Current Biology, Vol. 13, R326–R337, April 15, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00239-2
Mechanisms of Protein Import into
Mitochondria
Kaye N. Truscott, Katrin Brandner and Nikolaus
Pfanner
Apart from a handful of proteins encoded by the mitochondrial genome, most proteins residing in this
organelle are nuclear-encoded and synthesised in the
cytosol. Thus, delivery of proteins to their final destination depends on a network of specialised import
components that form at least four main translocation
complexes. The import machinery ensures that proteins earmarked for the mitochondrion are recognised and delivered to the organelle, transported
across membranes, sorted to the correct compartment and assisted in overcoming energetic barriers.
Introduction
Protein trafficking is a vital cellular process in which
proteins are transported from their site of synthesis to
locations within cells where they function. In eukaryotes
these locations are numerous, as cells are organised
into several compartments formed from single or multiple membrane boundaries. Nuclear-encoded proteins,
which are synthesised in the cytosol, are transported
for example to organelles such as mitochondria, the
endoplasmic reticulum (ER), peroxisomes, the nucleus
and chloroplasts. A small number of proteins are
encoded by the genomes of mitochondria and chloroplasts, and these add to the complexity of protein trafficking pathways. Here we shall discuss recent
advances in our understanding of the mechanisms of
import of nuclear-encoded proteins into mitochondria,
and also the export pathway to the inner membrane
that is used by mitochondrially encoded proteins.
Because of the way the mitochondrion is divided
into four subcompartments — the two aqueous
chambers (intermembrane space and matrix) and two
membranes (the outer and inner membranes) — there
are multiple pathways for protein translocation into this
organelle. Most of the ~1,000 mitochondrial proteins
are encoded by the nuclear genome, synthesised in
the cytosol on free ribosomes as precursor proteins
and then transported into or across mitochondrial
membranes with the aid of a handful of distinct complexes (Figure 1). Targeting information resides within
the protein itself; this may take the form of an aminoterminal extension or ‘presequence’, though for many
membrane proteins it is contained within the mature
protein [1–4]. The transfer of mitochondrial proteins
from their site of synthesis in the cytosol to the surface
of the mitochondrial outer membrane, where translocation begins, mainly occurs in a post-translational
Institut für Biochemie und Molekularbiologie, Universität
Freiburg, Hermann-Herder-Straße 7, D-79104 Freiburg,
Germany. E-mail: [email protected]
Review
manner — that is, the fully synthesized protein contacts the mitochondrial import machinery [1–3,5,6].
However, a co-translational mechanism of translocation cannot be excluded for some precursor proteins
[1–3,5,6].
To date, a single ‘translocase of the outer membrane’
(TOM) complex has been described, which is the only
known entry point into mitochondria for nuclear-encoded
proteins. After crossing the outer membrane at the TOM
complex, precursor proteins segregate, as there are two
structurally and functionally distinct ‘translocases of the
inner membrane’ (TIMs): the TIM23 and TIM22 complexes, each of which guides the import of subsets of
proteins to internal compartments of mitochondria.
All proteins with an amino-terminal presequence are
imported via the TIM23 translocase [7]. The transport
of presequences across the membrane occurs
through an aqueous pore, while the membrane potential across the inner membrane provides a driving
force for this energy-requiring process [8–11]. In addition, the ATP-dependent action of matrix heat shock
protein 70 (mtHsp70), together with its helper proteins
Tim44 and Mge1, drives to completion the unidirectional movement of precursor proteins into the mitochondrial matrix [12–15].
Some inner membrane proteins, which only contain
internal targeting signals, are imported with the assistance of the pore-forming TIM22 complex in a membrane potential-dependent manner [16–21]. The
mitochondrial export machinery is responsible for the
insertion of proteins into the inner membrane from the
matrix (Figure 1). The action of this machinery resembles protein translocation processes in bacteria.
Targeting Signals
Two types of mitochondrial targeting signal can
currently be described: presequences and internal
signals. Presequences direct precursor proteins via
the TOM complex to the TIM23 translocase, where
they are sorted to the matrix, inner membrane or
intermembrane space (Figure 2). One would expect
presequences to have a consensus amino acid
sequence, but rather they consist of a handful of
subtle distinguishing properties at the level of the
primary and secondary structure. They generally have
a high content of basic, hydrophobic and hydroxylated amino acids, and a length of about 10–80 amino
acids [22]. An amphipathic α helix with one positive
and one hydrophobic face is another common feature
that is important for receptor recognition [1–3,23].
Once they are exposed to the matrix, presequences are often removed by a processing peptidase, as they are not necessary for protein function.
