Protein Import to Mito Chondrai

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Mechanisms of Protein Import intoMitochondria

Review

Kaye N. Truscott, Katrin Brandner and Nikolaus

Pfanner

Apart from a handful of proteins encoded by the mito-

chondrial genome, most proteins residing in this

organelle are nuclear-encoded and synthesised in the

cytosol. Thus, delivery of proteins to their final desti-

nation depends on a network of specialised import

components that form at least four main translocation

complexes. The import machinery ensures that pro-

teins earmarked for the mitochondrion are recog-

nised and delivered to the organelle, transported

across membranes, sorted to the correct compart-

ment and assisted in overcoming energetic barriers.

Introduction

Protein trafficking is a vital cellular process in whichproteins are transported from their site of synthesis tolocations within cells where they function. In eukaryotesthese locations are numerous, as cells are organisedinto several compartments formed from single or multi-ple membrane boundaries. Nuclear-encoded proteins,which are synthesised in the cytosol, are transportedfor example to organelles such as mitochondria, theendoplasmic reticulum (ER), peroxisomes, the nucleusand chloroplasts. A small number of proteins are

encoded by the genomes of mitochondria and chloro-plasts, and these add to the complexity of protein traf-ficking pathways. Here we shall discuss recentadvances in our understanding of the mechanisms ofimport of nuclear-encoded proteins into mitochondria,and also the export pathway to the inner membranethat is used by mitochondrially encoded proteins.

Because of the way the mitochondrion is dividedinto four subcompartments — the two aqueouschambers (intermembrane space and matrix) and twomembranes (the outer and inner membranes) — thereare multiple pathways for protein translocation into thisorganelle. Most of the ~1,000 mitochondrial proteinsare encoded by the nuclear genome, synthesised in

the cytosol on free ribosomes as precursor proteinsand then transported into or across mitochondrialmembranes with the aid of a handful of distinct com-plexes (Figure 1). Targeting information resides withinthe protein itself; this may take the form of an amino-terminal extension or ‘presequence’, though for manymembrane proteins it is contained within the matureprotein [1–4]. The transfer of mitochondrial proteinsfrom their site of synthesis in the cytosol to the surfaceof the mitochondrial outer membrane, where translo-cation begins, mainly occurs in a post-translational

manner — that is, the fully synthesized protein con-tacts the mitochondrial import machinery [1–3,5,6].However, a co-translational mechanism of transloca-tion 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 onlyknown entry point into mitochondria for nuclear-encodedproteins. After crossing the outer membrane at the TOMcomplex, precursor proteins segregate, as there are twostructurally and functionally distinct ‘translocases of theinner membrane’ (TIMs): the TIM23 and TIM22 com-plexes, each of which guides the import of subsets ofproteins to internal compartments of mitochondria.

All proteins with an amino-terminal presequence areimported via the TIM23 translocase [7]. The transportof presequences across the membrane occursthrough an aqueous pore, while the membrane poten-tial across the inner membrane provides a drivingforce for this energy-requiring process [8–11]. In addi-tion, the ATP-dependent action of matrix heat shockprotein 70 (mtHsp70), together with its helper proteinsTim44 and Mge1, drives to completion the unidirec-tional movement of precursor proteins into the mito-chondrial matrix [12–15].

Some inner membrane proteins, which only containinternal targeting signals, are imported with the assis-tance of the pore-forming TIM22 complex in a mem-brane potential-dependent manner [16–21]. The

mitochondrial export machinery is responsible for theinsertion of proteins into the inner membrane from thematrix (Figure 1). The action of this machinery resem-bles protein translocation processes in bacteria.

Targeting Signals

Two types of mitochondrial targeting signal cancurrently be described: presequences and internalsignals. Presequences direct precursor proteins viathe TOM complex to the TIM23 translocase, wherethey are sorted to the matrix, inner membrane or intermembrane space (Figure 2). One would expectpresequences to have a consensus amino acidsequence, but rather they consist of a handful of

subtle distinguishing properties at the level of theprimary and secondary structure. They generally havea high content of basic, hydrophobic and hydroxy-lated amino acids, and a length of about 10–80 aminoacids [22]. An amphipathic α helix with one positiveand one hydrophobic face is another common featurethat is important for receptor recognition [1–3,23].

Once they are exposed to the matrix, prese-quences are often removed by a processing pepti-dase, as they are not necessary for protein function.In comparison, proteins which only contain internaltargeting signals are directed to either the inner mem-brane, intermembrane space or outer membrane,depending on the nature of the signal — which, todate, has not been clearly defined (Figure 2). The

Current Biology, Vol. 13, R326–R337, April 15, 2003, ©2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0960-9822(03)00239-2

Institut für Biochemie und Molekularbiologie, UniversitätFreiburg, Hermann-Herder-Straße 7, D-79104 Freiburg,Germany. E-mail: [email protected]



internal targeting information, best characterised for members of the carrier family of the mitochondrialinner membrane, is hidden in the mature proteinamongst amino acid residues important for foldingand function [2–4]. The targeting information seems tobe spread throughout the length of the protein [4,24].

