitochondria has emerged as an important mechanism involved in the regulation of mitochondrial quantity and quality. In this review, we highlight the intricate connections between mitochondrial energy metabolism and mitochondrial autophagic degradation by showing the importance of mitochondrial bioenergetics in this process and illustrating the role of mitophagy in mitochondrial patho-physiology. Furthermore, we discuss how energy metabolism could coordinate the biogenesis and degradation of this organelle.This article is part of a Special Issue entitled: Mitophagy. Copyright © 2015. Published by Elsevier B.V.
- "This depletion negatively impacts physiological processes, including PINK1/PARKIN-induced mitophagy. For example, ATP is a crucial co-factor in protein import , and a decrease in mitochondrial ATP after CCCP treatment could alter both the import of PINK1 and its degradation. PINK1 is a serine/threonine-protein kinase, and ATP levels might be a crucial trigger for both kinase activity and regulation. "
[Show abstract] [Hide abstract] ABSTRACT: The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.
- "In this regard, a previous report shows that over-expression or knockout of Mfn2 modulates and affects mRNA levels of several mitochondrial proteins [51,52]. We have described an arachidonic acid  generation/exportation system that includes an Acyl- CoA synthetase 4 (Acsl4) [38,40,56]. Acsl4 is anchored at the MAM (mitochondrial associated-membrane) structures  and its activity determines the production rate of AA which is necessary for StAR gene expression . "
[Show abstract] [Hide abstract] ABSTRACT: Mitochondria are involved in many essential cellular activities. These broad functions explicate the need for the well-orchestrated biogenesis of mitochondrial proteins to avoid death and pathological consequences, both in unicellular and more complex organisms. Yeast as a model organism has been pivotal in identifying components and mechanisms that drive the transport and sorting of nuclear-encoded mitochondrial proteins. The machinery components that are involved in the import of mitochondrial proteins are generally evolutionarily conserved within the eukaryotic kingdom. However, topological and functional differences have been observed. We review the similarities and differences in mitochondrial translocases from yeast to human. Additionally, we provide a systematic overview of the contribution of mitochondrial import machineries to human pathologies, including cancer, mitochondrial diseases, and neurodegeneration.
- "Its interaction with TIM23 is supported by the presence of Mgr2 (Fig. 1C). In addition to Tim21 and Mgr2, Tim17 was also shown to play an important role in the lateral sorting of membrane precursor proteins [6,2627282930. The presequence-containing precursor proteins undergo various maturation steps . "