Biochimica et Biophysica Acta 1833 (2013) 304–313

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Review

Unique components of the plant mitochondrial protein import apparatus☆

Owen Duncan, Monika W. Murcha, James Whelan ⁎ARC Centre of Excellence in Plant Energy Biology, Bayliss Building M316, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia

Abbreviations: TOM, translocase of the outer mitochmembrane; SAM, sorting and assembly machinery; TIMchondrial membrane; MIA, mitochondrial inter membraERV, essential for respiration and vegetative growth; Mpeptidase; PreP, presequence protease☆ This article is part of a Special Issue entitled: ProteinMitochondria and Plastids.⁎ Corresponding author. Tel.: +61 8 6488 1149; fax:

E-mail address: jim.whelan@uwa.edu.au (J. Whelan)

0167-4889/$ – see front matter © 2012 Elsevier B.V. Aldoi:10.1016/j.bbamcr.2012.02.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 January 2012Received in revised form 21 February 2012Accepted 23 February 2012Available online 1 March 2012

Keywords:Protein importMitochondrial biogenesisTOMTIMPlant mitochondrionPlant specific

The basic mitochondrial protein import apparatus was established in the earliest eukaryotes. Over the subse-quent course of evolution and the divergence of the plant, animal and fungal lineages, this basic import apparatushas been modified and expanded in order to meet the specific needs of protein import in each kingdom. In theplant kingdom, the arrival of the plastid complicated the process of protein trafficking and is thought to havegiven rise to the evolution of a number of unique components that allow specific and efficient targeting of mito-chondrial proteins from their site of synthesis in the cytosol, to their final location in the organelle. This includesthe evolution of two unique outer membrane import receptors, plant Translocase of outer membrane 20 kDasubunit (TOM20) and Outer membrane protein of 64 kDa (OM64), the loss of a receptor domain from an ances-tral import component, Translocase of outermembrane 22 kDa subunit (TOM22), evolution of unique features inthe disulfide relay systemof the intermembrane space, and the addition of an extramembrane spanning domainto another ancestral component of the inner membrane, Translocase of inner membrane 17 kDa subunit(TIM17). Notably, many of these components are encoded by multi-gene families and exhibit differential sub-cellular localisation and functional specialisation. This article is part of a Special Issue entitled: Protein Importand Quality Control in Mitochondria and Plastids.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

All mitochondria are thought to have descended from a single en-dosymbiotic event ~2 billion years ago [1]. Over the course of evolu-tion, much of the genetic material from the endosymbiont wastransferred to the nuclear genome [2], thereby creating a requirementfor the transport and import of these proteins back to their site offunction in the organelle. The mechanisms and many of the core com-ponents involved in this process are likely to have been established inthe earliest eukaryotes [3]. This is evidenced by the conservation ofmany of the central pore forming components in animals, plantsand fungi; including the translocase of the outer membrane (TOM)pore TOM40 [4], the sorting and assembly machinery (SAM) coresubunit SAM50 [5] and the translocase of the inner membrane(TIM) complexes TIM17:23 and TIM22, all of which are conservedacross the eukaryotic kingdoms [3] (Fig. 1). However, in the esti-mated 2 billion years following the common origins of these proteins,

ondrial membrane; OM, outer, translocase of the inner mito-ne space import and assembly;PP, mitochondrial processing

Import and Quality Control in

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the conserved core components have diversified and additional, line-age specific components have evolved to fulfil the specific requirementsof mitochondrial protein import in the varying conditions and cytosolicenvironments across these three kingdoms [6].

The arrival of the plastid in the plant lineage, as long as 1.5 billionyears ago [7], is thought to have posed a new challenge to the previ-ously established mitochondrial protein import apparatus [8]. Similarto the mitochondria, much of the plastid genetic material was subse-quently transferred to the nucleus [8] and this introduced complicationsin the process of protein trafficking [8]. Nuclear encoded mitochondrialand plastid proteins are commonly targeted by the presence of anN-terminal, cleavable presequence. These presequences share severalcommon features such as a region enriched in basic amino acids and aC-terminal domain predicted to form amphipathic α-helices [9,10]. Ithas been demonstrated that such signals are similar enough that theinclusion of a plastid targeting sequence from plants is capable of effi-ciently targeting proteins to mitochondria in yeast [11]. The selectivepressure to maintain the specificity and efficiency of protein traffickingin the face of this additional complexity is thought to have led to theevolution of a number of unique features in the plant mitochondrialprotein import apparatus that form the subject of this review. The avail-ability of whole genome sequence data has greatly facilitated compara-tive analysis of the mitochondrial protein import apparatus withinplants, as well as in comparison to other model organisms such asyeast [12,13]. Whilst this review principally deals with the mitochon-drial import apparatus of Arabidopsis, other species are also addressedwhere relevant.

20

Outer membrane

Inner membrane

TOM40

TIM23

7

9

OM64

METAXIN

17

ERV1

21

50

SAM50

44

1618

TIM9:10

TIM8:13 MIA40

756

TIM22

9

20N

C

CytochromeBc1 complex

MPPα

MPPβPreP

Fig. 1. The mitochondrial protein import apparatus of plants. Conserved components found in members of all of the eukaryotic lineages are shown in yellow. Plant specific com-ponents are shown in green and include — TOM20, TOM9, OM64 and the C-terminal regions of TIM17. Components which have been shown to be essential for normal plant de-velopment are outlined in red and presequence interacting domains are shown in blue. Abbreviations: TOM — translocase of the outer mitochondrial membrane, OM — outermembrane, SAM — sorting and assembly machinery, TIM — translocase of the inner mitochondrial membrane MIA — mitochondrial inter membrane space import and assembly,ERV — essential for respiration and vegetative growth, MPP mitochondrial processing peptidase, PreP — presequence protease.

