Unique components of the plant mitochondrial protein import apparatus

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

a r t i c l e

i n f o

Article history: Received 4 January 2012 Received in revised form 21 February 2012 Accepted 23 February 2012 Available online 1 March 2012 Keywords: Protein import Mitochondrial biogenesis TOM TIM Plant mitochondrion Plant specific

a b s t r a c t The basic mitochondrial protein import apparatus was established in the earliest eukaryotes. Over the subsequent course of evolution and the divergence of the plant, animal and fungal lineages, this basic import apparatus has been modified and expanded in order to meet the specific needs of protein import in each kingdom. In the plant kingdom, the arrival of the plastid complicated the process of protein trafficking and is thought to have given rise to the evolution of a number of unique components that allow specific and efficient targeting of mitochondrial proteins from their site of synthesis in the cytosol, to their final location in the organelle. This includes the evolution of two unique outer membrane import receptors, plant Translocase of outer membrane 20 kDa subunit (TOM20) and Outer membrane protein of 64 kDa (OM64), the loss of a receptor domain from an ancestral import component, Translocase of outer membrane 22 kDa subunit (TOM22), evolution of unique features in the disulfide relay system of the inter membrane space, and the addition of an extra membrane spanning domain to 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 subcellular localisation and functional specialisation. This article is part of a Special Issue entitled: Protein Import and 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 endosymbiotic event ~ 2 billion years ago [1]. Over the course of evolution, much of the genetic material from the endosymbiont was transferred to the nuclear genome [2], thereby creating a requirement for the transport and import of these proteins back to their site of function in the organelle. The mechanisms and many of the core components involved in this process are likely to have been established in the earliest eukaryotes [3]. This is evidenced by the conservation of many of the central pore forming components in animals, plants and fungi; including the translocase of the outer membrane (TOM) pore TOM40 [4], the sorting and assembly machinery (SAM) core subunit SAM50 [5] and the translocase of the inner membrane (TIM) complexes TIM17:23 and TIM22, all of which are conserved across the eukaryotic kingdoms [3] (Fig. 1). However, in the estimated 2 billion years following the common origins of these proteins,

Abbreviations: TOM, translocase of the outer mitochondrial membrane; OM, outer membrane; 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 ☆ This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. ⁎ Corresponding author. Tel.: + 61 8 6488 1149; fax: + 61 8 6488 1148. E-mail address: [email protected] (J. Whelan). 0167-4889/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2012.02.015

the conserved core components have diversified and additional, lineage specific components have evolved to fulfil the specific requirements of mitochondrial protein import in the varying conditions and cytosolic environments across these three kingdoms [6]. The arrival of the plastid in the plant lineage, as long as 1.5 billion years ago [7], is thought to have posed a new challenge to the previously established mitochondrial protein import apparatus [8]. Similar to the mitochondria, much of the plastid genetic material was subsequently transferred to the nucleus [8] and this introduced complications in the process of protein trafficking [8]. Nuclear encoded mitochondrial and plastid proteins are commonly targeted by the presence of an N-terminal, cleavable presequence. These presequences share several common features such as a region enriched in basic amino acids and a C-terminal domain predicted to form amphipathic α-helices [9,10]. It has been demonstrated that such signals are similar enough that the inclusion of a plastid targeting sequence from plants is capable of efficiently targeting proteins to mitochondria in yeast [11]. The selective pressure to maintain the specificity and efficiency of protein trafficking in the face of this additional complexity is thought to have led to the evolution of a number of unique features in the plant mitochondrial protein import apparatus that form the subject of this review. The availability of whole genome sequence data has greatly facilitated comparative analysis of the mitochondrial protein import apparatus within plants, as well as in comparison to other model organisms such as yeast [12,13]. Whilst this review principally deals with the mitochondrial import apparatus of Arabidopsis, other species are also addressed where relevant.

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16 MPPα

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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 components 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 development are outlined in red and presequence interacting domains are shown in blue. Abbreviations: TOM — translocase of the outer mitochondrial membrane, OM — outer membrane, 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.

2. Outer membrane Specific recognition of mitochondrial preproteins and the initiation of their import are processes that have been intensively studied in Saccharomyces cerevisiae (yeast), Neurospora crassa and Rattus norvegicus (rat) [14–16]. In the general import pathway, mitochondrial proteins are targeted by the presence of an N-terminal presequence, which is specifically recognised by receptors on the outer membrane and translocated through the TOM complex. The TOM complex (Fig. 1) is the principal outer membrane import complex and with few exceptions [17] all mitochondrial proteins are thought to pass through the pore forming TOM40 subunit [18,19]. In yeast, this complex is composed of TOM40, the secondary receptor and central organiser, TOM22, the small TOMs, TOM5, 6 and 7, which regulate the formation and function of the complex, and the two cytosolic facing receptor subunits TOM20 and TOM70 [18,20]. Studies on the various presequence interacting components in yeast have led to a model in which precursors are recognised and passed through the TOM40 pore to the inner membrane or inter membrane space by a system of increasing affinity. Each subsequent import component in the binding chain interacts with the presequence with greater affinity than the last, allowing the transport of mitochondrial proteins across the outer membrane in an ATP independent manner [21,22]. In yeast, mutant strains lacking either of the two principle import receptors TOM20 or TOM70 are viable but are seen to grow slowly and import nuclear encoded proteins at rates considerably lower than wild type, indicating that these components are important but not crucial for viability [23]. Yeast TOM20 is anchored to the outer mitochondrial membrane by an N-terminal α-helically anchored protein with a large cytoplasmic domain consisting of a tetratricopeptide (TPR) repeat motif