In comparison, proteins which only contain internal
targeting signals are directed to either the inner membrane, intermembrane space or outer membrane,
depending on the nature of the signal — which, to
date, has not been clearly defined (Figure 2). The
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Figure 1. Translocation complexes of
Proteins with
Proteins with internal
mitochondria.
presequence
targeting signals (eg. carrier)
+++
Four types of translocation complex are
N
currently known in mitochondria, three for
the import of precursor proteins into interC
nal compartments — TOM, TIM23 and
TIM22 — and one for the export of preR
Cytosol
cursor proteins from the matrix into the
R
inner membrane (Export). Precursor proTOM
teins with either an amino-terminal precomplex
OM
sequence or internal targeting signals
bind to a specific receptor (R), which
IMS
+
directs their import via a general import
pore (GIP) of the translocase of the outer
Export
TIM23
TIM22
∆ψ Carrier
membrane (TOM). Precursor proteins with
complex
IM
complex
complex
a presequence are directed to the TIM23
–
complex for import across the inner memMatrix
brane, a process which requires the memmt Hsp70
∆ψ) and the ATP-driven
brane potential (∆
ATP
action of mtHsp70. If they are destined for
Matrix
the inner membrane, nuclear-encoded
protein
Mitochondrial-encoded
precursor proteins with only internal targeting signals are guided to the TIM22
protein
Current Biology
complex by Tim factors of the intermembrane space. Membrane insertion at the
TIM22 complex is dependent on the membrane potential. Mitochondrially encoded proteins that are synthesised in the matrix and
some imported proteins are exported to the inner membrane with the assistance of the export machinery. OM, outer membrane; IMS,
intermembrane space; IM, inner membrane.
internal targeting information, best characterised for
members of the carrier family of the mitochondrial
inner membrane, is hidden in the mature protein
amongst amino acid residues important for folding
and function [2–4]. The targeting information seems to
be spread throughout the length of the protein [4,24].
The Importance of Surface Receptors for Outer
Membrane Translocation
Three proteins of the mitochondrial outer membrane
TOM machinery act as receptors for the recognition of
targeting signals: Tom70, Tom20 and Tom22 (Figure
3). Each has a large cytosolic domain anchored to the
membrane, with a single transmembrane span. Tom22
also has a small, functional carboxy-terminal domain
which is exposed to the intermembrane space. Tom22
forms part of a very stable general import pore (GIP)
complex of the outer membrane, which additionally
contains the pore-forming protein Tom40 and accessory proteins Tom5, Tom6 and Tom7 (Figure 3)
[25–28]. Tom70 and Tom20 are the primary receptors:
they are the first Tom proteins to make contact with
precursor proteins, but show preferences for different
precursor types. They are both only loosely associated with the GIP complex [25,26,28,29].
Tom70 is the major receptor for precursor proteins
that contain internal targeting information, such as
members of the metabolite carriers of the inner mitochondrial membrane [1–3]. It binds to multiple segments within the carrier proteins, though an amino
acid recognition motif has not been identified [4,30].
The carrier proteins consist of three related modules,
each containing two transmembrane domains connected by a hydrophilic loop, such that a carrier
monomer contains six transmembrane domains. Each
repeat module of a carrier contributes targeting information, so that a carrier precursor binds to several
molecules of the Tom70 receptor simultaneously [24].
Interestingly, it has recently been shown that Tom70
is not only a receptor for precursor proteins, but also
a docking point for cytosolic chaperones directly
involved in the import pathway [31]. The transfer of
newly synthesised Tom70-dependent precursor proteins from the cytosol to the receptor can now be
described. After synthesis, the precursor protein
forms a proteinaceous high molecular weight complex
[32], which in mammals contains Hsp90 and Hsp70
and in yeast Hsp70 (Figure 3) [31]. This is the first time
Hsp90 has been implicated in a mitochondrial import
pathway. Previous studies indicated that Hsp70 and
other cytosolic factors such as mitochondrial import
stimulating factor (MSF) [33-36] can also stimulate
protein import into mitochondria, although it remains
open if MSF plays a role in protein import in vivo [6].
An association with chaperones probably prevents
aggregation of the precursor and permits presentation
of the protein in a conformation conducive for receptor recognition.
The cargo precursor protein is then transferred to
the Tom70 receptor, an event that requires the
docking of Hsp70 to the receptor [31]. Binding of
Hsp90 and Hsp70 to Tom70 is mediated by a specialised tetratricopeptide repeat (TPR) domain in
Tom70. The determinants for selective chaperone
docking are apparently contained within Tom70. As
has been previously described, the transport of carriers to subsequent import stages requires hydrolysis of
ATP [32]: the authors propose that ATPase cycling,
most likely by the chaperones, leads to release of the
precursor proteins from Tom70.
The main function of Tom20 is to bind precursor
proteins with presequences. The hydrophobic face of
the helical presequence is predicted to interact with
Tom20 [30]. The structure of a presequence in a
complex with a soluble portion of Tom20 clearly
shows that the helical presequence indeed fits into a
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Non-cleavable precursors
A Precursors
with
N-terminal
presequences
B Inner C Intermembrane D Outer
membrane
space
membrane
carrier
proteins
proteins
proteins
Cytosol
TOM
OM
TOM
IMS
IM
Export
complex
TOM
TOM
+
TIM
23
TIM22
∆ψ
–
Matrix
E Mitochondrial-encoded
inner membrane
proteins
Current Biology
groove formed by Tom20 in which hydrophobic interactions predominantly occupy the interface [23].