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2. Outer membrane

Specific recognition of mitochondrial preproteins and the initia-tion of their import are processes that have been intensively studiedin Saccharomyces cerevisiae (yeast), Neurospora crassa and Rattusnorvegicus (rat) [14–16]. In the general import pathway, mitochon-drial proteins are targeted by the presence of anN-terminal presequence,which is specifically recognised by receptors on the outermembrane andtranslocated through the TOMcomplex. The TOMcomplex (Fig. 1) is theprincipal outer membrane import complex and with few exceptions[17] all mitochondrial proteins are thought to pass through the poreforming TOM40 subunit [18,19]. In yeast, this complex is composed ofTOM40, the secondary receptor and central organiser, TOM22, thesmall TOMs, TOM5, 6 and 7, which regulate the formation and functionof the complex, and the two cytosolic facing receptor subunits TOM20and TOM70 [18,20]. Studies on the various presequence interactingcomponents in yeast have led to a model in which precursors are recog-nised and passed through the TOM40 pore to the inner membrane orintermembrane space by a systemof increasing affinity. Each subsequentimport component in the binding chain interacts with the presequencewith greater affinity than the last, allowing the transport ofmitochondrialproteins across the outer membrane in an ATP independent manner[21,22]. In yeast, mutant strains lacking either of the two principle importreceptors TOM20 or TOM70 are viable but are seen to grow slowly andimport nuclear encoded proteins at rates considerably lower than wildtype, indicating that these components are important but not crucial forviability [23]. Yeast TOM20 is anchored to the outer mitochondrial mem-brane by an N-terminal α-helically anchored protein with a large cyto-plasmic domain consisting of a tetratricopeptide (TPR) repeat motif

capable of directly interacting with nuclear encoded mitochondrial pro-teins via N-terminal targeting signals [16,24]. In yeast, TOM70, likeTOM20, functions as an import receptor, though it does so through inter-action with the cytosolic chaperones HSP90 and HSP70 [25]. TOM70contains a total of seven TPR domains divided into two functional groups.Thefirst three from theN-terminal form a dicarboxylate clamp,which in-teractswith the cytosolic chaperones, passing the inboundmitochondrialprotein to the core domain of TOM70, which recognises targeting signalsinternal to the preprotein [26].Multiple TOM70dimers are then recruitedto the complex and the protein is passed through the import pore in anATP dependent manner [25].

Interestingly, whilst orthologues to TOM20 or TOM70 of animalsand fungi are absent from the plant mitochondrial outer membrane,proteins of similar size and function are found (see below), suggest-ing that TOM20 and TOM70 in yeast and animals evolved subsequentto the divergence of the eukaryotic lineages [27]. Whilst this mayexplain the absence of these receptors in plants, the core, conservedcomponents of the outermembrane import apparatus similarly demon-strate divergent structures in plants. Most notably, the cytosolic recep-tor domain of yeast and animal TOM22 [28] has been lost from the plantTOM22 protein, suggesting that a fundamentally different presequencebinding chain is operational in plants [4]. Similarly, amino acid residuesin yeast TOM40 that have been shown to be important for the transferof preproteins between the TOM and the TIM17:23 complexes [29]are absent from Arabidopsis TOM40. It has been proposed that thesedifferences in the outer membrane protein complexes of plants occurredas a result of both the independent origins of the components involved,and the greater need for specificity in the recognition of bona fide mito-chondrial proteins in the presence of plastid proteins [8].

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2.1. Plant TOM20

The protein named TOM20 in Arabidopsiswas first discovered by di-rect biochemical studies [30]. Based on the assumption that the plantmitochondrial protein import receptor would share common featureswith the animal and fungal TOM20, a list of candidate proteinswas iden-tified by protease treating intact mitochondria isolated from Solanumtuberosum (potato), allowing the discovery of outer membrane proteinswith large cytoplasmic domains [30]. A 23 kDa protein was identified inthis screen and tested for its role in protein import. Pre-incubation ofisolated mitochondria with antibodies raised to the cytosolic domain,followed by in vitro protein import showed a 30%–40% decrease in therate of import of a range of mitochondrial proteins [30]. The apparentmolecular weight of the protein along with the functional data resultedin this protein being named TOM20 [30]. Three proteins orthologous topotato TOM20 were later identified in the TOM complex of Arabidopsismitochondria by separation of mitochondrial outer membrane samplesby 2 dimension blue native/SDS-PAGE [31]. Subsequent studies of theseproteins, termed TOM20-2, TOM20-3 and TOM20-4, showed them tohave both distinct and common roles in the recognition and uptake ofnuclear encodedmitochondrial proteins. In addition, the import of a sub-set of nuclear encodedmitochondrial proteinswas 5-fold lower thanwildtype levels in the absence of TOM20. It was also observed that theseproteins are not essential, with only small delays in plant growth and de-velopment resulting from their absence [32].