The Importance of Surface Receptors for Outer

Membrane Translocation

Three proteins of the mitochondrial outer membraneTOM machinery act as receptors for the recognition oftargeting signals: Tom70, Tom20 and Tom22 (Figure3). Each has a large cytosolic domain anchored to themembrane, with a single transmembrane span. Tom22also has a small, functional carboxy-terminal domainwhich is exposed to the intermembrane space. Tom22forms part of a very stable general import pore (GIP)complex of the outer membrane, which additionallycontains the pore-forming protein Tom40 and acces-sory 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 withprecursor proteins, but show preferences for different

precursor types. They are both only loosely associ-ated with the GIP complex [25,26,28,29].Tom70 is the major receptor for precursor proteins

that contain internal targeting information, such asmembers of the metabolite carriers of the inner mito-chondrial membrane [1–3]. It binds to multiple seg-ments within the carrier proteins, though an aminoacid recognition motif has not been identified [4,30].The carrier proteins consist of three related modules,each containing two transmembrane domains con-nected by a hydrophilic loop, such that a carrier monomer contains six transmembrane domains. Eachrepeat module of a carrier contributes targeting infor-mation, so that a carrier precursor binds to severalmolecules of the Tom70 receptor simultaneously [24].

Interestingly, it has recently been shown that Tom70is not only a receptor for precursor proteins, but alsoa docking point for cytosolic chaperones directlyinvolved in the import pathway [31]. The transfer ofnewly synthesised Tom70-dependent precursor pro-teins from the cytosol to the receptor can now bedescribed. After synthesis, the precursor proteinforms 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 timeHsp90 has been implicated in a mitochondrial importpathway. Previous studies indicated that Hsp70 andother cytosolic factors such as mitochondrial importstimulating factor (MSF) [33-36] can also stimulateprotein import into mitochondria, although it remainsopen if MSF plays a role in protein import in vivo [6]. An association with chaperones probably preventsaggregation of the precursor and permits presentationof the protein in a conformation conducive for recep-tor recognition.

The cargo precursor protein is then transferred tothe Tom70 receptor, an event that requires thedocking of Hsp70 to the receptor [31]. Binding of

Hsp90 and Hsp70 to Tom70 is mediated by a spe-cialised tetratricopeptide repeat (TPR) domain inTom70. The determinants for selective chaperonedocking are apparently contained within Tom70. Ashas been previously described, the transport of carri-ers to subsequent import stages requires hydrolysis of ATP [32]: the authors propose that ATPase cycling,most likely by the chaperones, leads to release of theprecursor proteins from Tom70.

The main function of Tom20 is to bind precursor proteins with presequences. The hydrophobic face ofthe helical presequence is predicted to interact withTom20 [30]. The structure of a presequence in acomplex with a soluble portion of Tom20 clearlyshows that the helical presequence indeed fits into a

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Figure 1. Translocation complexes ofmitochondria.

Four types of translocation complex arecurrently known in mitochondria, three for the import of precursor proteins into inter-nal compartments — TOM, TIM23 and

TIM22 — and one for the export of pre-cursor proteins from the matrix into theinner membrane (Export). Precursor pro-teins with either an amino-terminal pre-sequence or internal targeting signalsbind to a specific receptor (R), whichdirects their import via a general importpore (GIP) of the translocase of the outer membrane (TOM). Precursor proteins witha presequence are directed to the TIM23complex for import across the inner mem-brane, a process which requires the mem-brane potential ( ∆ψ ) and the ATP-drivenaction of mtHsp70. If they are destined for the inner membrane, nuclear-encodedprecursor proteins with only internal tar-geting signals are guided to the TIM22

complex by Tim factors of the intermem-brane space. Membrane insertion at theTIM22 complex is dependent on the membrane potential. Mitochondrially encoded proteins that are synthesised in the matrix andsome imported proteins are exported to the inner membrane with the assistance of the export machinery. OM, outer membrane; IMS,intermembrane space; IM, inner membrane.

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groove formed by Tom20 in which hydrophobic inter-actions predominantly occupy the interface [23].Tom22 is the secondary receptor for mitochondrialprotein import, accepting both precursor types fromthe primary receptors but mainly precursors with apresequence [37]. The handing over of precursor pro-teins from the primary receptors to Tom22 is possiblypromoted by interactions between their respectivecytosolic domains [27]. While we do not yet know thestructure of Tom22, biochemical evidence indicates

that presequences bind to Tom22 via ionic interac-tions probably formed between the positively chargedface of the helical presequence and a negativelycharged surface on Tom22 [30,38].

