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FEBS Letters 588 (2014) 2484–2495

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Review Mitochondrial translocases for survival and wellbeing

Anna Magdalena Sokol a, Malgorzata Eliza Sztolsztener a, Michal Wasilewski a, Eva Heinz b,c, ⇑ Agnieszka Chacinska a,

a Laboratory of Mitochondrial Biogenesis, International Institute of Molecular and Cell Biology, Warsaw 02-109, Poland b Department of Microbiology, Monash University, Melbourne, Victoria 3800, Australia c Victorian Bioinformatics Consortium, Monash University, Melbourne, Victoria 3800, Australia

article info abstract

Article history: Mitochondria are involved in many essential cellular activities. These broad functions explicate the Received 28 April 2014 need for the well-orchestrated biogenesis of mitochondrial to avoid death and pathological Revised 15 May 2014 consequences, both in unicellular and more complex organisms. Yeast as a model organism has Accepted 15 May 2014 been pivotal in identifying components and mechanisms that drive the transport and sorting of Available online 24 May 2014 nuclear-encoded mitochondrial proteins. The machinery components that are involved in the Edited by Wilhelm Just import of mitochondrial proteins are generally evolutionarily conserved within the eukaryotic king- dom. However, topological and functional differences have been observed. We review the similari- ties and differences in mitochondrial translocases from yeast to human. Additionally, we provide a Keywords: Cancer systematic overview of the contribution of mitochondrial import machineries to human patholo- HIF1a gies, including cancer, mitochondrial diseases, and neurodegeneration. Mitochondrial disease Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Neurodegeneration p53 Protein biogenesis and import

1. Introduction mitochondrial compartments. This trafficking is based on sorting signals included within the precursors’ primary or secondary Mitochondria are semi-autonomous organelles that possess structures [5–8]. their own DNA and translational machinery. This is the legacy from their mitochondrial ancestor, the endosymbiotic proteobacterium 2. Principles of mitochondrial protein import: lessons from [1]. Mitochondrial DNA encodes only a handful of proteins that yeast are central to the process of oxidative phosphorylation. Therefore, the mitochondrial proteome, which consists of approximately 800 Extensive studies of mitochondrial protein import using the (in yeast) to 1500 (in mammals) proteins, needs to be substantially model yeast Saccharomyces cerevisiae uncovered the components complemented with nuclear-encoded proteins [2,3]. Upon synthe- of different translocases and provided detailed mechanistic and sis on cytosolic ribosomes, immature mitochondrial precursor pro- topological information about their function and interplay [5–8]. teins require chaperone-assisted transfer to the mitochondrial The translocase of the outer mitochondrial membrane (TOM) com- surface. The sorting of precursors within mitochondria is an intri- plex is a general entry gate for mitochondrial precursor proteins cate process because of the complex architecture of the organelle that are synthesized in the cytosol (Fig. 1A). Its components recog- [4]. Mitochondria are surrounded by two membranes that are nize a precursor protein and pass it to specialized import machin- highly divergent with regard to their function and lipid and protein eries. The precursors of b-barrel proteins are sorted to the OMM by composition. Both the outer mitochondrial membrane (OMM) and sorting and assembly machinery (SAM; Fig. 1A). The import of inner mitochondrial membrane (IMM) define two aqueous mito- small IMS proteins with multiple cysteine residues occurs because chondrial subcompartments, the intermembrane space (IMS) and of the thiol-disulfide exchange via the mitochondrial intermem- the matrix. To reach their destined mitochondrial milieu, precursor brane space assembly (MIA) pathway (Fig. 1B). The translocase of proteins are driven by various import machineries to different the inner mitochondrial membrane 23 (TIM23) complex directs presequence-containing precursors to the matrix, IMM, or IMS ⇑ Corresponding author. (Fig. 1C). Metabolite carriers that reside in the IMM and contain E-mail address: [email protected] (A. Chacinska). multiple internal transmembrane signals are embedded into the

http://dx.doi.org/10.1016/j.febslet.2014.05.028 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495 2485