In yeast and animals, TOM20 is an N-terminally anchored protein,unlike plant TOM20, which is anchored by its C-terminus. This reversalcontinues through its structure with the plant protein effectively mir-roring the domain arrangement of the animal and fungal TOM20 pro-teins; including the transmembrane domain, a disordered region andthe presequence binding TPR domain [15]. When compared to plants,the TOM20 proteins from yeast (and animals) do not exhibit significantsimilarities at the primary sequence level and as such, are thought to bea remarkable example of convergent evolution [33]. Both proteins havebeen shown to perform similar functions [23,32], however recent stud-ies into the nature of presequences recognised by the plant TOM20 haverevealed that this is achieved through different mechanisms [34]. Thepresequence binding domain of TOM20 in plants consists of two sepa-rate TPRmotifs. Thesemotifs differ from the classical TPRmotif, definedas having α-helical lengths of 34-residues between turns in that theseextend for 43 and 44 residues whilst maintaining the same inter helicalangles as the classical motif [33]. These two TPR motifs form two bind-ing sites, predominantly hydrophobic in nature, which are separated bya distance of ~20 Å [34]. Studies examining the binding between plantTOM20 and the presequence of the plant alternative oxidase, the NADHbinding subunit of complex 1 and ribosomal protein 10 demonstratedthat each of the plant presequences also had two binding regions separat-ed by a flexible linker segment [34]. This presequence configuration is incontrast to that observed in animals and fungi, which have a single bind-ing domain [16,24], suggesting that this difference may be an adaptationallowing the specific recognition ofmitochondrial presequences amongststructurally similar plastid presequences in the cytoplasm.

In yeast, TOM20 and TOM70 are known as accessory receptorsthat are free to dissociate from the TOM complex [18]. Under gentlesolubilisation conditions such as treatment with 1% (w/v) digitonin,these receptors are commonly found in lower molecular weightsub-complexes [18]. Similar treatment of Arabidopsis mitochondriashowed that unlike yeast TOM20, plant TOM20 is an integral memberof the TOM complex, remaining tightly associated with TOM40, evenwith digitonin concentrations as high as 10% (w/v) [32]. Despite theintegral nature of TOM20, the TOM complex of Arabidopsis is stable inthe absence of all three of the expressed TOM20 isoforms [32]. Of thethree isoforms, TOM20-2 exhibits the largest divergence from the con-sensus, both in terms of function and primary sequence [32]. This iso-form has an extended glycine rich linker segment inserted between thetransmembrane domain and presequence binding site, suggesting that

it may have additional roles compared to the other isoforms. This isalso evidenced by the formation of an additional, higher molecularweight complex containing TOM20-2 when digitonin solubilised mito-chondria are separated on blue native gels and a subtle delayed develop-mental phenotype of plants lacking TOM20-2, which is not observedacross plants lacking in the other isoforms of TOM20 [32].

2.2. TOM9/TOM22

In yeast, TOM22 and TOM40 are the only TOM proteins essentialfor viability [35,36]. Cross linking experiments show that in yeastwith an induced deficiency in the TOM22 cytosolic domain, precursorbinding to TOM20 and TOM70 is not inhibited, but presequence bind-ing to TOM40 is strongly reduced, indicating that TOM22 is importantin transferring incoming mitochondrial proteins from the primary re-ceptor to the import pore [37]. Evidence for this role is also supportedby the observation that the cytosolic domain of TOM22 can specificallybind mitochondrial presequences, as well as interact with both TOM20and TOM70 [22,37]. In addition to its role as a secondary receptor,TOM22 has two other functions; as an organiser of the TOM complexand as an extra step in the presequence binding chain [21,38,39]. Thetransmembrane segment has been shown to act as an organiser of theTOM complex with mutants lacking this domain failing to assemblecomplete TOM complexes [37]. An additional domain located on theintermembrane space side of the TOM22 transmembrane domain actsas a trans-site receptor, and can bind presequences on the other sideof the membrane for release into the inter membrane space or transferto the TIM23 complex [21,38,39].

The absence of a 22 kDa TOM subunit in plant mitochondria cameas a surprise given the essential nature of yeast TOM22, howeverTOM9 was subsequently identified as the plant orthologue ofTOM22 [31,40]. Plant TOM9 is a truncated form of the yeast TOM22protein, consisting of the transmembrane and trans-site domain, butlacking the large cytoplasmic region [8,40]. The absence of the cyto-solic domain has led to the proposal that, following the acquisitionof the plastid, the simple electrostatic interaction of “TOM22” with in-coming mitochondrial proteins may not have been sufficiently specificto ensure rejection of plastid proteins from the mitochondrial importapparatus [8]. This shares a common theme with the independent ori-gins and more complicated nature of the presequence binding site ofplant TOM20. Indeed, studies into the nature of a TOM9/TOM20 interac-tion show that this receptor complex operates with a substantively dif-ferent mechanism compared to yeast and animal systems [34]. YeastTOM22 has been shown to directly bind presequences, interactingwith the hydrophilic side of the amphipathic α-helix subsequently orconcurrently to the interaction between TOM20 and the hydrophobicside [22]. In plant systems, TOM9, lacking the receptor domain, fails tointeract with mitochondrial presequences, however, it has beenshown to interact with the hydrophobic binding sites of plant TOM20with a similar affinity to that of presequences [34]. This study led tothe proposal of a model for plant protein import, in which the cytosolicdomain of TOM9 normally occupies the binding site of TOM20. This in-teraction is displaced by mitochondrial presequences allowing them tobe bound by TOM20, thus increasing the concentration of mitochondri-al proteins near the protein import pore in a highly specific manner[34].

2.3. OM64

The discovery of a ‘plastid’ import receptor on the outer mem-brane of mitochondria in a subset of vascular plants was unexpected[41]. Whilst the absence of an orthologue to yeast TOM70 suggeststhat a receptor for cytosolic chaperonesmay be required for the deliveryof hydrophobic mitochondrial proteins, OM64 is 67% identical to theplastid localised TOC64 [41]. The TOC64 family of Arabidopsis consistsof three proteins, each of which contains similarities to two different

Wild type

OM64 Homozygous TOM20-2 HeterozygousTOM20-3 HomozygousTOM20-4 Homozygous

A

B

Gene t-DNA insertion

Mutant

Parental genotype

Normal Non viable

38

39

38

48

39

40.4 10.8

12

11

12

8

11

Average

Fig. 2. The combination of null mutations in Arabidopsis OM64 and each of the expressedTOM20genes results in defective seed development. A) Silique producedby parental planthomozygous for insertions in TOM20-3, 4 and OM64, and heterozygous in TOM20-2.B) Harvested seed from wild type and plants of the same genotype grown concurrently.Three batches of 50 seeds harvested from these plants were tallied to determine theratio of shrivelled (aborted) to normal seed.

