Life, 51: 345 350, 2001

c 2001 IUBMB
Copyright
1521-6543/01 $12.00 + .00
IUBMB

Review Article
Translocation of Proteins into Mitochondria
Nicholas J. Hoogenraad and Michael T. Ryan
Department of Biochemistry, La Trobe University, 3086 Melbourne, Australia

Summary
The translocase of the outer mitochondrial membrane (TOM)
is composed of receptors, a channel protein, and its modulators
that function together to import proteins into mitochondria. Although the import pathway of proteins directed to the mitochondrial matrix has been well characterized, recent studies into the
import pathway taken by proteins into the other submitochondrial
compartments have broadened our understanding into the way the
TOM machinery recognizes, interacts, and translocates proteins.
IUBMB Life, 51: 345 350, 2001
Keywords

Import; mitochondria; TIM; TOM; traf cking.

INTRODUCTION
Mitochondria are essential for the viability of eukaryotic
cells. Whereas they are the main source of respiratory energy,
cells in culture can survive the complete depletion of mitochondrial DNA, which encodes 13 polypeptides of the oxidative phosphorylation machinery. This suggests that mitochondria are involved in essential activities besides the production
of ATP. In fact, mitochondria are the major source of damaging reactive oxygen species and contain key regulators of programmed cell death (apoptosis). There has also been an emerging realization that mitochondrial dysfunction is at the center
of medical conditions ranging from early onset inborn errors of
metabolism to later onset neuromuscular disorders and a wide
range of unexpected phenotypes including Parkinsons Disease
and diabetes (1).
Mitochondria grow and divide by a process of accretion of
proteins encoded in the nucleus and mitochondria, followed by
binary ssion. The 1,000 nuclear encoded proteins need to be
imported into mitochondria (2, 3). Proteins are rst synthesized
in the cytosol on free ribosomes as precursors with signals that
direct them to one of the four mitochondrial subcompartments
the outer membrane, intermembrane space (IMS), inner
Received 28 June 2001; accepted 13 August 2001.
Address correspondence to Nicholas J. Hoogenraad. Fax: 61-39479-2467. E-mail: N.Hoogenraad@latrobe.edu.au

membrane, and matrix. A typical matrix-targeted precursor contains an N-terminal presequence 15 to 40 amino acids long that
is rich in basic and hydroxylated amino-acids and has the potential to form an amphipathic -helix. Whereas some inner
membrane and intermembrane space proteins also possess such
a presequence, others do not and are like all outer-membrane
proteins in that they contain their often cryptic mitochondrial
targeting signal(s) within the sequence of the mature protein.

Protein Import Into the Mitochondrial Matrix

Precursors imported into the matrix and inner membrane must
interact with two separate multicomponent membrane translocation machineriesa TOM1 machinery of the outer membrane
and a TIM machinery of the inner membrane (Fig. 1). Two separate and functionally distinct TIM machineries are so far known
and each of these contain channel proteins and accompanying
subunits. The Tim23 complex contains the integral membrane
proteins Tim23 and Tim17 along with a population of matrix
Hsp70 that interacts via the peripheral membrane protein Tim44.
The Tim23 complex is responsible for the import of all matrixtargeted precursors along with those precursors destined for the
inner membrane that, like matrix precursors, contain N-terminal
targeting signals. Tim22, which shares homology with Tim23,
exists in a separate complex that contains Tim54, Tim18, and
Tim12 plus a subpopulation of the intermembrane space proteins
Tim9 and Tim10. This complex is involved in the insertion of
inner membrane proteins such as metabolite carriers that generally contain multiple membrane spanning domains and possess
internal mitochondrial targeting signals (4).
Although two different TIM machineries exist, only one TOM
complex is involved in the translocation of almost all precursors
across or into the outer mitochondrial membrane. The import
of the cytochrome c precursor seems to be an exception (see
later). The TOM complex must therefore possess unique properties for the recognition of both N-terminal and internal targeting
1 Abbreviations: AAC, ADP/ATP carrier; IMS, intermembrane space; PAGE,

polyacrylamide gel electrophoresis ; TIM, translocase of the inner membrane;

TOM, translocase of the outer membrane; Tomx, Tom subunit where x is its
monomeric molecular weight in kDa.