Tom22 is the secondary receptor for mitochondrial
protein import, accepting both precursor types from
the primary receptors but mainly precursors with a
presequence [37]. The handing over of precursor proteins from the primary receptors to Tom22 is possibly
promoted by interactions between their respective
cytosolic domains [27]. While we do not yet know the
structure of Tom22, biochemical evidence indicates
that presequences bind to Tom22 via ionic interactions probably formed between the positively charged
face of the helical presequence and a negatively
charged surface on Tom22 [30,38].
Signal recognition does not stop with the major
receptors. A receptor-like protein called Tom5 acts at
the tertiary level influencing the import of precursors
with both presequences and internal targeting signals
[39]. The proximity of Tom5 at the entrance of the
translocation pore allows perfect positioning of the
incoming precursor for entry into the translocation
channel of the outer membrane. Targeting signal
recognition thus occurs at two to three levels to bring
most mitochondrial proteins to the translocation pore
(Figure 3).
Translocation at the Outermost Membrane
Whether proteins traverse membranes completely
or become embedded in them, aqueous proteinconducting channels are generally required. In
mitochondria, the cation–selective GIP complex of the
outer membrane is formed from the essential integral
membrane protein Tom40 [40,41]. Unlike the single
aqueous pore through the ER translocon, the GIP
complex contains two to three pores, most likely
completely constructed from Tom40 but held in the
complex through interactions with Tom22 [26,27,42].
In yeast, devoid of Tom22, the GIP complex fails to
assemble to the full-sized ~400 kDa core complex,
Figure 2. Sorting of precursor proteins in
mitochondria.
There are four compartments in mitochondria to which proteins are sorted. (A) Typically a precursor protein with an
amino-terminal presequence is directed
across the TOM and TIM23 machinery into
the matrix. When additional sorting signals
are present in the protein, sorting to the
inner membrane or intermembrane space
occurs at the TIM23 complex [118,119].
(B) Non-cleavable inner membrane proteins
such as metabolic carriers are imported
into mitochondria via the TOM complex
then directed to the TIM22 complex for
membrane insertion. (C) The import of
some intermembrane space proteins only
depends on the TOM machinery. (D) Likewise outer membrane proteins utilise the
TOM complex. (E) Sorting of the handful of
mitochondrially encoded proteins to the
inner membrane is facilitated by export
complexes. Also some nuclear-encoded
proteins that are imported into the matrix
are then sorted to the inner membrane by
the export machinery [105,120].
and the remaining components are found in a 100 kDa
complex consisting of Tom40 and small proteins
Tom5, Tom6 and Tom7 [27].
Several proteins of different types and locations have
been found in association with the TOM complex or to
be inhibited in their import when the Tom40 channel is
blocked, indicating the broad capacity of this translocase for transporting all types of nuclear-encoded mitochondrial proteins [7,32,43–46]. Tom6 and Tom7 are
integral membrane proteins that play a structural role in
the organisation and stability of the TOM complex
[47,48]. Tom6 facilitates interaction of the single TOM
channel complex of 100 kDa with Tom22 to form the full
sized GIP complex [25,27,49]. Tom7 is at least partially
antagonistic to Tom6. Tom7 favours dissociation of the
TOM complex and also influences sorting of proteins to
the outer membrane [48,49].
The multiple pore arrangement of the GIP complex
is intriguing, but why has it evolved in this way when
other translocases have only a single pore? It may
relate to the fact that import into mitochondria occurs
post-translationally: imported proteins are synthesised
in their entirety before any portion is translocated. For
polytopic membrane proteins, a pair of transmembrane α helices separated by a hydrophilic loop (a
helical hairpin) may insert into one pore while a
second helical hairpin in the same protein inserts into
a second TOM pore, allowing simultaneous import of
multiple domains. Such a mechanism may promote
efficient membrane insertion and subsequent assembly. An en bloc import mechanism would require considerable flexibility of the translocation complex in
order to allow either lateral movement of the protein
into the membrane or movement through the translocase to the intermembrane space.
With respect to its size a single TOM pore is wide
enough (~20 Å) to carry two closely packed transmembrane α helices at the same time [26,41,50]. At
least for the ADP/ATP carrier (AAC), import across the
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Figure 3. The translocase of the
mitochondrial outer membrane.
The TOM translocase consists of the
primary receptors Tom70 and Tom20, and
the GIP complex formed from Tom40,
Tom22, Tom5, Tom6 and Tom7. A precursor with internal targeting signals associates with cytosolic chaperones Hsp70
and Hsp90 in the cytosol. The precursor
then binds to its main receptor Tom70
when the chaperones also dock onto this
receptor. In an ATP-dependent manner
the precursor is transferred to Tom5 then
subsequently passes through the translocation channel formed by Tom40. A
backup receptor route via Tom20 and
Tom22 is also possible for this precursor
type. Precursor proteins with presequences on the other hand bind first to
Tom20 and then are subsequently transferred to Tom22, Tom5 and the Tom40
translocation channel. In addition the presequence can bind to the carboxy-terminal domain of Tom22 prior to further
import via the TIM23 complex.