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polypeptide classes: amidases and a three-fold repeat of a TPR motif[42]. The amidase-like domain of the plastid localised TOC64-III hasbeen inactivated by a point mutation in the active site and no longerplays an enzymatic role [42]. However the intermembrane space do-main of TOC64-III has been shown to directly interact with the plastidpreproteins [43]. The three TPR repeat motif located on the cytosolicside of the outer envelope plays a different role in plastid import, inter-acting with the cytosolic chaperone HSP90 for the delivery of nuclearencoded organelle proteins to the plastid import apparatus [44]. De-tailed analysis of this 3-TPR domain revealed few differences betweenthe plastid andmitochondrial localised forms, however residues locatedon the face opposite the chaperone interaction site were found to havediverged [45]. These differences may facilitate interaction with othercomponents of the import apparatus rather than specify the types ofproteins bound [45]. Blue native PAGE analysis of digitonin solubilisedmitochondria followed by immunodetection of the mitochondrial OM64showed that under these conditions, OM64 is not integrated into theTOM complex, suggesting that the interaction with other outer mem-brane components is transitory [32].

Functional studies have shown that depletion of OM64 affects theimport of some mitochondrial proteins [32]. This defect however, likethe removal of TOM20, does not result in any severe phenotypic abnor-malities [32]. As mentioned in the Introduction, the absence of eitherTOM20 or TOM70 in yeast has a deleterious effect, though not lethal;however the absence of both proteins was lethal [23]. Furthermore, re-cent, unpublished work constructing an Arabidopsismutant line absentin all of the TOM20 genes and OM64, results in an early embryo-lethalphenotype (Fig. 2). Thus, whilst the natures of the protein import recep-tors differ in terms of orthology between plants and yeast, both systemsrequire at least one import receptor, either an interacting chaperone ordirect presequence binding type in order to deliver incoming proteinsto the TOM40 pore.

2.4. Metaxin

Metaxin, like TOM20, OM64 is an outer membrane protein, how-ever, unlike TOM20 or OM64, the removal of metaxin results in asevere delayed developmental phenotype in Arabidopsis [32]. Plantslacking metaxin were seen to develop more slowly, remain smallerthan wild type, display lesions on developing and mature leaves andaccumulate higher levels of starch than wild type plants [32]. Therole of metaxin in protein import was first implicated through studiesin mammalian systems in which the addition of the cytosolic domainof metaxin to in vitro import assays reduced the rates of proteinuptake [46]. Subsequent studies in Arabidopsis have demonstrated asimilar effect. The cytosolic domain of metaxin has been shown todirectly and specifically interact with the presequences of a subset ofprecursors by both immunoprecipitation and yeast-2-hybrid interactionassays and the removal of metaxin was seen to result in dramaticallyreduced rates of import [32]. Despite these advances, the role of metaxinin mitochondrial protein import remains somewhat ambiguous. The ab-sence of metaxin results in lower abundance of other proteins involvedin import such as TOM20, OM64 and TIM17-2, making the direct effectof its absence difficult to distinguish from secondary effects [32].Whilst import is dramatically reduced when metaxin is absent, it isnot able to compensate for the loss of the other known import recep-tors, TOM20 and OM64 as evidenced by the absence of viable offspringfrom TOM20/OM64 knock out crosses (Fig. 2).

3. Intermembrane space

The mitochondrial intermembrane space (IMS) houses severalproteins involved in various import pathways. The tiny TIM proteins,consisting of TIM8, 9, 10 and 13 (Fig. 1) are involved in the import ofmitochondrial carrier proteins into the inner membrane and β-barrelproteins into the outer membrane [20]. Proteins involved in the

Mitochondrial Import and Assembly (MIA) pathway consisting ofMIA40 and ERV1 are also located in the IMS [47]. These are responsiblefor the oxidative folding and maturation of IMS proteins through theformation of disulphide bonds between cysteine residues in the dis-ulphide relay system [47].

3.1. The tiny TIMs

The tiny TIMs are a highly conserved protein family with TIM8, 9,10 and 13 being identified in a wide range of eukaryotic lineages, in-cluding plants [12]. TIM9 and 10 have been shown to be essential foryeast viability and form a hexameric chaperone complex that guidesprecursor proteins from the TOM complex to the inner membrane[48–50]. TIM8 and TIM13 form a similar chaperone complex, whichhas been shown to be involved in the import of TIM23, however,this function is not essential for yeast viability [50]. To date, plantsare the only species for which a biochemical reconstitution assay ofthe tiny TIMs has been described [51]. In these assays, outer mem-brane ruptured mitochondria were depleted of IMS proteins, whichwere then fractionated by anion-exchange chromatography beforereintroduction to in vitro import assays with the IMS depleted mito-chondria [51]. This procedure revealed that the import of the two