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HOOGENRAAD AND RYAN

4-Color
art

Figure 1. Import of proteins into mitochondria. Typical matrix-targeted precursors (blue) are targeted to the mitochondrial outer
membrane via positive N-terminal targeting signals, where they are mainly recognized by the receptor Tom20. Some precursors
destined for the inner membrane, such as AAC (red), contain internal targeting signals and bind to the receptor Tom70. Overlapping
speci cities between the receptors for precursors may be observed. The precursors are transferred to other members of the TOM
machinery (green) before their translocation across the Tom40 channel. There, the pathways diverge. Matrix-targeted precursors
engage with the Tim23 complex and are translocated into the matrix in a membrane potential dependent manner. Some of these
precursors contain stop-transfer signals and are instead released into the inner membrane. If proteolytically processed by an IMS
protease, they are released into the IMS. AAC associates with Tim9 and Tim10 in the IMS before binding to the Tim22 complex
and inserting into the inner membrane in a membrane potential dependent manner.
motifs and to have the ability to distinguish those hydropho bic membrane proteins for insertion into the outer membrane
from those that must bypass the outer membrane and be targeted
and inserted into the inner membrane. In addition, mechanisms
must be in place to ensure translocation occurs in a unidirectional manner. Whereas precursor-binding sites are present on
the intermembrane space (or trans-) side of the outer membrane,
precursors must still rst insert, travel along, and then exit the
TOM channel before such interactions can take place.

The TOM Machinery

Members of the TOM machinery that have been characterized in detail are the receptors Tom70, Tom20, and Tom22, the
channel protein Tom40, and the three small Tom subunits associated with Tom40, termed Tom7, Tom6, and Tom5. The Tom
subunits interact with one another to form a large multimeric
complex. Puri cation of a histidine-tagged form of Tom22 from
mitochondria isolated from the fungus Neurospora crassa led to
the isolation of a complex containing Tom70, Tom40, Tom22,

Tom20, Tom6, and Tom7 (5). A Tom5 homologue was not identi ed but may be present in low amounts. In yeast, a 400-kDa
TOM complex has been readily observed by Blue Native PAGE
and contains Tom40, Tom22, Tom7, Tom6, and Tom5 (6). Under
gentle solubilization conditions, Tom20 can also be found associated with this complex (7 ). The mammalian TOM complex
contains homologous subunits and migrates slightly faster than
the yeast complex on Blue Native PAGE (8).

Interactions of Precursors with TOM Receptors

The Tom70 receptor is a homo-dimer that seems to be the
least stably associated subunit of the TOM complex (5, 6, 9).
Most studies concerning the receptor function of Tom70 have
focused on its ability to form a stable interaction with the precursor of the ADP/ATP carrier (AAC) in the absence of ATP
(10, 11). The AAC precursor contains three modules each with
an internal mitochondrial targeting signal. These different modules contribute separate Tom70 binding sites that can lead to the
formation of a complex consisting of the AAC precursor with

MITOCHONDRIAL PROTEIN IMPORT

three Tom70 dimers (9). How ATP is utilized in releasing the

precursor from Tom70 is not known.
The Tom20 receptor seems to be involved in the early steps
of precursor recognition. The structure of the human Tom20
receptor domain in complex with the targeting signal peptide
of the matrix-destined precursor aldehyde dehydrogenase, revealed a cleft that bound mainly to the hydrophobi c face of the
amphipathic helical presequence (12). Such binding leaves the
positively charged face of the presequence open to contact with
possibly other TOM members such as Tom22. This may be important in aiding the movement of the precursor into the Tom40
channel.