Proteins with internal
targeting signals
Hsp70
outer membrane can occur in a partially folded state
in which helical hairpins traverse the outer membrane
[24,32]. In comparison, import into the ER translocon
occurs largely co-translationally, so that the translocation machinery is presented with polypeptide segments step by step as they are synthesised. For
polytopic membrane proteins, lateral release from the
ER translocon may occur simultaneously once all of
the transmembrane segments have gathered in the
one translocon pore [51]. This may be necessary for
correct intramolecular assembly. Indeed the active
translocon is larger than the TOM pores, with an
estimated diameter of 40–60 Å, and so could accommodate multiple transmembrane segments [52].
Assembly of Translocation Components
All mitochondrial translocation components are
nuclear-encoded. This means that they must also be
imported and assembled into multisubunit membrane
complexes following synthesis in the cytosol on free
ribosomes. Is a specialised machinery required for this
process, or do the standard pre-existing translocation
components suffice? At least for the essential poreforming protein Tom40, the evidence indicates that a
combination of these possibilities leads to its correct
import and assembly [49].
The Tom40 precursor is first recognised by the TOM
import machinery, as for any other precursor. It is
targeted to the outer membrane mainly via recognition
by the receptors Tom20 and Tom22. It is then transferred to a unique sorting and assembly complex
which is distinct from the mature TOM complex [49].
Upon release from this assembly complex, the Tom40
precursor is found in a partial GIP complex in association with Tom6. Assembly to the mature GIP complex
from this intermediate occurs upon incorporation of
Tom22 and Tom7. The TOM components that join in
the late assembly of Tom40 to form the GIP complex
also occur as free endogenous components, indicating
Hsp90
Proteins with
presequence
+++
N
ATP
70
5
7
Tom40
22 20
6
Cytosol
OM
IMS
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that the mature complex is structurally dynamic and
can exchange its subunits. Tom7 also appears to play
a role in the disassembly of the GIP complex.
The Import Pathway for Precursors with a
Presequence
All precursor proteins that have a presequence are
targeted to a specialised inner membrane translocation machinery — the TIM23 complex (Figure 4). The
presequence is not only important for translocation of
this subset of precursors across the outer membrane,
but is also required for initiation of import at the inner
membrane. The presequence is recognised by components of the TIM23 complex and also responds to a
large electrical field at the inner membrane which
actively promotes import [7–9,53].
What molecular events occur when the presequence emerges in the intermembrane space? A key
player in the process is the multifunctional translocation component Tim23. Tim23 is a pore-forming integral protein of the inner membrane, which also has an
amino-terminal hydrophilic receptor-like domain that
extends into the intermembrane space [3,9–11,54].
Tim23’s hydrophilic domain incorporates a high-affinity binding site for presequence-containing precursor
proteins, continuing the chain of precursor recognition
events that begins with the outer membrane TOM
receptors [9,54].
On its way to the matrix, a presequence-containing
preprotein makes contact with several binding sites
contributed by Tom20, Tom22 (cytosolic and intermembrane space domains), Tom5, Tom40 and then
finally Tim23 (Figures 3 and 4). This chain of events
was previously coined the ‘acid chain hypothesis’
because of the ionic nature of the interactions thought
to be responsible for preprotein recognition and
binding [54,55]. But as hydrophobic interactions have
also been shown to be involved, the pathway has
recently been renamed the ‘binding chain hypothesis’
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Precursor
protein
Tim50
+
Tim23
17
∆ψ
Tim44
ATP
–
IMS
IM
Matrix
mtHs
p70
MPP
Mg
+
+
+
e1
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Figure 4. The TIM23 translocase of the mitochondrial inner
membrane.
The TIM23 translocase consists of the membrane proteins
Tim23, Tim17 and Tim50, and peripherally associated Tim44
which provides a membrane anchor for mtHsp70 and its cofactor Mge1. Precursor proteins are received from the TOM
complex via binding to Tim50 and the intermembrane space
domain of Tim23. They are then translocated through an
aqueous pore formed by the membrane domain of Tim23 and
possibly Tim17. The membrane potential supports the transport
of presequences through the channel. The energy derived from
ATP hydrolysis by mtHsp70 drives protein translocation to
completion, a process dependent on Tim44 and Mge1. Finally
presequences are removed by the mitochondrial processing
peptidase (MPP).
to cover all the various non-covalent interactions that
may actually be involved [28]. The driving force for
movement along the pathway is believed to be, first
the increasing affinity of the binding sites as the presequence-containing precursor moves along the
import pathway, and then the pulling force of the
electrical membrane potential [8,54].
The carboxy-terminal domain of Tim23 is embedded in the membrane, forming a pore as determined
by in vitro reconstitution studies with the purified
protein [10]. The properties of this pore reflect those
expected for a presequence translocase, including
cation-selectivity and sensitivity to presequences. The
relatively narrow pore diameter of the Tim23 channel
(~13 Å), which can accommodate one α-helical chain,
may be necessary to prevent a major leakage of ions
across this energy-transducing membrane during preprotein transport. Tim23’s amino-terminal domain
influences the presequence sensitivity of the channel
and its selectivity filter [10]. Thus, interdomain communication is probably critical for adjusting the
translocation channel properties in response to an
incoming presequence. Tim23 is tightly associated
with Tim17, an integral membrane protein with
sequence and topological similarities to Tim23’s
membrane domain [3,7,11,56,57]. While Tim17 is
intimately linked to the transport of presequencecontaining precursors, its precise function continues
to mystify.