capable of directly interacting with nuclear encoded mitochondrial proteins via N-terminal targeting signals [16,24]. In yeast, TOM70, like TOM20, functions as an import receptor, though it does so through interaction with the cytosolic chaperones HSP90 and HSP70 [25]. TOM70 contains a total of seven TPR domains divided into two functional groups. The first three from the N-terminal form a dicarboxylate clamp, which interacts with the cytosolic chaperones, passing the inbound mitochondrial protein to the core domain of TOM70, which recognises targeting signals internal to the preprotein [26]. Multiple TOM70 dimers are then recruited to the complex and the protein is passed through the import pore in an ATP dependent manner [25]. Interestingly, whilst orthologues to TOM20 or TOM70 of animals and fungi are absent from the plant mitochondrial outer membrane, proteins of similar size and function are found (see below), suggesting that TOM20 and TOM70 in yeast and animals evolved subsequent to the divergence of the eukaryotic lineages [27]. Whilst this may explain the absence of these receptors in plants, the core, conserved components of the outer membrane import apparatus similarly demonstrate divergent structures in plants. Most notably, the cytosolic receptor domain of yeast and animal TOM22 [28] has been lost from the plant TOM22 protein, suggesting that a fundamentally different presequence binding chain is operational in plants [4]. Similarly, amino acid residues in yeast TOM40 that have been shown to be important for the transfer of preproteins between the TOM and the TIM17:23 complexes [29] are absent from Arabidopsis TOM40. It has been proposed that these differences in the outer membrane protein complexes of plants occurred as a result of both the independent origins of the components involved, and the greater need for specificity in the recognition of bona fide mitochondrial proteins in the presence of plastid proteins [8].

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2.1. Plant TOM20 The protein named TOM20 in Arabidopsis was first discovered by direct biochemical studies [30]. Based on the assumption that the plant mitochondrial protein import receptor would share common features with the animal and fungal TOM20, a list of candidate proteins was identified by protease treating intact mitochondria isolated from Solanum tuberosum (potato), allowing the discovery of outer membrane proteins with large cytoplasmic domains [30]. A 23 kDa protein was identified in this screen and tested for its role in protein import. Pre-incubation of isolated mitochondria with antibodies raised to the cytosolic domain, followed by in vitro protein import showed a 30%–40% decrease in the rate of import of a range of mitochondrial proteins [30]. The apparent molecular weight of the protein along with the functional data resulted in this protein being named TOM20 [30]. Three proteins orthologous to potato TOM20 were later identified in the TOM complex of Arabidopsis mitochondria by separation of mitochondrial outer membrane samples by 2 dimension blue native/SDS-PAGE [31]. Subsequent studies of these proteins, termed TOM20-2, TOM20-3 and TOM20-4, showed them to have both distinct and common roles in the recognition and uptake of nuclear encoded mitochondrial proteins. In addition, the import of a subset of nuclear encoded mitochondrial proteins was 5-fold lower than wild type levels in the absence of TOM20. It was also observed that these proteins are not essential, with only small delays in plant growth and development 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 reversal continues through its structure with the plant protein effectively mirroring the domain arrangement of the animal and fungal TOM20 proteins; including the transmembrane domain, a disordered region and the presequence binding TPR domain [15]. When compared to plants, the TOM20 proteins from yeast (and animals) do not exhibit significant similarities at the primary sequence level and as such, are thought to be a remarkable example of convergent evolution [33]. Both proteins have been shown to perform similar functions [23,32], however recent studies into the nature of presequences recognised by the plant TOM20 have revealed that this is achieved through different mechanisms [34]. The presequence binding domain of TOM20 in plants consists of two separate TPR motifs. These motifs differ from the classical TPR motif, defined as having α-helical lengths of 34-residues between turns in that these extend for 43 and 44 residues whilst maintaining the same inter helical angles as the classical motif [33]. These two TPR motifs form two binding sites, predominantly hydrophobic in nature, which are separated by a distance of ~20 Å [34]. Studies examining the binding between plant TOM20 and the presequence of the plant alternative oxidase, the NADH binding subunit of complex 1 and ribosomal protein 10 demonstrated that each of the plant presequences also had two binding regions separated by a flexible linker segment [34]. This presequence configuration is in contrast to that observed in animals and fungi, which have a single binding domain [16,24], suggesting that this difference may be an adaptation allowing the specific recognition of mitochondrial presequences amongst structurally similar plastid presequences in the cytoplasm. In yeast, TOM20 and TOM70 are known as accessory receptors that are free to dissociate from the TOM complex [18]. Under gentle solubilisation conditions such as treatment with 1% (w/v) digitonin, these receptors are commonly found in lower molecular weight sub-complexes [18]. Similar treatment of Arabidopsis mitochondria showed that unlike yeast TOM20, plant TOM20 is an integral member of the TOM complex, remaining tightly associated with TOM40, even with digitonin concentrations as high as 10% (w/v) [32]. Despite the integral nature of TOM20, the TOM complex of Arabidopsis is stable in the absence of all three of the expressed TOM20 isoforms [32]. Of the three isoforms, TOM20-2 exhibits the largest divergence from the consensus, both in terms of function and primary sequence [32]. This isoform has an extended glycine rich linker segment inserted between the transmembrane domain and presequence binding site, suggesting that