Signal recognition does not stop with the major receptors. A receptor-like protein called Tom5 acts atthe tertiary level influencing the import of precursorswith both presequences and internal targeting signals[39]. The proximity of Tom5 at the entrance of thetranslocation pore allows perfect positioning of theincoming precursor for entry into the translocationchannel of the outer membrane. Targeting signalrecognition thus occurs at two to three levels to bringmost mitochondrial proteins to the translocation pore

(Figure 3).Translocation at the Outermost Membrane

Whether proteins traverse membranes completelyor become embedded in them, aqueous protein-conducting channels are generally required. Inmitochondria, the cation–selective GIP complex of theouter membrane is formed from the essential integralmembrane protein Tom40 [40,41]. Unlike the singleaqueous pore through the ER translocon, the GIPcomplex contains two to three pores, most likelycompletely constructed from Tom40 but held in thecomplex through interactions with Tom22 [26,27,42].In yeast, devoid of Tom22, the GIP complex fails toassemble to the full-sized ~400 kDa core complex,

and the remaining components are found in a 100 kDacomplex consisting of Tom40 and small proteinsTom5, Tom6 and Tom7 [27].

Several proteins of different types and locations havebeen found in association with the TOM complex or tobe inhibited in their import when the Tom40 channel isblocked, indicating the broad capacity of this translo-case for transporting all types of nuclear-encoded mito-chondrial proteins [7,32,43–46]. Tom6 and Tom7 areintegral membrane proteins that play a structural role in

the organisation and stability of the TOM complex[47,48]. Tom6 facilitates interaction of the single TOMchannel complex of 100 kDa with Tom22 to form the fullsized GIP complex [25,27,49]. Tom7 is at least partiallyantagonistic to Tom6. Tom7 favours dissociation of theTOM complex and also influences sorting of proteins tothe outer membrane [48,49].

The multiple pore arrangement of the GIP complexis intriguing, but why has it evolved in this way whenother translocases have only a single pore? It mayrelate to the fact that import into mitochondria occurspost-translationally: imported proteins are synthesisedin their entirety before any portion is translocated. For polytopic membrane proteins, a pair of transmem-

brane α helices separated by a hydrophilic loop (ahelical hairpin) may insert into one pore while asecond helical hairpin in the same protein inserts intoa second TOM pore, allowing simultaneous import ofmultiple domains. Such a mechanism may promoteefficient membrane insertion and subsequent assem-bly. An en bloc import mechanism would require con-siderable flexibility of the translocation complex inorder to allow either lateral movement of the proteininto the membrane or movement through the translo-case to the intermembrane space.

With respect to its size a single TOM pore is wideenough (~20 Å) to carry two closely packed trans-membrane α helices at the same time [26,41,50]. Atleast for the ADP/ATP carrier (AAC), import across the

Figure 2. Sorting of precursor proteins inmitochondria.

There are four compartments in mitochon-dria to which proteins are sorted. (A) Typi-cally a precursor protein with anamino-terminal presequence is directed

across the TOM and TIM23 machinery intothe matrix. When additional sorting signalsare present in the protein, sorting to theinner membrane or intermembrane spaceoccurs at the TIM23 complex [118,119].(B) Non-cleavable inner membrane proteinssuch as metabolic carriers are importedinto mitochondria via the TOM complexthen directed to the TIM22 complex for membrane insertion. (C) The import ofsome intermembrane space proteins onlydepends on the TOM machinery. (D) Like-wise outer membrane proteins utilise theTOM complex. (E) Sorting of the handful ofmitochondrially encoded proteins to theinner membrane is facilitated by exportcomplexes. Also some nuclear-encoded

proteins that are imported into the matrixare then sorted to the inner membrane bythe export machinery [105,120].

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inner membraneproteins

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outer membrane can occur in a partially folded statein which helical hairpins traverse the outer membrane[24,32]. In comparison, import into the ER transloconoccurs largely co-translationally, so that the translo-cation machinery is presented with polypeptide seg-ments step by step as they are synthesised. For polytopic membrane proteins, lateral release from theER translocon may occur simultaneously once all ofthe transmembrane segments have gathered in theone translocon pore [51]. This may be necessary for

correct intramolecular assembly. Indeed the activetranslocon is larger than the TOM pores, with anestimated diameter of 40–60 Å, and so could accom-modate multiple transmembrane segments [52].

Assembly of Translocation Components

All mitochondrial translocation components arenuclear-encoded. This means that they must also beimported and assembled into multisubunit membranecomplexes following synthesis in the cytosol on freeribosomes. Is a specialised machinery required for thisprocess, or do the standard pre-existing translocationcomponents suffice? At least for the essential pore-forming protein Tom40, the evidence indicates that a

combination of these possibilities leads to its correctimport and assembly [49].The Tom40 precursor is first recognised by the TOM

import machinery, as for any other precursor. It istargeted to the outer membrane mainly via recognitionby the receptors Tom20 and Tom22. It is then trans-ferred to a unique sorting and assembly complexwhich is distinct from the mature TOM complex [49].Upon release from this assembly complex, the Tom40precursor is found in a partial GIP complex in associa-tion with Tom6. Assembly to the mature GIP complexfrom this intermediate occurs upon incorporation ofTom22 and Tom7. The TOM components that join inthe late assembly of Tom40 to form the GIP complexalso occur as free endogenous components, indicating

that the mature complex is structurally dynamic andcan exchange its subunits. Tom7 also appears to playa role in the disassembly of the GIP complex.