Recently, it was reported that the extreme aminoterminal region of Tim23 also spans the outer membrane [58]. This could effectively bring the outer and
inner membranes into close contact to promote efficient import. The missing link, however, is evidence
for a direct contact between the TIM23 complex and
the TOM machinery. With the recent discovery of a
new translocation component, Tim50, we may be
coming closer to understanding the true nature of
TIM–TOM associations in organello.
The discovery of Tim50 came several years after the
last novel member of the TIM23 translocase had been
identified, and it had been thought that perhaps all the
activities of the complex were performed by known
components, even though there were indications that
addditional Tims exist [59–61]. But last year, two different biochemical strategies [62,63] brought to light
Tim50, an essential and highly conserved component
of the TIM23 translocase. In one approach [62], a tag
was placed on the amino-terminal domain of Tim23 in
vivo, and the intact translocase was isolated by affinity
chromatography after mitochondrial solubilisation in a
mild detergent. Isolated proteins were identified by
mass spectrometry, revealing Tim50. In the second
approach [63], a preprotein was arrested during import
into mitochondria. Site-specific photocrosslinking was
performed and cross-linked products were affinity
purified via a tag on the preprotein. Identification of
Tim50 forming part of the cross-link product was again
achieved by mass spectrometry.
Tim50 is an integral membrane protein with a large
carboxy-terminal domain protruding into the intermembrane space [62,63]. Selective depletion of Tim50
from yeast cells revealed that Tim50 plays a crucial
role in the import of presequence-containing precursor
proteins destined for the mitochondrial matrix [62,63],
but Tim50 depletion had only a mild effect on the
import of precursors with additional inner membrane
sorting signals [62]. Precisely how Tim50 influences the
presequence pathway has yet to be determined, but
there are some tantalising possibilities.
Tim50 provides a functional link between the TOM
machinery and the Tim23 channel. A key feature of
Tim50 is that it binds to the part of Tim23’s aminoterminal domain exposed in the intermembrane space,
and so it can guide precursor proteins directly to the
channel protein. Tim50 also comes into direct contact
with precursor proteins during their import. Tim50 may
have chaperone-like properties and assist the classical matrix–targeted precursor proteins in the transfer
across the intermembrane space and into the inner
membrane channel.
Once precursors emerge in the mitochondrial matrix,
their amino-terminal presequences are generally
cleaved off by the dimeric mitochondrial processing
peptidase, a zinc-dependent metalloendopeptidase
[64,65]. The high-resolution structure of mitochondrial
processing peptidase surprisingly showed that the presequence is in an extended conformation when bound
to the peptidase, allowing access to the polypeptide
backbone [66], as opposed to the helical conformation
of the presequence when bound to the receptor
Tom20 [23]. This indicates that conformational
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Figure 5. Models of the action of
mtHsp70 in protein translocation.
(A) Pulling: mtHsp70 associated with the
import machinery via Tim44 binds the
incoming precursor protein and then
undergoes a conformational change as a
consequence of ATP hydrolysis which
draws the protein further into the matrix.
(B) Trapping: multiple mtHsp70s bind to
the precursor protein as it emerges in the
matrix and prevent its backsliding in the
translocation channel, providing favourable
conditions for productive import.
A Pulling
B Trapping
TOM
OM
+
∆ψ
TIM
23
TOM
IMS
IM
+
∆ψ
TIM
23
–
–
mtHsp70
mtHsp70
ATP
ATP
+
+
+
+
+
+
Current Biology
changes within the presequence itself are a way in
which recognition by different translocation components can be accommodated.
Some precursors are additionally processed by a
second matrix peptidase, mitochondrial intermediate
peptidase, which removes an octapeptide behind the
mitochondrial processing peptidase cleavage site [64].
A further processing peptidase, inner membrane peptidase, has its active site on the intermembrane space
side. Both inner membrane peptidase subunits, Imp1
and Imp2, are homologous to the bacterial leader peptidase [67], and they remove the sorting sequences of
some proteins that are directed to the inner membrane or intermembrane space [67,68]. Recently it was
shown that, upon cleavage by mitochondrial processing peptidase, presequence peptides can be
degraded by a novel mitochondrial zinc metalloendoprotease, presequence protease [69].
Energetics of Import of Precursor Proteins with a
Presequence
Two sources of energy are required for the translocation of precursor proteins across the mitochondrial
inner membrane. The first to come into effect is the
∆ψ), which plays two roles in
membrane potential (∆
precursor import. It directly stimulates the channel
protein Tim23 [9,10] and imparts an electrophoretic
effect on presequences [8,53]. Most likely, the
positively charged presequences are forced through
the translocation channel as a result of an electrophoretic effect of ∆ψ (negative on the matrix side)
[8]. Thus it is not surprising that the magnitude of ∆ψ
differentially influences the import efficiency of precursor proteins that vary considerably in the composition of their presequence. Interestingly, the membrane
potential, by pulling upon the presequence, can even
support the unfolding of precursor domains situated
outside of mitochondria [70].
The second source of energy comes from matrix ATP.