it may have additional roles compared to the other isoforms. This is also evidenced by the formation of an additional, higher molecular weight complex containing TOM20-2 when digitonin solubilised mitochondria are separated on blue native gels and a subtle delayed developmental phenotype of plants lacking TOM20-2, which is not observed across plants lacking in the other isoforms of TOM20 [32]. 2.2. TOM9/TOM22 In yeast, TOM22 and TOM40 are the only TOM proteins essential for viability [35,36]. Cross linking experiments show that in yeast with an induced deficiency in the TOM22 cytosolic domain, precursor binding to TOM20 and TOM70 is not inhibited, but presequence binding to TOM40 is strongly reduced, indicating that TOM22 is important in transferring incoming mitochondrial proteins from the primary receptor to the import pore [37]. Evidence for this role is also supported by the observation that the cytosolic domain of TOM22 can specifically bind mitochondrial presequences, as well as interact with both TOM20 and TOM70 [22,37]. In addition to its role as a secondary receptor, TOM22 has two other functions; as an organiser of the TOM complex and as an extra step in the presequence binding chain [21,38,39]. The transmembrane segment has been shown to act as an organiser of the TOM complex with mutants lacking this domain failing to assemble complete TOM complexes [37]. An additional domain located on the intermembrane space side of the TOM22 transmembrane domain acts as a trans-site receptor, and can bind presequences on the other side of the membrane for release into the inter membrane space or transfer to the TIM23 complex [21,38,39]. The absence of a 22 kDa TOM subunit in plant mitochondria came as a surprise given the essential nature of yeast TOM22, however TOM9 was subsequently identified as the plant orthologue of TOM22 [31,40]. Plant TOM9 is a truncated form of the yeast TOM22 protein, consisting of the transmembrane and trans-site domain, but lacking the large cytoplasmic region [8,40]. The absence of the cytosolic domain has led to the proposal that, following the acquisition of the plastid, the simple electrostatic interaction of “TOM22” with incoming mitochondrial proteins may not have been sufficiently specific to ensure rejection of plastid proteins from the mitochondrial import apparatus [8]. This shares a common theme with the independent origins and more complicated nature of the presequence binding site of plant TOM20. Indeed, studies into the nature of a TOM9/TOM20 interaction show that this receptor complex operates with a substantively different mechanism compared to yeast and animal systems [34]. Yeast TOM22 has been shown to directly bind presequences, interacting with the hydrophilic side of the amphipathic α-helix subsequently or concurrently to the interaction between TOM20 and the hydrophobic side [22]. In plant systems, TOM9, lacking the receptor domain, fails to interact with mitochondrial presequences, however, it has been shown to interact with the hydrophobic binding sites of plant TOM20 with a similar affinity to that of presequences [34]. This study led to the proposal of a model for plant protein import, in which the cytosolic domain of TOM9 normally occupies the binding site of TOM20. This interaction is displaced by mitochondrial presequences allowing them to be bound by TOM20, thus increasing the concentration of mitochondrial 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 membrane of mitochondria in a subset of vascular plants was unexpected [41]. Whilst the absence of an orthologue to yeast TOM70 suggests that a receptor for cytosolic chaperones may be required for the delivery of hydrophobic mitochondrial proteins, OM64 is 67% identical to the plastid localised TOC64 [41]. The TOC64 family of Arabidopsis consists of three proteins, each of which contains similarities to two different

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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 has been inactivated by a point mutation in the active site and no longer plays an enzymatic role [42]. However the intermembrane space domain of TOC64-III has been shown to directly interact with the plastid preproteins [43]. The three TPR repeat motif located on the cytosolic side of the outer envelope plays a different role in plastid import, interacting with the cytosolic chaperone HSP90 for the delivery of nuclear encoded organelle proteins to the plastid import apparatus [44]. Detailed analysis of this 3-TPR domain revealed few differences between the plastid and mitochondrial localised forms, however residues located on the face opposite the chaperone interaction site were found to have diverged [45]. These differences may facilitate interaction with other components of the import apparatus rather than specify the types of proteins bound [45]. Blue native PAGE analysis of digitonin solubilised mitochondria followed by immunodetection of the mitochondrial OM64 showed that under these conditions, OM64 is not integrated into the TOM complex, suggesting that the interaction with other outer membrane components is transitory [32]. Functional studies have shown that depletion of OM64 affects the import of some mitochondrial proteins [32]. This defect however, like the removal of TOM20, does not result in any severe phenotypic abnormalities [32]. As mentioned in the Introduction, the absence of either TOM20 or TOM70 in yeast has a deleterious effect, though not lethal; however the absence of both proteins was lethal [23]. Furthermore, recent, unpublished work constructing an Arabidopsis mutant line absent in all of the TOM20 genes and OM64, results in an early embryo-lethal phenotype (Fig. 2). Thus, whilst the natures of the protein import receptors differ in terms of orthology between plants and yeast, both systems require at least one import receptor, either an interacting chaperone or direct presequence binding type in order to deliver incoming proteins to the TOM40 pore.