Protein Translocation Through the TOM Machinery

Electrophysiological measurements of reconstituted Tom40
and shows a preferindicate that it is has a pore size of 22 A
ence for cations (13). The size estimate correlates with import
gold pardata, which showed that a precursor attached to a 20-A
ticle could translocate across the outer membrane but not one
gold particle (14) and is supported by early
attached to a 26-A
ndings that precursors must be relatively unfolded to translocate across the mitochondrial membranes (15). However, it does
seem that some precursors can translocate across the TOM machinery in a somewhat less extended state. It was recently found
that the ADP-ATP carrier (AAC) precursor destined for the inner membrane could translocate across the Tom40 channel and
make contact with components of the IMS while having each of
its N- and C-termini fused to the cytosolic protein, dihydrofolate
reductase (9). This suggests that the AAC precursor translocates
in a loop form. Tom40 also appears to be targeted and inserted
into the outer membrane via a similar mechanism (16, 17, and
see later).
The channel activity of reconstituted Tom40 was similar to
the activity observed with the TOM complex measured from isolated outer membrane vesicles; however, in this case the channel was found to be more often in a closed state (13). This
indicates that the channel may be regulated by other components of the TOM complex. Indeed, electrophysiological measurements of mitochondria from a yeast mutant lacking Tom22
revealed that channel activity resembled that of Tom40 by itself. The TOM complex in mitochondria lacking Tom22 was
shifted in size on native PAGE from 400 kDa to 100 kDa
although it still contained Tom40 and the small Tom subunits
(18). The drastic reduction in size of the complex cannot be accounted for by the loss of four Tom22 subunits predicted to be
present (19). The 400-kDa TOM complex appears to consist of
multiple pores. Thus, analysis of the puri ed N. crassa TOM
complex by electron microscopy showed that up to three central pits are present (5). The complex puri ed under conditions
that removed Tom20 and Tom70 revealed a complex containing two pits (20). Although it has not been directly demonstrated, these pits most likely represent the protein translocation
channels formed by Tom40. Thus, Tom22 is involved in connecting individual Tom40 channels, with its associated small

347

Tom subunits, together. The transmembrane domain of Tom22

was found to be essential for oligomerization. Tom22 is involved
in regulation of the TOM channeleither through its cytosolic
receptor domain, via its transmembrane anchor or a combination
of both. It has been reported that removal of the Tom22 cytosolic domain directly alters the structural arrangement of Tom40
oligomers (21) while a region of the cytosolic domain is essential for protein import (22, 23). Why would the TOM channel
require regulation? It may be that Tom40 translocates precursors productively through structural arrangements of the TOM
complex that in uences its gating properties. Indeed, precursor
binding sites have been reported to exist on both the cytosolic
(cis-) and IMS (trans-) sides of Tom40 (24).

The Role of trans -binding Sites on Protein Translocation

A key area of study into protein translocation is to de ne the
driving forces for polypeptide movement through membrane
channels. Although the membrane potential along with the action of matrix Hsp70 and ATP hydrolysis is known to be required for precursor translocation across the inner membrane
(3), the driving force for translocation across the mitochondrial
outer membrane is not yet clear. For precursors destined for the
matrix or inner membrane, membrane potential/Hsp70 action
may also be suf cient to drive the complete precursor across
the outer membrane channel. The membrane potential has been
suggested to act as an electrophoretic force by attracting the positively charged N-terminal targeting signals into the negatively
charged matrix. Still, a mechanism must be in place to allow
the initial movement of the precursor in a forward direction
across the Tom40 channel so that it can engage with the TIM
machinery. This is especially evident for a number of precursors destined for the intermembrane space that do not require a
membrane potential, or the action of ATP for their translocation.
The movement of some precursors from TOM receptors, across
the TOM complex and into the IMS, has been suggested to take
place in a similar manner to that thought to be utilized by the
membrane potential. The acid-chain hypothesis predicts that
the basic targeting signals of precursors bind to acidic domains
of import components with sequential increasing af nity. Such
acidic patches are predicted to be present on the cytosolic domains of Tom20, Tom22, and Tom5, the IMS domains of Tom22
and Tim23. Although this hypothesis has been supported experimentally using soluble domains of these import subunits (25), a
number of recent reports question the signi cance of the results.
The structural characterization of Tom20 bound to a targeting signal revealed that this interaction is largely hydrophobi c
(12). In addition, precursors could be imported into mitochondria at almost wild-type rates even when the IMS domain of
yeast Tom22 is missing (26), whereas the IMS domain of human Tom22 lacks acidic patches (27). Tom40 has also been
suggested to be a major trans-binding site and precursor binding is not salt-sensitive (24). Finally, the acid-chain hypothesis
cannot account for the translocation of those precursor proteins
that lack typical N-terminal targeting signals.