The Import Pathway for Precursors with a

Presequence

All precursor proteins that have a presequence aretargeted to a specialised inner membrane transloca-tion machinery — the TIM23 complex (Figure 4). Thepresequence 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 com-ponents of the TIM23 complex and also responds to alarge electrical field at the inner membrane whichactively promotes import [7–9,53].

What molecular events occur when the prese-quence emerges in the intermembrane space? A keyplayer in the process is the multifunctional transloca-tion component Tim23. Tim23 is a pore-forming inte-gral protein of the inner membrane, which also has anamino-terminal hydrophilic receptor-like domain thatextends into the intermembrane space [3,9–11,54].Tim23’s hydrophilic domain incorporates a high-affin-

ity binding site for presequence-containing precursor proteins, continuing the chain of precursor recognitionevents that begins with the outer membrane TOMreceptors [9,54].

On its way to the matrix, a presequence-containingpreprotein makes contact with several binding sitescontributed by Tom20, Tom22 (cytosolic and inter-membrane space domains), Tom5, Tom40 and thenfinally Tim23 (Figures 3 and 4). This chain of eventswas previously coined the ‘acid chain hypothesis’because of the ionic nature of the interactions thoughtto be responsible for preprotein recognition andbinding [54,55]. But as hydrophobic interactions havealso been shown to be involved, the pathway hasrecently been renamed the ‘binding chain hypothesis’

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Figure 3. The translocase of themitochondrial outer membrane.

The TOM translocase consists of theprimary receptors Tom70 and Tom20, andthe GIP complex formed from Tom40,

Tom22, Tom5, Tom6 and Tom7. A precur-sor with internal targeting signals associ-ates with cytosolic chaperones Hsp70and Hsp90 in the cytosol. The precursor then binds to its main receptor Tom70when the chaperones also dock onto thisreceptor. In an ATP-dependent manner the precursor is transferred to Tom5 thensubsequently passes through the trans-location channel formed by Tom40. A backup receptor route via Tom20 andTom22 is also possible for this precursor type. Precursor proteins with prese-quences on the other hand bind first toTom20 and then are subsequently trans-ferred to Tom22, Tom5 and the Tom40translocation channel. In addition the pre-

sequence can bind to the carboxy-termi-nal domain of Tom22 prior to further import via the TIM23 complex.

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to cover all the various non-covalent interactions thatmay actually be involved [28]. The driving force for movement along the pathway is believed to be, firstthe increasing affinity of the binding sites as the pre-sequence-containing precursor moves along theimport pathway, and then the pulling force of theelectrical membrane potential [8,54].

The carboxy-terminal domain of Tim23 is embed-ded in the membrane, forming a pore as determinedby in vitro reconstitution studies with the purifiedprotein [10]. The properties of this pore reflect thoseexpected for a presequence translocase, includingcation-selectivity and sensitivity to presequences. Therelatively 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 ionsacross this energy-transducing membrane during pre-protein transport. Tim23’s amino-terminal domaininfluences the presequence sensitivity of the channeland its selectivity filter [10]. Thus, interdomain com-munication is probably critical for adjusting thetranslocation channel properties in response to anincoming presequence. Tim23 is tightly associatedwith Tim17, an integral membrane protein withsequence and topological similarities to Tim23’smembrane domain [3,7,11,56,57]. While Tim17 isintimately linked to the transport of presequence-containing precursors, its precise function continuesto mystify.

Recently, it was reported that the extreme amino-terminal region of Tim23 also spans the outer mem-brane [58]. This could effectively bring the outer andinner membranes into close contact to promote effi-cient import. The missing link, however, is evidence

for a direct contact between the TIM23 complex andthe TOM machinery. With the recent discovery of anew translocation component, Tim50, we may becoming closer to understanding the true nature ofTIM–TOM associations in organello.

The discovery of Tim50 came several years after thelast novel member of the TIM23 translocase had beenidentified, and it had been thought that perhaps all theactivities of the complex were performed by knowncomponents, even though there were indications thataddditional Tims exist [59–61]. But last year, two dif-ferent biochemical strategies [62,63] brought to lightTim50, an essential and highly conserved componentof the TIM23 translocase. In one approach [62], a tagwas placed on the amino-terminal domain of Tim23 in

vivo, and the intact translocase was isolated by affinitychromatography after mitochondrial solubilisation in amild detergent. Isolated proteins were identified bymass spectrometry, revealing Tim50. In the secondapproach [63], a preprotein was arrested during importinto mitochondria. Site-specific photocrosslinking wasperformed and cross-linked products were affinitypurified via a tag on the preprotein. Identification ofTim50 forming part of the cross-link product was againachieved by mass spectrometry.