Parental genotype

A

Gene

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

t-DNA insertion

OM64 TOM20-2 TOM20-3 TOM20-4

B

Homozygous Heterozygous Homozygous Homozygous

Wild type

2.4. Metaxin Metaxin, like TOM20, OM64 is an outer membrane protein, however, unlike TOM20 or OM64, the removal of metaxin results in a severe delayed developmental phenotype in Arabidopsis [32]. Plants lacking metaxin were seen to develop more slowly, remain smaller than wild type, display lesions on developing and mature leaves and accumulate higher levels of starch than wild type plants [32]. The role of metaxin in protein import was first implicated through studies in mammalian systems in which the addition of the cytosolic domain of metaxin to in vitro import assays reduced the rates of protein uptake [46]. Subsequent studies in Arabidopsis have demonstrated a similar effect. The cytosolic domain of metaxin has been shown to directly and specifically interact with the presequences of a subset of precursors by both immunoprecipitation and yeast-2-hybrid interaction assays and the removal of metaxin was seen to result in dramatically reduced rates of import [32]. Despite these advances, the role of metaxin in mitochondrial protein import remains somewhat ambiguous. The absence of metaxin results in lower abundance of other proteins involved in import such as TOM20, OM64 and TIM17-2, making the direct effect of its absence difficult to distinguish from secondary effects [32]. Whilst import is dramatically reduced when metaxin is absent, it is not able to compensate for the loss of the other known import receptors, TOM20 and OM64 as evidenced by the absence of viable offspring from TOM20/OM64 knock out crosses (Fig. 2).

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12

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8

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Fig. 2. The combination of null mutations in Arabidopsis OM64 and each of the expressed TOM20 genes results in defective seed development. A) Silique produced by parental plant homozygous 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 the ratio of shrivelled (aborted) to normal seed.

Mitochondrial Import and Assembly (MIA) pathway consisting of MIA40 and ERV1 are also located in the IMS [47]. These are responsible for the oxidative folding and maturation of IMS proteins through the formation of disulphide bonds between cysteine residues in the disulphide 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, including plants [12]. TIM9 and 10 have been shown to be essential for yeast viability and form a hexameric chaperone complex that guides precursor proteins from the TOM complex to the inner membrane [48–50]. TIM8 and TIM13 form a similar chaperone complex, which has been shown to be involved in the import of TIM23, however, this function is not essential for yeast viability [50]. To date, plants are the only species for which a biochemical reconstitution assay of the tiny TIMs has been described [51]. In these assays, outer membrane ruptured mitochondria were depleted of IMS proteins, which were then fractionated by anion-exchange chromatography before reintroduction to in vitro import assays with the IMS depleted mitochondria [51]. This procedure revealed that the import of the two

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carrier proteins tested was positively correlated with the presence of TIM9 and TIM10, showing a direct functional role for these proteins in the efficient import and assembly of carrier pathway proteins [51]. A unique feature of carrier protein import in plant mitochondria is the existence of cleavable presequences [52]. In yeast, carrier proteins do not require the presence of N-terminal presequences, instead relying on multiple internal signals for interaction with components of the import apparatus [53]. Studies have shown that removal of this plant presequence does not affect import ability and that the presequence alone does not facilitate protein targeting to the mitochondria [54,55]. Further analysis showed that the presence of IMS proteins stimulates the import of carrier proteins containing N-terminal cleavable presequences to a greater extent than those with only internal targeting signals [56]. This raises the possibility that plants may contain additional import components that specifically recognise the N-terminal cleavable presequence of plant carrier proteins. Differences in the processing of these signals have also been observed for carrier proteins in plants [52]. N-terminal presequences from plant carrier proteins were first processed by the mitochondrial processing peptidase (MPP) on the matrix side of the inner membrane, and the remaining presequence was subsequently processed by an unknown serine protease in the IMS of Arabidopsis [52]. In contrast, presequences can only be processed at the site of MPP cleavage in yeast, with no further modifications required, and thus a unique two-step processing pathway was identified in plant mitochondria [52].

complex IV of the mitochondrial respiratory chain via TIM21 [65]. These supercomplexes are thought to form in order to promote efficient translocation at contact sites, utilising the proton motive force at cristae junctions [65,66]. The second form of the TIM17:23 complex, termed TIM23-PAM, functions in the transfer of proteins through the inner membrane to the matrix [61,67]. This complex consists of TIM17, TIM23, TIM50 and the presequence translocase associated motor (PAM) complex, mainly comprising of mtHSP70, TIM44 and its associated cochaperones; PAM16, PAM17 and PAM18 [61,67]. Plants appear to have orthologues to all of the components of the TIM17:23 translocase observed in yeast [20,66], with the addition of several plant specific features. In plants, the TIM17 protein differs from the yeast counterpart in that the plant TIM17 contains a C-terminal extension of 143 amino acids [56]. This C-terminal extension is present in all higher plant species and must be removed in order to complement yeast TIM17 deletion strains [56]. The C-terminal extension of Arabidopsis TIM17 has been shown to link the inner and outer mitochondrial membranes and exhibits presequence binding properties, however, the exact role for this C-terminal extension is still largely unknown [12,68,69]. In contrast, the yeast TIM23 has been shown to span the IMS, linking the inner and outer membranes and it has been shown to direct precursor proteins to the TIM17:23 pore [70]. In plants, TIM23 appears to lack any extension and only contains the four transmembrane spanning regions characteristic of a translocation pore of the inner membrane [69]. 4.2. The TIM22 complex