Completion of preprotein import into the mitochondrial
matrix absolutely requires the ATP-dependent action of
the molecular chaperone mtHsp70. Tim44, an adaptor
protein located at the TIM23 translocase, is capable of
switching the folding function of mtHsp70 to an essential role in protein translocation. Through binding to
mtHsp70, Tim44 effectively localises a fraction of the
total population of matrix mtHsp70 at the site of protein
translocation [71–74]. The domains of mtHsp70 which
are important for its folding function — an amino-terminal ATPase domain and a peptide-binding domain —
are likewise essential for the translocation process.
Positioned at the outlet of the import channel, mtHsp70
binds to both the presequence and mature segments of
precursor proteins, which are presented in an unfolded
state as they emerge from the translocation channel
[75]. The energy derived from ATP hydrolysis by
mtHsp70 drives protein translocation to completion. The
co-chaperone Mge1 promotes the exchange of
nucleotide from mtHsp70 after hydrolysis of ATP so that
further reaction cycles may occur.
One of the most hotly debated topics in mitochondrial protein import has been the mechanism by which
mtHsp70 action supports protein translocation. It is
clear the precursor protein must be in an unfolded,
linear conformation in order to pass through both the
outer and inner membrane translocation channels
[10,50,76]. But as translocation can occur post-translationally, some precursor proteins can first form
folded domains. It is clear that mtHsp70 promotes the
unfolding of these folded domains when the amino-terminal segment of the precursor protein is long enough
to reach the matrix, but here the controversy begins.
It has been suggested that mtHsp70 actively pulls
on translocating precursor proteins to facilitate the
unfolding of protein domains on the outside of a mitochondrion, promoting active transport of precursor
proteins into the mitochondrial matrix [13,77–79]. It is
believed that an ATP-induced conformational change
in mtHsp70 generates a pulling force on the preprotein, levering it into the matrix. This is the essence of
the motor (pulling) model, one of two ideas describing
mtHsp70 action (Figure 5). The alternative Brownian
ratchet (trapping) model posits that mtHsp70 merely
binds to the polypeptide chain, providing a bulky
obstruction that effectively prevents the backsliding of
the precursor protein, which moves by Brownian
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Stage I
Hsp70
Hsp90
Precursor
Stage II
Tom
70
Cytosol
ATP
TOM
OM
IMS
IM
Matrix
+
∆ψ
–
Stage III
Tim9-10
TIM
22
Stage IV
no ATP
Stage V
Figure 6. Import of carrier proteins into
the inner membrane.
Stage I: the newly synthesised precursor
protein binds to molecular chaperones
which deliver the precursor to the translocation machinery. Stage II: the precursor binds to the receptor Tom70. Release
from the receptor is an ATP-dependent
process that allows the precursor to
transfer to the TOM complex. Stage III:
the precursor passes through the translocation channels as hairpin loops making
contact with the Tim9–Tim10 complex of
the intermembrane space. Stage IV: the
precursor inserts into the inner membrane
in a membrane potential-dependent
manner, assisted by the TIM22 complex.
Stage V: the precursor assembles into a
functional carrier dimer in the inner membrane.
Current Biology
motion (Figure 5) [80–82]. In the original trapping
model, folded cytosolic domains were envisaged as
unfolding spontaneously to completion as a result of
mtHsp70 binding (trapping) in the matrix. The model
was modified later to account for the interaction of
mtHsp70 with Tim44 at the membrane (targeted
Brownian ratchet).
Which of these two seemingly contrasting ideas,
pulling or trapping, is correct? As with many controversies, each model probably addresses different
characteristics of the same underlying mechanism.
Indeed, in light of recent findings one unifying model
appears to be emerging which encompasses aspects
of both [78,82,83]. A passive trapping mechanism by
mtHsp70 is sufficient for the import of precursor proteins that are loosely folded or have completely
unfolded spontaneously [84]. Huang et al. [85] showed
that the mitochondrial import machinery can promote
active unfolding of precursor proteins by unravelling
folded domains from their amino terminus. Conformational changes of mtHsp70 (pulling) would promote
import by capturing spontaneous small unfolding fluctuations — thus preventing local refolding — and
provide an additional input of energy to overcome the
activation barrier of unfolding. Indeed it has been
shown that for folded precursor domains, but not for
loosely folded preproteins, efficient mtHsp70–Tim44
interaction is required for import [79,84], indicating
that a pulling mechanism is needed in the case of
tightly folded proteins. Thus a fully active mitochondrial import motor is likely to act by using a combination
of both passive trapping and active pulling.
The Carrier Import Pathway
The ADP/ATP carrier is one of the most abundant
mitochondrial proteins. It is a member of a large family
of related proteins responsible for transport of
numerous metabolites across the inner mitochondrial
membrane. With respect to protein trafficking, the
most notable features of this protein family are the lack
of a presequence for targeting, and the highly
hydrophobic nature of the six transmembrane spans in
each subunit. While the transport of carrier proteins
through the cytosol and across the outer membrane is
facilitated co-operatively by molecular chaperones and
the TOM complex, as outlined in previous sections,
their transport to the inner membrane depends entirely
on a specialised translocase, the TIM22 complex
(Figure 6).