Tim50 is an integral membrane protein with a largecarboxy-terminal domain protruding into the inter-membrane space [62,63]. Selective depletion of Tim50from 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 theimport of precursors with additional inner membranesorting signals [62]. Precisely how Tim50 influences thepresequence pathway has yet to be determined, butthere are some tantalising possibilities.

Tim50 provides a functional link between the TOMmachinery and the Tim23 channel. A key feature ofTim50 is that it binds to the part of Tim23’s amino-terminal domain exposed in the intermembrane space,and so it can guide precursor proteins directly to thechannel protein. Tim50 also comes into direct contactwith precursor proteins during their import. Tim50 may

have chaperone-like properties and assist the classi-cal 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 generallycleaved off by the dimeric mitochondrial processingpeptidase, a zinc-dependent metalloendopeptidase[64,65]. The high-resolution structure of mitochondrialprocessing peptidase surprisingly showed that the pre-sequence is in an extended conformation when boundto the peptidase, allowing access to the polypeptidebackbone [66], as opposed to the helical conformationof the presequence when bound to the receptor Tom20 [23]. This indicates that conformational

Figure 4. The TIM23 translocase of the mitochondrial inner membrane.

The TIM23 translocase consists of the membrane proteinsTim23, Tim17 and Tim50, and peripherally associated Tim44which provides a membrane anchor for mtHsp70 and its cofac-tor Mge1. Precursor proteins are received from the TOMcomplex via binding to Tim50 and the intermembrane spacedomain of Tim23. They are then translocated through anaqueous pore formed by the membrane domain of Tim23 andpossibly Tim17. The membrane potential supports the transportof presequences through the channel. The energy derived from

ATP hydrolysis by mtHsp70 drives protein translocation tocompletion, a process dependent on Tim44 and Mge1. Finallypresequences are removed by the mitochondrial processingpeptidase (MPP).

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changes within the presequence itself are a way inwhich recognition by different translocation compo-nents can be accommodated.

Some precursors are additionally processed by asecond matrix peptidase, mitochondrial intermediatepeptidase, which removes an octapeptide behind themitochondrial processing peptidase cleavage site [64]. A further processing peptidase, inner membrane pep-tidase, has its active site on the intermembrane spaceside. Both inner membrane peptidase subunits, Imp1and Imp2, are homologous to the bacterial leader pep-tidase [67], and they remove the sorting sequences ofsome proteins that are directed to the inner mem-brane or intermembrane space [67,68]. Recently it was

shown that, upon cleavage by mitochondrial process-ing peptidase, presequence peptides can bedegraded by a novel mitochondrial zinc metalloendo-protease, presequence protease [69].

Energetics of Import of Precursor Proteins with a

Presequence

Two sources of energy are required for the transloca-tion of precursor proteins across the mitochondrialinner membrane. The first to come into effect is themembrane potential ( ∆ψ ), which plays two roles inprecursor import. It directly stimulates the channelprotein Tim23 [9,10] and imparts an electrophoreticeffect on presequences [8,53]. Most likely, the

positively charged presequences are forced throughthe translocation channel as a result of an elec-trophoretic 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 pre-cursor proteins that vary considerably in the composi-tion of their presequence. Interestingly, the membranepotential, by pulling upon the presequence, can evensupport the unfolding of precursor domains situatedoutside of mitochondria [70].

The second source of energy comes from matrix ATP.Completion of preprotein import into the mitochondrialmatrix absolutely requires the ATP-dependent action ofthe molecular chaperone mtHsp70. Tim44, an adaptor protein located at the TIM23 translocase, is capable of

switching the folding function of mtHsp70 to an essen-tial role in protein translocation. Through binding tomtHsp70, Tim44 effectively localises a fraction of thetotal population of matrix mtHsp70 at the site of proteintranslocation [71–74]. The domains of mtHsp70 whichare important for its folding function — an amino-termi-nal ATPase domain and a peptide-binding domain —are likewise essential for the translocation process.Positioned at the outlet of the import channel, mtHsp70binds to both the presequence and mature segments ofprecursor proteins, which are presented in an unfoldedstate as they emerge from the translocation channel[75]. The energy derived from ATP hydrolysis bymtHsp70 drives protein translocation to completion. The

co-chaperone Mge1 promotes the exchange ofnucleotide from mtHsp70 after hydrolysis of ATP so thatfurther reaction cycles may occur.