348

HOOGENRAAD AND RYAN

Pfanner and Geissler (3) have now modi ed the acid-chain

hypothesis to accommodate precursors that lack typical positively charged targeting signals. The binding chain hypothesis relies on precursors still having higher af nities for receptors
further along the import pathway although the import subunits
are predicted to act cooperatively in binding precursors. Electrostatic interactions are still most likely to be the predominant
force between members of the import machinery and those precursors that follow a matrix-targeting pathway. This is supported
by the nding that a negatively charged domain is also present
on a region of Tim23 that is exposed to the intermembrane space.
Precursors with positively charged N-terminal targeting signals
have been shown to bind to the intermembrane space domain
of Tim23 through electrostatic interactions (25). Recently, it has
been suggested that a portion of the N-terminal domain of Tim23
that faces the IMS is actually embedded in the outer membrane,
thereby fusing both membranes together (28). Whereas Tim23
did not seem to be in complex with the TOM machinery, it was
suggested that Tim23 lacking this outer membrane anchor led
to the reduction of mitochondrial contact sites. Other proteins
have also recently been suggested to make contact between the
outer and inner membranes and include members of the ssion
fusion machinery, Fzo1p (29) and MMM1p (30). Early models,
which suggested that protein import into the mitochondrial matrix occurred at mitochondrial contact sites, may now need to be
revisited.

Targeting Pathways Diverge After the TOM Complex

The interaction of precursors with the IMS domain of Tim23
may act as a trigger to open the Tim23 channel thereby allowing
the precursor to enter the matrix through the initial action of
the membrane potential. A population of mt-Hsp70 associated
with the inner membrane functions to translocate the precursor
further into the matrix in a process that is still under debate,
but the process involves preventing precursors from slipping out
of the matrix as they undergo a state of unfolding in order to
translocate across the TOM and TIM machinery (3).
What is the case for translocation of non matrix-targeted precursors? A number of other precursors follow a similar route
taken by matrix-targeted precursors, but they most often diverge
from the pathway at the Tim23 complex. These precursors contain transmembrane anchors following the N-terminal targeting
signal that function as stop-transfer signals. These stop-transfer
signals lead to the precursors arrest and movement into the inner
membrane or, alternatively, its release into the IMS following
proteolytic removal of the membrane anchor (Fig. 1).
For the multimembrane spanning metabolite (carrier) proteins of the inner membrane, a different import pathway exists. The ADP/ATP carrier precursor can translocate almost fully
into the intermembrane space in the absence of a membrane potential (10). However, this potential is required for its insertion
into the inner membrane via its interaction with the TIM22 complex. In addition, a set of small zinc- nger proteins are involved

in carrier precursor translocation. An IMS complex of Tim10

and Tim9 binds to the AAC precursor as it emerges from the
TOM complex (4). It is still not known whether the small Tim
proteins act in pulling the precursor across the TOM channel,
however ATP is not needed for their action. Nevertheless, their
binding may act to prevent backsliding of the precursor into
the cytosol and through biased Brownian movements, lead to
its complete translocation into the IMS. The small Tim proteins
probably act to chaperone these hydrophobi c precursors while
they are in the IMS, thereby preventing their aggregation prior
to their interaction with the inner membrane.
Although some precursor proteins destined for the IMS contain N-terminal targeting signals and stop-transfer signals and
thus diverge from the matrix targeting pathway at the level of the
inner membrane, others lack obvious targeting signals and do
not require a membrane potential or ATP for their import. Deletion analysis of the cytochrome c heme lyase precursor revealed
that its targeting signal resides in a highly conserved region towards its C-terminus (31). This precursor still engages with the
TOM machinery and can be transported into vesicles containing
the TOM complex (32). The precursors of the small Tim proteins also seem to be translocated across the TOM machinery
although in vitro studies indicated that they did not require Tom
receptors for their import but rather Tom5 (33). Cytochrome
c, an IMS protein, of focus recently for its role in apoptosis
following its export from the mitochondria, also follows what
seems to be a unique pathway for its import into mitochondria.
No evidence exists for the precursor of cytochrome c (termed
apocytochrome c) interacting with the TOM complex. Moreover, some ndings indicate that apocytochrome c (which lacks
heme addition) translocates directly across the outer membrane
and is covalently modi ed by cytochrome c heme lyase, which
acts as a trans-binding site, resulting in its retention within the
IMS (2). It will be of interest to determine whether precursors
such as the small Tim proteins, are trapped in the IMS via their
chelation with zinc and their subsequent folding.
Precursors that must be inserted into the mitochondrial outer
membrane also contain targeting signals that enable them to
engage with TOM receptors. Some proteins such as Tom22
and Bcl-2 contain single transmembrane domains and are Cterminally (or tail-) anchored. In such cases, the mitochondrial
targeting information may be separate from the information
specifying topology. For Tom22, it was found that the membrane anchor plus an adjacent positively-charged sequence in
the cytosolic domain were necessary for targeting and insertion
(22, 23). Mutations in this positively-charged region prevented
mitochondrial localization. Tom20 is required for the import of
Tom22, yet it binds to precursors mainly via hydrophobi c interactions (12, 34). Although hydrophilic interactions with Tom20
may also play a role an alternative is that the Tom22 import signal forms an amphipathic -helix containing both a positivelycharged and a hydrophobi c face (22). Whether this domain represents the region through which Tom20 and Tom22 interact
in complex form is not known, although binding of Tom20 to