When a carrier protein spans the TOM complex
through the translocation pore formed by Tom40, it is
still associated with the primary receptors Tom70 and
Tom20 [45]. It is at this point that carrier proteins make
contact with the first members of the TIM22 translocation machinery and thus diverge from the pathway
taken by proteins with a presequence [18,19]. The
carrier precursor interacts with a soluble 70 kDa heterooligomeric complex consisting of the essential subunits
Tim9 and Tim10 [18,19,86,87]. This interaction occurs in
a membrane potential-independent manner and is necessary to facilitate the release of the carrier from the
primary receptors and hence permit subsequent movement of the protein across the outer membrane into the
intermembrane space [18,19,45,88].
The purified Tim9–Tim10 complex has been shown
to bind to the hydrophobic transmembrane spans of
carrier proteins, rather than matrix exposed loops, and
so it may act in a chaperone-like manner to prevent
aggregation of these proteins when they are exposed
to the aqueous intermembrane space [89]. But a
carrier translocation intermediate entirely associated
with the Tim9–Tim10 complex alone may not exist at
any point in the translocation pathway as the translocating carrier has been found to make contact with
the inner membrane while still associated with the
outer membrane TOM machinery [90]. Thus transport
from the outer membrane to the inner membrane
occurs in a stepwise manner.
Contact of translocating carriers with the inner
membrane is facilitated by the membrane-associated
components of the TIM22 complex. This large complex
consists of three integral membrane proteins, Tim22,
Tim54 and Tim18, and a small percentage of peripherally associated Tim9 and Tim10 subunits as well as
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Table 1. Mitochondrial export machinery.
Component
Location and orientation
Proposed function
References
Oxa1
Integral inner membrane protein,
Five TM segments, Nout–Cin
General insertion complex for export of mitochondrial
and nuclear-encoded precursor proteins. Functions
independently of Mba1
[105,107,109–114]
Mba1
Peripherally associated with
inner membrane from matrix side
Required for the efficient membrane insertion of both
mitochondrial and nuclear-encoded precursor proteins.
A putative receptor that overlaps in substrate
specificity with Oxa1
[105,114]
Cox18
Integral inner membrane protein
C-terminal export of cytochrome c oxidase
subunit 2. Interaction partner of Mss2 and Pnt1
[116]
Pnt1
Integral inner membrane,
Two TM segments, Nin–Cin
C-terminal export of cytochrome c oxidase subunit 2
[115]
Mss2
Peripherally associated with inner
membrane from matrix side
C-terminal export of cytochrome c oxidase subunit 2
[117]
TM, transmembrane; N, amino terminus; C, carboxyl terminus; in, facing into matrix; out, facing into intermembrane space; Oxa1, oxidase
assembly; Mba1, multi-copy bypass of AFG3; Cox18, cytochrome c oxidase subunit 18; Pnt1, pentamidine resistance protein.
another member of the Tim9/Tim10 protein family,
Tim12 [16–19,21,86,87,91,92]. The mechanism by which
these small Tims assist the translocation process at the
inner membrane is far from understood, but their
function is essential for insertion of hydrophobic proteins at the TIM22 complex [18,19,93]. While Tim22
forms the insertion pores (see below), the roles of
Tim54 and Tim18 are not yet known, although Tim54 is
required to maintain the structural integrity of the TIM22
complex, which remains unassembled in its absence
[17,20].
The insertion of membrane proteins facilitated by the
TIM22 complex is absolutely dependent on the membrane potential. When there is no membrane potential,
the carrier protein accumulates in the intermembrane
space associated with the soluble Tim9–Tim10 complex
and also weakly associates with the membrane associated TIM22 complex [16,18,21,90]. But under conditions
of moderate to low membrane potential, the carrier
protein arrests at the TIM22 membrane complex (stage
IV) (Figure 6) [21]. It is only when the membrane potential is high that translocation proceeds to facilitate the
completion of membrane insertion followed by the
assembly of monomers to a functional dimer of carrier
proteins (stage V) [21].
The central player in this process is the essential
protein Tim22. Like Tim17 and the carboxy-terminal
domain of Tim23, Tim22 has four predicted membrane
spanning domains, and it exhibits sequence similarity
to these other proteins [16]. In vitro reconstitution of
purified Tim22 showed that it forms a cation-selective
channel activated by both membrane potential and a
targeting signal [20]. Greatest stimulation of the
channel is achieved with high membrane potential in
the presence of a carrier signal peptide [20], conditions
that promote efficient import in intact mitochondria
[21]. This was the first evidence that the translocation
of these hydrophobic proteins requires a translocation
pore for their insertion into the inner membrane, rather
than direct insertion into the lipid phase assisted by
translocation components.
Electron microscopy of the purified TIM22 complex
revealed two stain-filled pits that looked likely to
represent import pores. The cross section of each pit
∼16 Å) is consistent with the pore size determined for
(∼
purified Tim22 [20,21]. Analysis of the purified TIM22
complex by electrophysiology showed that it indeed
functions as a twin-pore translocase. A high membrane potential and an internal signal peptide together
stimulate gating transitions of one pore in the complex
while closing the second pore, indicating coordinated
action and regulation of the pores. Thus, the two pores
do not function as independent entities, but cooperate
in the insertion of precursor proteins that contain multiple hydrophobic segments [21]. Under physiological
conditions, the membrane potential most likely promotes membrane insertion through an electrophoretic
effect on positively charged regions of the precursor,
and also through direct activation of the channel.