3.2. The Mia40–Erv1 pathway The disulphide relay system has been characterised in yeast and to a lesser extent in plants [57–59]. This pathway describes the import of proteins located in the IMS that contain conserved cysteine residues in twin CX(9)C or CX(3)C motifs [60]. This pathway consists of only two proteins, MIA40 and ERV1, both of which are essential for yeast viability [57,61–63]. MIA40 acts as a receptor for preproteins; transferring them from the outer membrane to the inter membrane space, where the conserved cysteine residues are oxidised by MIA40, which is subsequently oxidised by ERV1 [47]. Similarly, plants contain MIA40 and ERV1 proteins, however, unlike yeast, plant MIA40 has been determined to be co-localised to both the mitochondria and peroxisomes [58]. In addition, the deletion of MIA40 in Arabidopsis does not result in an embryo lethal phenotype, or in the disruption of the disulphide relay system, whilst disruption of ERV1 does result in an embryo lethal phenotype [64]. This demonstrates that the plant disulphide relay system can operate without MIA40 and that this pathway is functional in the presence of ERV1 alone [64]. 4. Inner membrane Proteins targeted to the inner membrane or the matrix are passed to the TIM22 or TIM17:23 complexes of the inner membrane (Fig. 1). Preproteins are either directly transferred from the outer membrane import channel or chaperoned through the IMS [20]. The TIM22 and TIM17:23 complexes consist of channel forming pores and associated proteins involved in the mechanisms of translocation and assembly [20]. 4.1. The TIM17:23 complex The TIM17:23 complex is the major translocation pore responsible for the import of the majority of the mitochondrial proteome. Two functionally and structurally distinct forms, which are in dynamic equilibrium have been characterised in yeast [20]. The initial form, TIM23-SORT, containing TIM23, TIM17, TIM50 and TIM21, is involved in the recognition of precursors from the TOM complex and insertion into the inner membrane [20]. This complex has been shown to form supercomplexes with the TOM40 channel or with complex III and

The TIM22 complex is responsible for the translocation of multispanning membrane proteins through and into the inner membrane. In yeast, this complex consists of 4 subunits, the TIM22 channel and accessory proteins TIM54, TIM18 and TIM12, of which only TIM12 is essential for yeast viability, most likely due to its linker role with the tiny TIMs [71–74]. Two identical genes on different chromosomes encode the Arabidopsis homologue for TIM22, and the encoded protein has been shown to have the ability to complement a yeast deletion strain of TIM22 [75]. Homologues to TIM54, 18 and 12 cannot be identified in any plant genome database [12] and as such, the TIM22 channel may function alone or with yet undetermined plant specific proteins. 4.3. Multi-gene families In Arabidopsis, TIM17, TIM23 and TIM22 belong to a large gene family consisting of 17 members, termed the preprotein and amino acid transporters (PRAT) [75,76]. These proteins are thought to originate from a single eubacterial ancestor, and are characterised by four transmembrane regions connected by three short hydrophilic loops, with a characteristic motif: [G/A]X2[F/Y}X10RX3Dx6[G/A/S]GX3G, where X is any amino acid [76]. The 17 loci encode 16 different PRAT proteins (two proteins are 100% identical) that exhibit significant diversity in size, structure and location [75]. The predicted length of these 16 proteins ranges from 133 to 261 aa. Furthermore, the PRAT domain is only present in 7 of the 16 predicted proteins, with 4 members containing a degenerate PRAT domain [75]. Of all 16 proteins: 11 were determined to be mitochondrial, 4 chloroplastic, and one targeted to both organelles [75]. The 11 PRAT proteins localised to the mitochondria fall into three subfamilies based on homology to yeast TIM17, TIM23 and TIM22, respectively. Three genes encode TIM17 (At1g20250, At2g37410 and At5g11690), 3 genes encode TIM23 (At1g72750, At1g17530 and At3g04800), and two genes encode TIM22 (At3g10110 and At1g18320) (Fig. 3). An additional three genes encode proteins that have no obvious yeast/mammalian counterparts but are largely homologous to the known Arabidopsis PRATs, these are termed as “unknown”, as they are yet to be functionally annotated (Fig. 3). Considering that, in yeast, only one gene encodes one protein, for each of the TIM17, TIM23 and

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yeast orthologs exist

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TIM17-1 At1g20350

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TIM22 At3g10110 At1g18320

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Unknown At2g42210

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Unknown At3g25120

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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 for TIM23 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 proteins are 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-terminal extensions is indicated in black.