308 O. Duncan et al. / Biochimica et Biophysica Acta 1833 (2013) 304–313

carrier proteins tested was positively correlated with the presence ofTIM9 and TIM10, showing a direct functional role for these proteinsin the efficient import and assembly of carrier pathway proteins[51]. A unique feature of carrier protein import in plant mitochondriais the existence of cleavable presequences [52]. In yeast, carrier pro-teins do not require the presence of N-terminal presequences, insteadrelying onmultiple internal signals for interactionwith components ofthe import apparatus [53]. Studies have shown that removal of thisplant presequence does not affect import ability and that the prese-quence alone does not facilitate protein targeting to the mitochondria[54,55]. Further analysis showed that the presence of IMS proteinsstimulates the import of carrier proteins containing N-terminal cleav-able presequences to a greater extent than those with only internaltargeting signals [56]. This raises the possibility that plants maycontain additional import components that specifically recognise theN-terminal cleavable presequence of plant carrier proteins. Differ-ences in the processing of these signals have also been observed forcarrier proteins in plants [52]. N-terminal presequences from plantcarrier proteins were first processed by the mitochondrial processingpeptidase (MPP) on the matrix side of the inner membrane, and theremaining presequence was subsequently processed by an unknownserine protease in the IMS of Arabidopsis [52]. In contrast, presequencescan only be processed at the site of MPP cleavage in yeast, with no fur-ther modifications required, and thus a unique two-step processingpathway was identified in plant mitochondria [52].

3.2. The Mia40–Erv1 pathway

The disulphide relay system has been characterised in yeast and to alesser extent in plants [57–59]. This pathway describes the import ofproteins located in the IMS that contain conserved cysteine residuesin twin CX(9)C or CX(3)C motifs [60]. This pathway consists of onlytwo proteins, MIA40 and ERV1, both of which are essential for yeastviability [57,61–63]. MIA40 acts as a receptor for preproteins; transfer-ring them from the outer membrane to the inter membrane space,where the conserved cysteine residues are oxidised by MIA40, whichis subsequently oxidised by ERV1 [47]. Similarly, plants contain MIA40and ERV1 proteins, however, unlike yeast, plant MIA40 has been deter-mined to be co-localised to both the mitochondria and peroxisomes[58]. In addition, the deletion of MIA40 in Arabidopsis does not resultin an embryo lethal phenotype, or in the disruption of the disulphiderelay system, whilst disruption of ERV1 does result in an embryo lethalphenotype [64]. This demonstrates that the plant disulphide relaysystem can operate without MIA40 and that this pathway is functionalin the presence of ERV1 alone [64].

4. Inner membrane

Proteins targeted to the inner membrane or the matrix are passedto the TIM22 or TIM17:23 complexes of the inner membrane (Fig. 1).Preproteins are either directly transferred from the outer membraneimport channel or chaperoned through the IMS [20]. The TIM22 andTIM17:23 complexes consist of channel forming pores and associatedproteins involved in the mechanisms of translocation and assembly[20].

4.1. The TIM17:23 complex

The TIM17:23 complex is the major translocation pore responsiblefor the import of the majority of the mitochondrial proteome. Twofunctionally and structurally distinct forms, which are in dynamicequilibrium have been characterised in yeast [20]. The initial form,TIM23-SORT, containing TIM23, TIM17, TIM50 and TIM21, is involvedin the recognition of precursors from the TOM complex and insertioninto the inner membrane [20]. This complex has been shown to formsupercomplexes with the TOM40 channel or with complex III and

complex IV of the mitochondrial respiratory chain via TIM21 [65].These supercomplexes are thought to form in order to promote efficienttranslocation at contact sites, utilising the proton motive force at cristaejunctions [65,66]. The second form of the TIM17:23 complex, termedTIM23-PAM, functions in the transfer of proteins through the innermem-brane to the matrix [61,67]. This complex consists of TIM17, TIM23,TIM50 and the presequence translocase associated motor (PAM) com-plex, mainly comprising of mtHSP70, TIM44 and its associated co-chaperones; PAM16, PAM17 and PAM18 [61,67].

Plants appear to have orthologues to all of the components of theTIM17:23 translocase observed in yeast [20,66], with the addition ofseveral plant specific features. In plants, the TIM17 protein differs fromthe yeast counterpart in that the plant TIM17 contains a C-terminalextension of 143 amino acids [56]. This C-terminal extension is presentin all higher plant species andmust be removed in order to complementyeast TIM17 deletion strains [56]. The C-terminal extension of ArabidopsisTIM17 has been shown to link the inner and outer mitochondrial mem-branes and exhibits presequence binding properties, however, the exactrole for this C-terminal extension is still largely unknown [12,68,69]. Incontrast, the yeast TIM23 has been shown to span the IMS, linking theinner and outer membranes and it has been shown to direct precursorproteins to the TIM17:23 pore [70]. In plants, TIM23 appears to lack anyextension and only contains the four transmembrane spanning regionscharacteristic of a translocation pore of the inner membrane [69].

4.2. The TIM22 complex

The TIM22 complex is responsible for the translocation of multi-spanning membrane proteins through and into the inner membrane.In yeast, this complex consists of 4 subunits, the TIM22 channel andaccessory proteins TIM54, TIM18 and TIM12, of which only TIM12 isessential for yeast viability, most likely due to its linker role withthe tiny TIMs [71–74]. Two identical genes on different chromosomesencode the Arabidopsis homologue for TIM22, and the encoded pro-tein has been shown to have the ability to complement a yeast dele-tion strain of TIM22 [75]. Homologues to TIM54, 18 and 12 cannot beidentified in any plant genome database [12] and as such, the TIM22channel may function alone or with yet undetermined plant specificproteins.