One of the most hotly debated topics in mitochon-drial protein import has been the mechanism by whichmtHsp70 action supports protein translocation. It isclear the precursor protein must be in an unfolded,linear conformation in order to pass through both theouter and inner membrane translocation channels[10,50,76]. But as translocation can occur post-trans-lationally, some precursor proteins can first formfolded domains. It is clear that mtHsp70 promotes theunfolding of these folded domains when the amino-ter-minal segment of the precursor protein is long enough

to reach the matrix, but here the controversy begins.It has been suggested that mtHsp70 actively pullson translocating precursor proteins to facilitate theunfolding of protein domains on the outside of a mito-chondrion, promoting active transport of precursor proteins into the mitochondrial matrix [13,77–79]. It isbelieved that an ATP-induced conformational changein mtHsp70 generates a pulling force on the prepro-tein, levering it into the matrix. This is the essence ofthe motor (pulling) model, one of two ideas describingmtHsp70 action (Figure 5). The alternative Brownianratchet (trapping) model posits that mtHsp70 merelybinds to the polypeptide chain, providing a bulkyobstruction that effectively prevents the backsliding ofthe precursor protein, which moves by Brownian

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Figure 5. Models of the action ofmtHsp70 in protein translocation.

(A) Pulling: mtHsp70 associated with theimport machinery via Tim44 binds theincoming precursor protein and thenundergoes a conformational change as a

consequence of ATP hydrolysis whichdraws the protein further into the matrix.(B) Trapping: multiple mtHsp70s bind tothe precursor protein as it emerges in thematrix and prevent its backsliding in thetranslocation channel, providing favourableconditions for productive import.

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motion (Figure 5) [80–82]. In the original trappingmodel, folded cytosolic domains were envisaged asunfolding spontaneously to completion as a result ofmtHsp70 binding (trapping) in the matrix. The modelwas modified later to account for the interaction ofmtHsp70 with Tim44 at the membrane (targetedBrownian ratchet).

Which of these two seemingly contrasting ideas,pulling or trapping, is correct? As with many contro-versies, each model probably addresses differentcharacteristics of the same underlying mechanism.Indeed, in light of recent findings one unifying modelappears to be emerging which encompasses aspects

of both [78,82,83]. A passive trapping mechanism bymtHsp70 is sufficient for the import of precursor pro-teins that are loosely folded or have completelyunfolded spontaneously [84]. Huang et al. [85] showedthat the mitochondrial import machinery can promoteactive unfolding of precursor proteins by unravellingfolded domains from their amino terminus. Conforma-tional changes of mtHsp70 (pulling) would promoteimport by capturing spontaneous small unfolding fluc-tuations — thus preventing local refolding — andprovide an additional input of energy to overcome theactivation barrier of unfolding. Indeed it has beenshown that for folded precursor domains, but not for loosely folded preproteins, efficient mtHsp70–Tim44

interaction is required for import [79,84], indicatingthat a pulling mechanism is needed in the case oftightly folded proteins. Thus a fully active mitochondr-ial import motor is likely to act by using a combinationof both passive trapping and active pulling.

The Carrier Import Pathway

The ADP/ATP carrier is one of the most abundantmitochondrial proteins. It is a member of a large familyof related proteins responsible for transport ofnumerous metabolites across the inner mitochondrialmembrane. With respect to protein trafficking, themost notable features of this protein family are the lackof a presequence for targeting, and the highlyhydrophobic nature of the six transmembrane spans in

each subunit. While the transport of carrier proteinsthrough the cytosol and across the outer membrane isfacilitated co-operatively by molecular chaperones andthe TOM complex, as outlined in previous sections,their transport to the inner membrane depends entirelyon a specialised translocase, the TIM22 complex(Figure 6).

When a carrier protein spans the TOM complexthrough the translocation pore formed by Tom40, it isstill associated with the primary receptors Tom70 andTom20 [45]. It is at this point that carrier proteins makecontact with the first members of the TIM22 transloca-tion machinery and thus diverge from the pathway

taken by proteins with a presequence [18,19]. Thecarrier precursor interacts with a soluble 70 kDa hetero-oligomeric complex consisting of the essential subunitsTim9 and Tim10 [18,19,86,87]. This interaction occurs ina membrane potential-independent manner and is nec-essary to facilitate the release of the carrier from theprimary receptors and hence permit subsequent move-ment of the protein across the outer membrane into theintermembrane space [18,19,45,88].

The purified Tim9–Tim10 complex has been shownto bind to the hydrophobic transmembrane spans ofcarrier proteins, rather than matrix exposed loops, andso it may act in a chaperone-like manner to preventaggregation of these proteins when they are exposed

to the aqueous intermembrane space [89]. But acarrier translocation intermediate entirely associatedwith the Tim9–Tim10 complex alone may not exist atany point in the translocation pathway as the translo-cating carrier has been found to make contact withthe inner membrane while still associated with theouter membrane TOM machinery [90]. Thus transportfrom the outer membrane to the inner membraneoccurs in a stepwise manner.