TIM22 proteins, 17 family members in Arabidopsis represent a large expansion of this transporter family and raise the possibility that these proteins have acquired new functions. Considerable diversity exists within these subfamilies. For example, the TIM17 subfamily (discussed in Section 4.1) consists of three genes, two of which contain the plant specific C-terminal extensions, whilst the third lacks this extension and encodes a degenerate PRAT domain (Fig. 3). A recent study in Trypanosoma brucei implicated TIM17 to be involved in tRNA import [77]. Whilst the T. brucei TIM17 does not contain the long C-terminal extension present in plants, sequence similarities are observed, hinting that a conservation of functions is also possible. Systematic liquid chromatography coupled to mass spectrometry (LC/MS) analysis of mitochondria separated by BN-PAGE showed that the TIM17-2 migrates in complex of 90 and 200 kDa [78]. This is similar to the yeast model that shows two distinct translocation complexes of TIM23-SORT and TIM23-PAM [78]. However, it is notable that TIM17-3 was observed to migrate at 110 kDa, distinct from other known TIM23 complex components, and possibly associated with TIM9 and TIM10, which also migrate as a complex of approximately 110 kDa [78]. Transcript abundance of TIM17-1 has been positively correlated with the mitochondrial stress response [79,80]. This transcript was 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 an inverse change in TIM17-2 abundance, suggesting diverse functions for this subfamily (Fig. 4). The expression of TIM17-1 is very low in all tissues and throughout development, suggesting that it may only be required in a stress responsive role (Fig. 4). Further supporting this is the finding that the TIM17-1 promoter region contains a binding site for WRKY transcription factors, which are well documented for their role in plant stress and defence [81]. A relationship between protein import and stress responses has previously been observed, with a number of environmental stresses being shown to differentially affect the rate of import of a number of nuclear encoded mitochondrial proteins [82]. The induction of TIM17-1 and a corresponding increase in protein abundance in response to stress may function to alter the rate of mitochondrial import in order to better meet the needs of the plant. The mitochondrial PRAT genes were seen to exhibit various levels of expression

throughout development and in a variety of tissue types within the gene family (Fig. 4). As import ability has previously been shown to be a regulated process throughout plant development, the observed differences in expression of these genes may serve to regulate mitochondrial biogenesis [83,84]. Determining the functions of the “unknown” plant specific Arabidopsis putative PRAT family members is of particular interest as this may give greater insight into the reason for the expansion of this family. The proteins encoded by At2g42210, At3g25120 and At5g49560 have no direct yeast or mammalian orthologue and whilst these do exhibit the characteristics of the PRAT transporters family, early investigations (outlined below) suggest that their functions maybe somewhat different (Fig. 3). The protein encoded by At2g42210 has been shown to be a component of complex I of the respiratory pathway [80,87], and surprisingly, in the same study, TIM22 was also identified alongside members of respiratory complex I [78]. In addition, another PRAT protein encoded by At2g26670 may also have links to complex I, as it is orthologous to a bovine complex I subunit termed B14.7. Although this protein has been identified in chloroplasts by immunodetection [85], independent in vitro and in vivo protein targeting experiments were inconclusive [75]. Thus, further investigation is necessary to reveal whether this is a mitochondrial location, and whether it is associated with complex I. 4.4. Mitochondrial processing peptidase Mitochondria contain a large number of peptidases involved in protein turnover. Several peptidases are involved in the protein import pathway either through removal of the targeting peptide, formation of peptide intermediates or degradation of the signal peptide after cleavage. The mitochondrial processing peptidase (MPP) is an ATP independent protease involved in cleavage of the targeting peptide from the translocating precursor protein for the majority of mitochondrial matrix proteins. In yeast and mammals, the MPP contains two structurally related components, MPPα and MPPβ both of which reside in the matrix [86]. In contrast, whilst the plant MPP exhibits high sequence similarity to the yeast MPP, the Arabidopsis MPP is unique as both MPPα and MPPβ subunits are located on the inner mitochondrial membrane, being integrated into the cytochrome bc1 complex [87,88]. Thus, the cytochrome

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callus cell culture sperm cell protoplast guard protoplast mesophyll protoplast seedling cotyledon hypocotyl radical imbibed seed inflorescence flower pistil petal sepal stamen anther pollen abscission zone pedicel silique replum seed stem node shoot apex cauline leaf rosette juvenile leaf adult leaf petiole senecent leaf hypocotyl stem shoot apex roots

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germinated seed seedling young rosetee developing rosette bolting young flower deveoped flower flowers and siliques mature siliques

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cold study 6 CaLcuV P. syringae cycloheximide rotenone (12h) syrinogolin early syrinogolin late dark/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 early genotoxic late salt study 2 early salt study 2 late

Fig. 4. Expression profiles of genes encoding the mitochondrial located preprotein and amino acid transporters (PRATs) gene family. Geneinvestigator profile showing the expression levels 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 represent expression level, and the colour intensity is shown as computed in Genevestigator after MAS5 normalization of data from Affymetrix microarrays [118].