In addition to carrier proteins, other polytopic
membrane proteins with internal targeting signals also
use the TIM22 translocation machinery for their
import. In the case of Tim23, import across the intermembrane space takes a slightly different route. Tim8
and Tim13, non-essential homologs of Tim9 and
Tim10, assist the transport of Tim23 across the intermembrane space, particularly under conditions of low
membrane potential [94–97]. The domains of Tim23 to
which the Tim8–Tim13 complex binds requires clarification, as independent reports are not completely
consistent [96-98]. The Tim9–Tim10 complex may also
play a role in the import of Tim23 [97]. Although Tim8
is non-essential in yeast, mutation of its homologue in
humans is responsible for the development of the
severe genetically inherited disease referred to as
deafness/dystonia syndrome [94,99,100].
The Export Machinery
Another major protein transport pathway in mitochondria involves the export of proteins from the matrix into
the inner membrane. Proteins encoded by the mitochondrial genome and synthesised in the matrix, as
well as a fraction of nuclear-encoded precursor proteins, take this route. Nuclear-encoded proteins must
first be imported into the matrix via the TOM and TIM23
complexes before engaging the export machinery for
Review
R334
insertion into the inner membrane. In budding yeast,
eight proteins are encoded by the mitochondrial
genome, seven of which are integral membrane components of respiratory chain complexes.
The mechanism of protein export from the matrix to
the inner membrane is poorly understood. It has
become increasingly evident that membrane insertion
from the matrix side is not a spontaneous event, but
requires the assistance of specialised translocation
components. The first component found to be involved
in the biogenesis of mitochondrial respiratory chain
complexes was Oxa1 (oxidase assembly 1) [101–103].
In fact, Oxa1 is a member of an evolutionary conserved
protein family with homologs in the inner membrane of
bacteria (YidC) and the thylakoid membrane of chloroplasts (Alb3) [104,105]. Each family member is known
to play a role in the membrane insertion of proteins
[105,106].
Oxa1 is a nuclear-encoded integral protein of the
inner membrane [107,108]. Isolation from Neurospora
crassa indicated that at least the stable core of the
translocase consists only of Oxa1, possibly as a
tetramer [109]. The translocase is believed to function
as a general membrane insertion machinery, as it is
not only required for the insertion of membrane
proteins that undergo amino-terminal tail export, but
also assists the import of other polytopic proteins in
which the amino terminus is retained in the matrix
[105,110–113]. It comes into direct contact with a
translocating precursor [113], but so far there is no
evidence to suggest that the translocase forms a pore
for membrane insertion.
When Oxa1 function is compromised in yeast cells,
the export of proteins is not completely blocked but
rather the severity of the export impairment appears
to be precursor-dependent [105]. This suggests that
backup import pathways maintain protein export function in mitochondria. Indeed, additional proteins have
recently been implicated in export pathways by
genetic and biochemical evidence [114–117]. Table 1
summarises all proteins found, so far, to be involved
in export pathways. The peripheral inner membrane
protein Mba1 is involved in amino-tail export and can
largely, but not completely, compensate for loss of
Oxa1 [114]. The proteins Cox18, Pnt1 and Mss2, not
parts of the Oxa1 translocase, together may constitute
a second translocation complex [115,116]. Cox18, a
weak homolog of Oxa1, has been implicated in
carboxy-tail export [116]. The composition and mode
of action of the export machinery is far from solved
and thus should provide rapid developments in mitochondrial protein transport in the near future.
Perspectives
The basic principles currently defining precursor protein
import into mitochondria are generally agreed upon. For
example, there is a need for intrinsic targeting signals
for protein recognition, molecular chaperones and
receptors to mediate protein–protein interactions,
aqueous channels to support transport across membranes and systems for generating protein movement
from stored chemical or electrical energy. What is currently difficult to fathom is the way in which the import
machinery collectively is able to sort the hundreds of
different proteins to the correct compartment. Are the
translocation channels highly regulated, capable of
changing their interior surface in response to incoming
precursors, thus leading to differential compartment
sorting? Perhaps there are as yet to be discovered
‘sorting proteins’ or complexes. Protein sorting most
likely results from a network of rapid and subtle
changes in translocation components in response to
targeting and sorting signals.
Furthermore, the molecular mechanisms by which
translocation components such as molecular chaperones, receptors and channel-forming proteins mediate
import are only partially understood. How are so many
different proteins recognised by so few import
components? Additional high resolution structures will
certainly help our understanding of these processes.
Finally further work is required to understand how
proteins are assembled into multisubunit complexes.
In some cases, specialised assembly components are
required to assist and it seems that large complexes
are structurally dynamic in order that new components can be incorporated.
Acknowledgements
We thank David A. Dougan for comments on the manuscript. Work of the authors’ laboratory was supported
by the Deutsche Forschungsgemeinschaft, SFB 388
and the Fonds der Chemischen Industrie/BMBF.
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