Contact of translocating carriers with the inner membrane is facilitated by the membrane-associatedcomponents of the TIM22 complex. This large complexconsists of three integral membrane proteins, Tim22,Tim54 and Tim18, and a small percentage of peripher-ally associated Tim9 and Tim10 subunits as well as

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Figure 6. Import of carrier proteins intothe inner membrane.

Stage I: the newly synthesised precursor protein binds to molecular chaperoneswhich deliver the precursor to the trans-location machinery. Stage II: the precur-

sor binds to the receptor Tom70. Releasefrom the receptor is an ATP-dependentprocess that allows the precursor totransfer to the TOM complex. Stage III:the precursor passes through the trans-location channels as hairpin loops makingcontact with the Tim9–Tim10 complex ofthe intermembrane space. Stage IV: theprecursor inserts into the inner membranein a membrane potential-dependentmanner, assisted by the TIM22 complex.Stage V: the precursor assembles into afunctional carrier dimer in the inner mem-brane.

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IMS

IM

OM

ATP

∆ψ

+

–

Tim9-10

no ATP

Stage I

Stage III

Stage IV Stage V

Hsp70Hsp90

Tom70

TOM

Cytosol

Matrix

Stage IIPrecursor

TIM22



another member of the Tim9/Tim10 protein family,Tim12 [16–19,21,86,87,91,92]. The mechanism by whichthese small Tims assist the translocation process at theinner membrane is far from understood, but their function is essential for insertion of hydrophobic pro-teins at the TIM22 complex [18,19,93]. While Tim22forms the insertion pores (see below), the roles ofTim54 and Tim18 are not yet known, although Tim54 isrequired to maintain the structural integrity of the TIM22complex, which remains unassembled in its absence[17,20].

The insertion of membrane proteins facilitated by theTIM22 complex is absolutely dependent on the mem-brane potential. When there is no membrane potential,

the carrier protein accumulates in the intermembranespace associated with the soluble Tim9–Tim10 complexand also weakly associates with the membrane associ-ated TIM22 complex [16,18,21,90]. But under conditionsof moderate to low membrane potential, the carrier protein arrests at the TIM22 membrane complex (stageIV) (Figure 6) [21]. It is only when the membrane poten-tial is high that translocation proceeds to facilitate thecompletion of membrane insertion followed by theassembly of monomers to a functional dimer of carrier proteins (stage V) [21].

The central player in this process is the essentialprotein Tim22. Like Tim17 and the carboxy-terminaldomain of Tim23, Tim22 has four predicted membrane

spanning domains, and it exhibits sequence similarityto these other proteins [16]. In vitro reconstitution ofpurified Tim22 showed that it forms a cation-selectivechannel activated by both membrane potential and atargeting signal [20]. Greatest stimulation of thechannel is achieved with high membrane potential inthe presence of a carrier signal peptide [20], conditionsthat promote efficient import in intact mitochondria[21]. This was the first evidence that the translocationof these hydrophobic proteins requires a translocationpore for their insertion into the inner membrane, rather than direct insertion into the lipid phase assisted bytranslocation components.

Electron microscopy of the purified TIM22 complexrevealed 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 TIM22complex by electrophysiology showed that it indeedfunctions as a twin-pore translocase. A high mem-brane potential and an internal signal peptide together stimulate gating transitions of one pore in the complexwhile closing the second pore, indicating coordinatedaction and regulation of the pores. Thus, the two poresdo not function as independent entities, but cooperatein the insertion of precursor proteins that contain mul-tiple hydrophobic segments [21]. Under physiologicalconditions, the membrane potential most likely pro-motes 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 polytopicmembrane proteins with internal targeting signals alsouse the TIM22 translocation machinery for their import. In the case of Tim23, import across the inter-membrane space takes a slightly different route. Tim8and Tim13, non-essential homologs of Tim9 andTim10, assist the transport of Tim23 across the inter-membrane space, particularly under conditions of lowmembrane potential [94–97]. The domains of Tim23 towhich the Tim8–Tim13 complex binds requires clarifi-cation, as independent reports are not completelyconsistent [96-98]. The Tim9–Tim10 complex may also

play a role in the import of Tim23 [97]. Although Tim8is non-essential in yeast, mutation of its homologue inhumans is responsible for the development of thesevere genetically inherited disease referred to asdeafness/dystonia syndrome [94,99,100].

The Export Machinery

Another major protein transport pathway in mitochon-dria involves the export of proteins from the matrix intothe inner membrane. Proteins encoded by the mito-chondrial genome and synthesised in the matrix, aswell as a fraction of nuclear-encoded precursor pro-teins, take this route. Nuclear-encoded proteins mustfirst be imported into the matrix via the TOM and TIM23complexes before engaging the export machinery for

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Table 1. Mitochondrial export machinery.