4.3. Multi-gene families

In Arabidopsis, TIM17, TIM23 and TIM22 belong to a large genefamily consisting of 17 members, termed the preprotein and aminoacid transporters (PRAT) [75,76]. These proteins are thought to originatefrom a single eubacterial ancestor, and are characterised by four trans-membrane regions connected by three short hydrophilic loops, with acharacteristic motif: [G/A]X2[F/Y}X10RX3Dx6[G/A/S]GX3G, where X isany amino acid [76]. The 17 loci encode 16 different PRAT proteins(two proteins are 100% identical) that exhibit significant diversity insize, structure and location [75]. The predicted length of these 16 pro-teins ranges from 133 to 261 aa. Furthermore, the PRAT domain isonly present in 7 of the 16 predicted proteins, with 4members contain-ing a degenerate PRAT domain [75]. Of all 16 proteins: 11 were deter-mined to be mitochondrial, 4 chloroplastic, and one targeted to bothorganelles [75].

The 11 PRAT proteins localised to the mitochondria fall into threesubfamilies based on homology to yeast TIM17, TIM23 and TIM22,respectively. Three genes encode TIM17 (At1g20250, At2g37410and At5g11690), 3 genes encode TIM23 (At1g72750, At1g17530 andAt3g04800), and two genes encode TIM22 (At3g10110 and At1g18320)(Fig. 3). An additional three genes encode proteins that have no obviousyeast/mammalian counterparts but are largely homologous to theknown Arabidopsis PRATs, these are termed as “unknown”, as they areyet to be functionally annotated (Fig. 3). Considering that, in yeast,only one gene encodes one protein, for each of the TIM17, TIM23 and

UnknownAt3g49560 TM TM TM TM

UnknownAt2g42210 TM TM TM TM

UnknownAt3g25120 TM TM TM TM

plantspecific

components

conserved PRAT domain

degenerate PRAT domain

nucleic acid binding motif

TM

yeast orthologs

plant specific

transmembrane domain

TM TM TM TM

TM TM TM TM

TM TM TM TM

TM TM TM TM

TM TM TM TM

TM TM TM TM

TM TM TM TM

TIM17-2At2g37410

TIM17-1At1g20350

TIM17-3At5g11690

TIM23-1At1g17530

TIM23-3At3g04800

TIM23-2At1g72750

TIM22At3g10110At1g18320

yeastorthologs

exist

218 aa

243 aa

133 aa

187 aa

188 aa

188 aa

173 aa

261 aa

159 aa

189 aa

Fig. 3. A schematic representation of the mitochondrial located PRATS (preprotein and amino acid transporters) gene family. Three genes encode for TIM17, 3 genes encode forTIM23 and 2 genes encode for TIM22. An additional 3 genes encode for proteins of yet uncharacterised functions. The 4 transmembrane (TM) regions characteristic of PRAT proteinsare indicated in grey. The conserved PRAT domains and the degenerative PRAT domains are indicated in red and blue respectively. The nucleic-acid binding motif of the C-terminalextensions is indicated in black.

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TIM22 proteins, 17 family members in Arabidopsis represent a large ex-pansion of this transporter family and raise the possibility that theseproteins have acquired new functions. Considerable diversity existswithin these subfamilies. For example, the TIM17 subfamily (discussedin Section 4.1) consists of three genes, two of which contain the plantspecific C-terminal extensions, whilst the third lacks this extensionand encodes a degenerate PRAT domain (Fig. 3). A recent study inTrypanosoma brucei implicated TIM17 to be involved in tRNA import[77].Whilst the T. brucei TIM17does not contain the long C-terminal ex-tension present in plants, sequence similarities are observed, hintingthat a conservation of functions is also possible.

Systematic liquid chromatography coupled to mass spectrometry(LC/MS) analysis of mitochondria separated by BN-PAGE showedthat the TIM17-2 migrates in complex of 90 and 200 kDa [78]. Thisis similar to the yeast model that shows two distinct translocationcomplexes of TIM23-SORT and TIM23-PAM [78]. However, it is notablethat TIM17-3 was observed to migrate at 110 kDa, distinct from otherknown TIM23 complex components, and possibly associated withTIM9 and TIM10, which also migrate as a complex of approximately110 kDa [78]. Transcript abundance of TIM17-1 has been positively cor-related with the mitochondrial stress response [79,80]. This transcriptwas induced by several stress treatments including high ultraviolet,high salt and cycloheximide (Fig. 4). Notably, several treatments,which show an increase/decrease in TIM17-1, are accompanied by aninverse change in TIM17-2 abundance, suggesting diverse functions forthis subfamily (Fig. 4). The expression of TIM17-1 is very low in alltissues and throughout development, suggesting that it may only berequired in a stress responsive role (Fig. 4). Further supporting this isthe finding that the TIM17-1 promoter region contains a binding sitefor WRKY transcription factors, which are well documented for theirrole in plant stress and defence [81]. A relationship between proteinimport and stress responses has previously been observed, with a num-ber of environmental stresses being shown to differentially affect therate of import of a number of nuclear encoded mitochondrial proteins[82]. The induction of TIM17-1 and a corresponding increase in proteinabundance in response to stress may function to alter the rate of mito-chondrial import in order to better meet the needs of the plant. Themi-tochondrial PRAT geneswere seen to exhibit various levels of expression

throughout development and in a variety of tissue typeswithin the genefamily (Fig. 4). As import ability has previously been shown to be a reg-ulated process throughout plant development, the observed differencesin expression of these genes may serve to regulate mitochondrial bio-genesis [83,84].

Determining the functions of the “unknown”plant specificArabidopsisputative PRAT family members is of particular interest as this may givegreater insight into the reason for the expansion of this family. The pro-teins encoded by At2g42210, At3g25120 and At5g49560 have no directyeast or mammalian orthologue and whilst these do exhibit the charac-teristics of the PRAT transporters family, early investigations (outlinedbelow) suggest that their functions maybe somewhat different (Fig. 3).The protein encoded by At2g42210 has been shown to be a componentof complex I of the respiratory pathway [80,87], and surprisingly, in thesame study, TIM22 was also identified alongside members of respiratorycomplex I [78]. In addition, another PRAT protein encoded by At2g26670mayalsohave links to complex I, as it is orthologous to a bovine complex Isubunit termed B14.7. Although this protein has been identified in chlo-roplasts by immunodetection [85], independent in vitro and in vivoprotein targeting experiments were inconclusive [75]. Thus, further in-vestigation is necessary to revealwhether this is amitochondrial location,and whether it is associated with complex I.