bc1 complex in Arabidopsis has two roles; mitochondrial electron transport and protein import [89]. With the exception of MPP, the characterisation of plant mitochondrial proteases is still in its infancy. Initial studies to date, suggest that plant proteases share some unique characteristics; Arabidopsis proteases are encoded by large multi-gene families, several proteases are dual targeted to the chloroplast and the mitochondria and functionally, some proteases are distinct from their yeast counterparts [90]. The presequence protease (PreP) was first identified and characterised in plants, and has been shown to be located in both mitochondria and chloroplasts [91]. In Arabidopsis, it was shown that PreP is involved in the degradation of the presequence peptide signal following import [91]. Insertional inactivation of the two genes encoding PreP in Arabidopsis resulted in a severe growth phenotype [92] and given that mammalian PreP has been linked to the degradation of amyloid-β a peptide linked to Alzheimer's disease, PreP is considered highly important in maintaining mitochondrial integrity [77,93,94]. Another group of proteases, AAA (ATPase Associated with diverse cellular Activities) proteases contain a number of family members, which exhibit a wide range of functions in a variety of species. In Arabidopsis, ten members of this gene family were found to be mitochondrial and initial functional characterisation suggests emerging structural differences to the yeast orthologues [95]. Two of these are related to the bacterial FtsH protease. These are membrane bound metalloproteases, each spanning the inner membrane twice and differ in their topology, with the proteolytic domain of the

i-AAA proteases facing the intermembrane space and the m-AAA proteases facing the matrix. Both of these subtypes are found in high molecular weight complexes with the i subtype forming complexes of ~1500 kDa [48] of unknown composition and the m subtype being found in complexes of 2 mDa in association with prohibitins [49]. Interestingly, the i-AAA ATPase of yeast (named Yme1) has been shown to be necessary for the import of the IMS located PNPase [96]. This activity is not dependent on its function in proteolysis; instead, Yme1 acts as a nonconventional translocation motor, pulling PNPase into the IMS after cleavage of its targeting signal by MPP [96]. The rhomboid protease family is another large multi-gene family, encoded by 12 genes in the Arabidopsis genome. Thus far, only RBL12 has been confirmed to be mitochondrial and has been shown to have different substrate specificity to its yeast counterpart, Pcp1 [97]. The large number of types of proteases and gene family members involved in mitochondrial biogenesis complicates their functional characterisation, though divergent roles are expected to be uncovered. 5. Outstanding questions It remains to be seen whether the components discussed above are the only unique components of the plant mitochondrial import apparatus. Evidence suggests that there are other, as yet uncharacterised components involved. This evidence includes the presence of multiple α-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 component capable of inserting such proteins is yet to be characterised and obvious candidates such as members of the TOM complex have been shown not to be involved [17]. Additionally, examination of the import of a dual targeted glutathione reductase showed that the import of this protein is independent of either TOM20 or OM64, indicating the possible presence 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]. SAM35 and SAM37 are thought to be related to human metaxin 1 and metaxin 2 respectively [100,101]. In Arabidopsis, the composition of the SAM complex is yet to be determined and whilst two genes encoding SAM50 have been identified, there is only one metaxin gene, raising the possibility of additional plant specific components in this complex, or that the single plant metaxin protein is responsible for the roles of SAM35 and SAM37 [102]. Basic Local Alignment Search Tool (BLAST) analysis of the Arabidopsis genome has so far failed to identify a homolog of the final subunit of this complex, yeast MDM10. MDM10 in yeast is a β-barrel protein that also associates with an ER protein, MMM1, and the outer 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 from the Arabidopsis genome and, as well as mediating the mitochondrial/ ER interaction, the complex has been shown to play a role in the assembly of β-barrel proteins, associating with both the TOM and SAM complexes [105]. The absence of genes encoding subunits of the ERMES complex in Arabidopsis certainly suggests that other, plant specific proteins are involved in the process of mitochondrial protein import and assembly. One of the major hurdles in determining which proteins, if any, are fulfilling the roles of apparently missing components is the complexity of the Arabidopsis genome. Whilst this is a relatively small and simple genome of 157 Mbp [106] compared to other plant genomes such as Paris japonica, which is 150 Gbp [107], accurately identifying distantly related proteins from the estimated ~35,000 translated Arabidopsis genes is not a simple task. Even with rigorous bioinformatic studies, reverse genetic approaches often require experimental analysis of numerous ‘false positives’ with no guarantee of success. Sub-organelle proteomic studies can greatly accelerate this process by reducing the numbers of candidate proteins for a given function, and also have the advantage of exposing unexpected functional and evolutionary relationships (for example [98]). The recent definition of the mitochondrial outer membrane proteome of Arabidopsis has revealed numerous components including several specific to plants, which are potentially involved in the processes of protein import and assembly of the outer membrane [98]. One candidate protein observed in the course of this research — anion transporting ATPase (At5g60730.1) is related to a bacterial arsenate transporter. An orthologue of this protein studied in yeast appears to have lost its transport function [108,109] and gained a role in inserting tail anchored proteins into the endoplasmic reticulum post-translationally [110]. The discovery of a mitochondrial located orthologue of this protein in Arabidopsis [98] suggests that in plants at least, this family may be involved in the insertion of outer mitochondrial membrane proteins into the lipid bi-layer. Although no proteins directly related to components of the ERMES complex were discovered, a previously unknown, plant specific β-barrel protein was localised to the outer mitochondrial membrane [98] and its role in protein import and endoplasmic reticulum interaction is currently under investigation. The functional significance of the differences observed in the MIA ERV1 pathway compared to this pathway in yeast, is as yet unclear, though a unique feature of plant mitochondria is the occurrence of the ascorbate biosynthesis pathway within the IMS [111]. Given that ascorbate has been shown to participate in oxidative folding [90], this process may be utilised by the MIA import pathway in plants. Whilst MIA40 insertional knockouts in Arabidopsis did not exhibit any changes in total ascorbate concentrations [58], plant MIA40 is not essential and thus the disulphide relay system can be carried out regardless of the presence