MITOCHONDRIAL PROTEIN IMPORT

Tom22 occurs via their cytosolic domains (18, 27 ). Indeed this

may be one mechanism whereby some precursors are scrutinized
before import by competing with Tom22 for binding to Tom20.
Other outer membrane proteins are orientated with their
N-termini facing the intermembrane space. For example, Tom70
contains a positively-charged N-terminal region that somewhat
resembles a matrix-targeting signal. However, it seems that the
transmembrane domain is also important for its targeting and
insertion (35). The hydrophobi c domain of precursors can therefore act as stop-transfer signals. Like Tom70, the precursor
of NADH-cytochrom e b5 contains a weak matrix-targeting Nterminal signal along with a hydrophobi c transmembrane domain. This precursor is sorted both to the IMS and to the outer
membrane. Mutations in its hydrophobi c domain resulted in
preferential targeting to the IMS, utilizing a stop-transfer pathway taken by some other IMS precursors (36).
The sorting of multiple-membrane spanning proteins such as
porin (VDAC) and the precursor of Tom40 to the outer membrane is even more complex. Both of these integral membrane
proteins are predicted to contain mainly -barrel structure and
do not contain clearly de ned hydrophobi c regions. The targeting signal of porin seems to reside in both N- and C-terminal
regions of the precursor (37), whereas Tom40 harbors internal
targeting signal(s) (16). A recent analysis into the import of
Tom40 uncovered a new pathway for its insertion into outer
membrane from the IMS compartment (17 ). Tom40 interacts
with Tom20 and Tom22 and is subsequently transferred across
the outer membrane where it becomes peripherally associated
on the inner face of the outer membrane with Tom5. There, it
inserts into the outer membrane and assembles into subcomplexes prior to forming the 400-kDa TOM complex (17 ). The
reason why Tom40 does not directly insert into the outer membrane from the cytosolic side is unclear, although its transport
across the Tom channel may be to promote rearrangements in
its structure and/or its release from cytosolic chaperones. Once
in the outer membrane, the endogenous Tom40 is predicted to
be relatively in exible due to the formation of hydrogen bonds
between adjacent -strands of the protein (38), which could
prevent precursors with hydrophobi c regions from escaping laterally into the outer membrane. This prediction does present
some problems for the insertion of precursors into the outer
membrane. It has been suggested that precursors destined for
the outer membrane do not insert into the Tom40 channel but
are rather redirected into the lipid bilayer following their interaction with the TOM complex (38). However, this pathway
may not be able to accommodate precursors that contain hydrophilic domains that need to be translocated in order to face
the intermembrane space. Whether porin also translocates fully
across the outer membrane before its re-insertion is yet to be
addressed. However, recent investigations have shown that following its binding to Tom20, porin associates with Tom22 and
Tom40 prior to its import (39).
Clearly, it will be important to analyze the puri ed TOM
complex in more detail and in association with precursors des-

349

tined for different mitochondrial subcompartments. These studies could also determine the functional relevance of multiple
channels within a single complex. Perhaps some asymmetry exists between the different channels of a single complex that enable the import machinery to discriminate between precursors
that must insert into the outer membrane and those that must
translocate across the outer membrane.
ACKNOWLEDGMENTS
We apologize to the many investigators whose work could
not be cited due to space limitations. We thank A. Johnston
for critically reading the manuscript. This work is supported by
grants from the Australian Research Council.
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