4.4. Mitochondrial processing peptidase

Mitochondria contain a large number of peptidases involved in pro-tein turnover. Several peptidases are involved in the protein importpathway either through removal of the targeting peptide, formation ofpeptide intermediates or degradation of the signal peptide after cleavage.The mitochondrial processing peptidase (MPP) is an ATP independentprotease involved in cleavage of the targeting peptide from the translo-cating precursor protein for the majority of mitochondrial matrix pro-teins. In yeast and mammals, the MPP contains two structurally relatedcomponents, MPPα and MPPβ both of which reside in the matrix [86].In contrast, whilst the plant MPP exhibits high sequence similarity tothe yeast MPP, the Arabidopsis MPP is unique as both MPPα and MPPβsubunits are located on the inner mitochondrial membrane, being inte-grated into the cytochrome bc1 complex [87,88]. Thus, the cytochrome

A B

C

calluscell culturesperm cellprotoplast

guard protoplastmesophyll protoplast

seedlingcotyledonhypocotyl

radicalimbibed seedinflorescence

flowerpistilpetalsepal

stamenantherpollen

abscission zonepedicelsiliquereplum

seedstemnode

shoot apexcauline leaf

rosettejuvenile leaf

adult leafpetiole

senecent leafhypocotyl

stemshoot apex

roots

germinated seedseedlingyoung roseteedeveloping rosetteboltingyoung flowerdeveoped flowerflowers and siliquesmature siliques

cold study 6CaLcuVP. syringaecycloheximiderotenone (12h)syrinogolin earlysyrinogolin latedark/21 C (220-280)dark/21 C (300-360)dark/32 C (220-280)dark/32 C (140-200)dark/4 C (140-200)dark/4 C (220-280)high light study 2 (3h)high light study 2 (8h)UV max 310nm (1h)UW max 310nm (6h)genotixic earlygenotoxic late salt study 2 earlysalt study 2 late

TIM

17-1

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17-2

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9560

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22

Fig. 4. Expression profiles of genes encoding the mitochondrial located preprotein and amino acid transporters (PRATs) gene family. Geneinvestigator profile showing the expressionlevels of the mitochondrial located PRAT gene family members throughout development (A), in a variety of tissues (B) and following various stress treatments (C). Heat maps representexpression level, and the colour intensity is shown as computed in Genevestigator after MAS5 normalization of data from Affymetrix microarrays [118].

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bc1 complex in Arabidopsis has two roles; mitochondrial electron trans-port and protein import [89].

With the exception of MPP, the characterisation of plant mitochon-drial proteases is still in its infancy. Initial studies to date, suggest thatplant proteases share some unique characteristics; Arabidopsis proteasesare encoded by large multi-gene families, several proteases are dual tar-geted to the chloroplast and the mitochondria and functionally, someproteases are distinct from their yeast counterparts [90]. The prese-quence protease (PreP) was first identified and characterised in plants,and has been shown to be located in both mitochondria and chloro-plasts [91]. In Arabidopsis, it was shown that PreP is involved in the deg-radation of the presequence peptide signal following import [91].Insertional inactivation of the two genes encoding PreP in Arabidopsisresulted in a severe growth phenotype [92] and given that mammalianPreP has been linked to the degradation of amyloid-β a peptide linked toAlzheimer's disease, PreP is considered highly important in maintainingmitochondrial integrity [77,93,94]. Another group of proteases, AAA(ATPase Associated with diverse cellular Activities) proteases contain anumber of family members, which exhibit a wide range of functions ina variety of species. In Arabidopsis, ten members of this gene familywere found to be mitochondrial and initial functional characterisationsuggests emerging structural differences to the yeast orthologues [95].Two of these are related to the bacterial FtsH protease. These are mem-brane bound metalloproteases, each spanning the inner membranetwice and differ in their topology, with the proteolytic domain of the

i-AAA proteases facing the intermembrane space and the m-AAA prote-ases facing thematrix. Both of these subtypes are found in highmolecularweight complexes with the i subtype forming complexes of ~1500 kDa[48] of unknown composition and the m subtype being found in com-plexes of 2 mDa in association with prohibitins [49]. Interestingly, thei-AAA ATPase of yeast (named Yme1) has been shown to be necessaryfor the import of the IMS located PNPase [96]. This activity is not depen-dent on its function in proteolysis; instead, Yme1 acts as a nonconven-tional translocationmotor, pulling PNPase into the IMS after cleavage ofits targeting signal byMPP [96]. The rhomboid protease family is anoth-er large multi-gene family, encoded by 12 genes in the Arabidopsisgenome. Thus far, only RBL12 has been confirmed to be mitochondrialand has been shown to have different substrate specificity to its yeastcounterpart, Pcp1 [97]. The large number of types of proteases andgene familymembers involved inmitochondrial biogenesis complicatestheir functional characterisation, thoughdivergent roles are expected tobe uncovered.

5. Outstanding questions

It remains to be seen whether the components discussed above arethe only unique components of the plant mitochondrial import appara-tus. Evidence suggests that there are other, as yet uncharacterisedcomponents involved. This evidence includes the presence of multipleα-helically anchored proteins in the outer membrane [98]. Although a

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variety of these proteins are present on the outer membrane, a compo-nent capable of inserting such proteins is yet to be characterised andobvious candidates such as members of the TOM complex have beenshown not to be involved [17]. Additionally, examination of the importof a dual targeted glutathione reductase showed that the import of thisprotein is independent of either TOM20 or OM64, indicating the possiblepresence of an additional outer membrane import receptor [32].