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of MIA40. Organisms lacking MIA40 have a different arrangement of cysteine motifs within ERV1 similar to the plant ERV1, and thus in plants, ERV1 may function independently in the oxidative folding of cysteine residues [58]. Recent characterisation of protein complexes in plant mitochondria has uncovered a number of expected and unexpected protein complex associations [65,112–114]. These include the formation of super complexes between 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 the Cyt bc1/COX supercomplex in yeast [113]. This occurrence has only recently been found in plants, with Arabidopsis TIM23 shown to interact with complex III of the respiratory chain, via TIM21, similar to the yeast model (Wang and Murcha, personal communication). In addition, evidence that Arabidopsis TIM23 interacts with complex I of the respiratory chain has also been obtained (Wang and Murcha, personal communication). As yeast lacks complex I and has always been the primary eukaryotic model for these types of studies, this interaction between complex I and TIM23 has never been previously reported. It has been suggested that there are around 4000 plant specific proteins encoded in the Arabidopsis genome [117], and with the specific challenges of mitochondrial protein import in plants, it seems likely that new components will be discovered as the various import complexes are better characterised. It is also worth noting that, whilst it has been shown that multiple isomers of import components can have overlapping functions [32], more detailed functional characterisation may uncover specialised roles for individual members that have evolved in response to the complexities specific to plants. Acknowledgements We would like to thank Dr. Reena Narsai (University of Western Australia) for discussion and comments on the manuscript. MWM is a recipient of the Australian Research Council APD Fellowship and an Australian Research Council Centre of Excellence Program grant CEO561495 to JW. References [1] M.W. Gray, G. Burger, B.F. Lang, The origin and early evolution of mitochondria, Genome Biol. 2 (2001) REVIEWS1018. [2] K.L. Adams, J.D. Palmer, Evolution of mitochondrial gene content: gene loss and transfer to the nucleus, Mol. Phylogenet. Evol. 29 (2003) 380–395. [3] T. Lithgow, A. Schneider, Evolution of macromolecular import pathways in mitochondria, hydrogenosomes and mitosomes, Philos. Trans. R. Soc. Lond. B Biol. Sci. 365 (2010) 799–817. [4] D. Macasev, J. Whelan, E. Newbigin, M.C. Silva-Filho, T.D. Mulhern, T. Lithgow, Tom22′, an 8-kDa trans-site receptor in plants and protozoans, is a conserved feature of the TOM complex that appeared early in the evolution of eukaryotes, Mol. Biol. Evol. 21 (2004) 1557–1564. [5] S.A. Paschen, T. Waizenegger, T. Stan, M. Preuss, M. Cyrklaff, K. Hell, D. Rapaport, W. Neupert, Evolutionary conservation of biogenesis of beta-barrel membrane proteins, Nature 426 (2003) 862–866. [6] P. Dolezal, V. Likic, J. Tachezy, T. Lithgow, Evolution of the molecular machines for protein import into mitochondria, Science 313 (2006) 314–318. [7] C.X. Chan, J. Gross, H.S. Yoon, D. Bhattacharya, Plastid origin and evolution: new models provide insights into old problems, Plant Physiol. 155 (2011) 1552–1560. [8] D. Macasev, E. Newbigin, J. Whelan, T. Lithgow, How do plant mitochondria avoid importing chloroplast proteins? Components of the import apparatus Tom20 and Tom22 from Arabidopsis differ from their fungal counterparts, Plant Physiol. 123 (2000) 811–816. [9] G. von Heijne, Mitochondrial targeting sequences may form amphiphilic helices, EMBO J. 5 (1986) 1335–1342. [10] G. von Heijne, J. Steppuhn, R.G. Herrmann, Domain structure of mitochondrial and chloroplast targeting peptides, Eur. J. Biochem. 180 (1989) 535–545. [11] E.C. Hurt, N. Soltanifar, M. Goldschmidt-Clermont, J.D. Rochaix, G. Schatz, The cleavable pre-sequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria, EMBO J. 5 (1986) 1343–1350. [12] C. Carrie, M.W. Murcha, J. Whelan, An in silico analysis of the mitochondrial protein import apparatus of plants, BMC Plant Biol. 10 (2010) 249. [13] R. Lister, O. Chew, M.N. Lee, J.L. Heazlewood, R. Clifton, K.L. Parker, A.H. Millar, J. Whelan, A transcriptomic and proteomic characterization of the Arabidopsis mitochondrial protein import apparatus and its response to mitochondrial dysfunction, Plant Physiol. 134 (2004) 777–789.

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