Component Location and orientation Proposed function References

Oxa1 Integral inner membrane protein, General insert ion complex for export of mitochondrial [105,107,109–114]Five TM segments, Nout–Cin and nuclear-encoded precursor proteins. Functions

independently of Mba1Mba1 Peripherally associated with Required for the efficient membrane insertion of both [105,114]

inner membrane from matrix side mitochondrial and nuclear-encoded precursor proteins. A putative receptor that overlaps in substratespecificity with Oxa1

Cox18 Integral inner membrane protein C-terminal export of cytochrome c oxidase [116]subunit 2. Interaction partner of Mss2 and Pnt1

Pnt1 Integral inner membrane, C-terminal export of cytochrome c oxidase subunit 2 [115]Two TM segments, Nin–Cin

Mss2 Peripherally associated with inner C-terminal export of cytochrome c oxidase subunit 2 [117]membrane from matrix side

TM, transmembrane; N, amino terminus; C, carboxyl terminus; in, facing into matrix; out, facing into intermembrane space; Oxa1, oxidaseassembly; Mba1, multi-copy bypass of AFG3; Cox18, cytochrome c oxidase subunit 18; Pnt1, pentamidine resistance protein.



insertion into the inner membrane. In budding yeast,eight proteins are encoded by the mitochondrialgenome, seven of which are integral membrane com-ponents of respiratory chain complexes.

The mechanism of protein export from the matrix to

the inner membrane is poorly understood. It hasbecome increasingly evident that membrane insertionfrom the matrix side is not a spontaneous event, butrequires the assistance of specialised translocationcomponents. The first component found to be involvedin the biogenesis of mitochondrial respiratory chaincomplexes was Oxa1 (oxidase assembly 1) [101–103].In fact, Oxa1 is a member of an evolutionary conservedprotein family with homologs in the inner membrane ofbacteria (YidC) and the thylakoid membrane of chloro-plasts (Alb3) [104,105]. Each family member is knownto play a role in the membrane insertion of proteins[105,106].

Oxa1 is a nuclear-encoded integral protein of theinner membrane [107,108]. Isolation from Neurospora

crassa indicated that at least the stable core of thetranslocase consists only of Oxa1, possibly as atetramer [109]. The translocase is believed to functionas a general membrane insertion machinery, as it isnot only required for the insertion of membraneproteins that undergo amino-terminal tail export, butalso assists the import of other polytopic proteins inwhich the amino terminus is retained in the matrix[105,110–113]. It comes into direct contact with atranslocating precursor [113], but so far there is noevidence to suggest that the translocase forms a porefor 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 appearsto be precursor-dependent [105]. This suggests thatbackup import pathways maintain protein export func-tion in mitochondria. Indeed, additional proteins haverecently been implicated in export pathways bygenetic and biochemical evidence [114–117]. Table 1summarises all proteins found, so far, to be involvedin export pathways. The peripheral inner membraneprotein Mba1 is involved in amino-tail export and canlargely, but not completely, compensate for loss ofOxa1 [114]. The proteins Cox18, Pnt1 and Mss2, notparts of the Oxa1 translocase, together may constitutea second translocation complex [115,116]. Cox18, aweak homolog of Oxa1, has been implicated in

carboxy-tail export [116]. The composition and modeof action of the export machinery is far from solvedand thus should provide rapid developments in mito-chondrial protein transport in the near future.

Perspectives

The basic principles currently defining precursor proteinimport into mitochondria are generally agreed upon. For example, there is a need for intrinsic targeting signalsfor protein recognition, molecular chaperones andreceptors to mediate protein–protein interactions,aqueous channels to support transport across mem-branes and systems for generating protein movementfrom stored chemical or electrical energy. What is cur-rently difficult to fathom is the way in which the import

machinery collectively is able to sort the hundreds ofdifferent proteins to the correct compartment. Are thetranslocation channels highly regulated, capable ofchanging their interior surface in response to incomingprecursors, thus leading to differential compartment

sorting? Perhaps there are as yet to be discovered‘sorting proteins’ or complexes. Protein sorting mostlikely results from a network of rapid and subtlechanges in translocation components in response totargeting and sorting signals.

Furthermore, the molecular mechanisms by whichtranslocation components such as molecular chaper-ones, receptors and channel-forming proteins mediateimport are only partially understood. How are so manydifferent proteins recognised by so few importcomponents? Additional high resolution structures willcertainly help our understanding of these processes.Finally further work is required to understand howproteins are assembled into multisubunit complexes.In some cases, specialised assembly components arerequired to assist and it seems that large complexesare structurally dynamic in order that new compo-nents can be incorporated.

Acknowledgements

We thank David A. Dougan for comments on the man-uscript. Work of the authors’ laboratory was supportedby the Deutsche Forschungsgemeinschaft, SFB 388and the Fonds der Chemischen Industrie/BMBF.

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