In yeast, the SAM complex consists of at least four proteins— SAM50,MDM10 and two related proteins — SAM35 and SAM37 [5,99]. SAM35and SAM37 are thought to be related to human metaxin 1 and metaxin2 respectively [100,101]. In Arabidopsis, the composition of the SAMcomplex is yet to be determined and whilst two genes encodingSAM50 have been identified, there is only one metaxin gene, raisingthe possibility of additional plant specific components in this complex,or that the single plant metaxin protein is responsible for the roles ofSAM35 and SAM37 [102]. Basic Local Alignment Search Tool (BLAST)analysis of the Arabidopsis genomehas so far failed to identify a homologof the final subunit of this complex, yeast MDM10. MDM10 in yeast is aβ-barrel protein that also associates with an ER protein,MMM1, and theouter membrane protein MDM12, to form a complex known as ERMES(endoplasmic reticulum mitochondria encounter structure) [103,104].The other subunits of this yeast ERMES complex are also absent fromthe Arabidopsis genome and, as well as mediating the mitochondrial/ER interaction, the complex has been shown to play a role in the assemblyof β-barrel proteins, associating with both the TOM and SAM complexes[105]. The absence of genes encoding subunits of the ERMES complex inArabidopsis certainly suggests that other, plant specific proteins are in-volved in the process of mitochondrial protein import and assembly.

One of the major hurdles in determining which proteins, if any, arefulfilling the roles of apparently missing components is the complexityof the Arabidopsis genome.Whilst this is a relatively small and simple ge-nome of 157 Mbp [106] compared to other plant genomes such as Parisjaponica, which is 150 Gbp [107], accurately identifying distantly relatedproteins from the estimated ~35,000 translatedArabidopsis genes is not asimple task. Even with rigorous bioinformatic studies, reverse geneticapproaches often require experimental analysis of numerous ‘false posi-tives’with no guarantee of success. Sub-organelle proteomic studies cangreatly accelerate this process by reducing the numbers of candidateproteins for a given function, and also have the advantage of exposingunexpected functional and evolutionary relationships (for example[98]). The recent definition of the mitochondrial outer membrane pro-teome of Arabidopsis has revealed numerous components includingseveral specific to plants, which are potentially involved in the process-es of protein import and assembly of the outer membrane [98]. Onecandidate protein observed in the course of this research— anion trans-porting ATPase (At5g60730.1) is related to a bacterial arsenate trans-porter. An orthologue of this protein studied in yeast appears to havelost its transport function [108,109] and gained a role in inserting tailanchored proteins into the endoplasmic reticulum post-translationally[110]. The discovery of a mitochondrial located orthologue of this pro-tein in Arabidopsis [98] suggests that in plants at least, this family maybe involved in the insertion of outer mitochondrial membrane proteinsinto the lipid bi-layer. Although no proteins directly related to compo-nents of the ERMES complex were discovered, a previously unknown,plant specific β-barrel protein was localised to the outer mitochondrialmembrane [98] and its role in protein import and endoplasmic reticu-lum interaction is currently under investigation.

The functional significance of the differences observed in the MIAERV1 pathway compared to this pathway in yeast, is as yet unclear,though a unique feature of plant mitochondria is the occurrence of theascorbate biosynthesis pathway within the IMS [111]. Given that ascor-bate has been shown to participate in oxidative folding [90], this processmay be utilised by the MIA import pathway in plants. Whilst MIA40 in-sertional knockouts in Arabidopsis did not exhibit any changes in totalascorbate concentrations [58], plant MIA40 is not essential and thusthe disulphide relay system canbe carried out regardless of the presence

ofMIA40. Organisms lackingMIA40have a different arrangement of cys-teine motifs within ERV1 similar to the plant ERV1, and thus in plants,ERV1 may function independently in the oxidative folding of cysteineresidues [58].

Recent characterisation of protein complexes in plant mitochondriahasuncovered anumber of expected andunexpectedprotein complex as-sociations [65,112–114]. These include the formation of super complexesbetween the outer and inner membrane translocation complexes [115]and unexpectedly, between the respiratory chain and import complexes.TIM17:23 has been shown to interact with both complex III [116] and theCyt bc1/COX supercomplex in yeast [113]. This occurrence has only re-cently been found in plants, with Arabidopsis TIM23 shown to interactwith complex III of the respiratory chain, via TIM21, similar to the yeastmodel (Wang and Murcha, personal communication). In addition, evi-dence that Arabidopsis TIM23 interacts with complex I of the respiratorychain has also been obtained (Wang and Murcha, personal communica-tion). As yeast lacks complex I and has always been the primary eukary-otic model for these types of studies, this interaction between complex Iand TIM23 has never been previously reported.

It has been suggested that there are around 4000 plant specificproteins encoded in the Arabidopsis genome [117], and with the spe-cific challenges of mitochondrial protein import in plants, it seemslikely that new components will be discovered as the various importcomplexes are better characterised. It is also worth noting that, whilstit has been shown thatmultiple isomers of import components can haveoverlapping functions [32], more detailed functional characterisationmay uncover specialised roles for individualmembers that have evolvedin response to the complexities specific to plants.

Acknowledgements

We would like to thank Dr. Reena Narsai (University of WesternAustralia) for discussion and comments on the manuscript. MWM isa recipient of the Australian Research Council APD Fellowship andan Australian Research Council Centre of Excellence Program grantCEO561495 to JW.

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