Fig. 1. Protein transport pathways into yeast Saccharomyces cerevisiae mitochondria. (A) General entry gate for mitochondrial precursor proteins and sorting into the outer mitochondrial membrane (OMM). Mitochondrial proteins are synthesized in the cytosol in precursor forms. Their delivery to the mitochondrial surface is assisted by cytosolic chaperones that maintain precursor proteins in an import-competent state. Precursors enter mitochondria through the translocase of the outer membrane (TOM), the general entry gate, and are further directed to their final mitochondrial destinations with assistance from specific sorting machineries. Precursor proteins that contain a-helical transmembrane segments are inserted into the OMM by the insertase of the mitochondrial outer membrane (MIM) for assembly. More complex precursors with b-barrel topology are transported via TOM into the intermembrane space (IMS) and handed over to the sorting and assembly machinery (SAM) with the help of small Tim chaperone complexes. (B) Oxidation-driven import into the intermembrane space (IMS). Cysteine-rich precursor proteins are delivered into the IMS via the mitochondrial intermembrane space assembly (MIA) pathway. Emerging from the TOM complex, they transiently bind Mia40 via disulfide bonds. Mia40 oxidizes and folds precursor proteins. The reoxidation of Mia40 is mediated by Erv1, which shuttles released electrons via cytochrome c to cytochrome c oxidase (complex IV). (C) Translocation to the matrix and inner mitochondrial membrane (IMM). Presequence-containing precursors are delivered to the matrix and inner membrane in an inner membrane electrochemical potential (Dw)-dependent manner via the presequence translocase TIM23. TIM23 is associated with the presequence translocase-associated motor (PAM) and transfers precursor proteins into the matrix in an ATP-dependent manner. The insertion of presequence-containing precursors into the inner membrane is driven by the TIM23SORT complex, marked by the presence of Tim21. Upon delivery into the matrix, the presequences are recognized and cleaved by matrix-processing peptidase (MPP). The hydrophobic precursors of carrier proteins are guided through the IMS by small TIM chaperone complexes that deliver precursors to the carrier translocase of the inner membrane, the TIM22 complex. The membrane insertion of carrier precursors via the TIM22 complex is driven by the electrochemical potential across the inner membrane. The mitochondrial protein export machinery (OXA) participates in the biogenesis of some cytosol-translated multispanning membrane proteins that are targeted to the IMM via the TIM22 and TIM23 translocases. eÀ, electron.

IMM by the carrier translocase of the inner mitochondrial mem- subunits perform important functions in maintaining the architec- brane 22 (TIM22) complex [5–8] (Fig. 1C). ture of the TOM complex and passage of precursor proteins. Tom20 and Tom70 are membrane-anchored proteins that are loosely asso- 2.1. Yeast outer membrane translocase: a closer look ciated with the TOM complex. Through their cytosol-exposed C-terminal domains, they serve as receptors for various classes of All mitochondrial precursors enter the organelle in an unfolded, incoming precursor proteins. Tom22 is a core subunit of TOM import-competent state via the TOM complex [9–11] (Fig. 1A). The and performs two essential functions. First, Tom22 maintains the central component and a channel-forming subunit of the TOM architecture of the TOM complex. Second, it provides further bind- complex is a membrane b-barrel protein, Tom40. Additional ing sites for mitochondrial precursor proteins on both sides of the 2486 A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495

OMM. The assembly and stability of TOM are also secured by precursor’s cysteine residue that forms a transient disulfide bond Tom5, Tom6, and Tom7. Additionally, Tom5 directly participates with the redox active Mia40. The efficient recognition of a precur- in the transfer of precursors [9–11]. Mitochondrial precursor pro- sor by Mia40 is promoted by the localization of Mia40 in close teins are recognized and transferred into the mitochondrial inte- proximity to TOM. The Mic60 (Fcj1/Mitofilin) protein, a component rior through the TOM complex and subsequently directed to of the mitochondrial contact site and cristae-organizing system their final submitochondrial location by respective downstream MICOS (MINOS/MitOS), plays a role in Mia40 positioning as it binds machineries. both Mia40 and TOM [19,21]. The formation of a transient complex between Mia40 and its 2.2. Biogenesis of b-barrel and a-helical proteins of the OMM substrates is an important initial step in the oxidation-coupled bio- genesis of IMS proteins [10,16–19] (Fig. 1B). Upon the transfer of a The OMM of mitochondria possesses a class of membrane pro- disulfide bond from Mia40 to the substrate, the CPC motif of Mia40 teins with b-barrel topology, such as Tom40 and porins (VDAC). is reduced and subsequently reoxidized by sulfhydryl oxidase Erv1 The machinery responsible for the assembly and OMM integration to be ready for the next round of substrate binding. This simple of b-barrel proteins is the SAM complex [5,9,10,12]. Initially, function of Erv1 in disulfide relay is well proven by in vitro and b-barrel precursors are translocated through TOM into the IMS structural data. However, in vivo experiments demonstrated that where they are assisted by the Tim9-Tim10 and Tim8-Tim13 chap- Erv1 forms a transient ternary complex with Mia40 and a substrate erone complexes to reach the SAM. Recognition by the SAM com- that promotes its efficient release in a fully oxidized form that typ- plex is driven by the signal embedded in the primary structure of ically contains two disulfide bonds. Electrons that are received in b-barrel substrate proteins, called b-signal. The SAM complex con- the course of IMS oxidation reactions are transferred from the pre- sists of three core components, Sam50, Sam35, and Sam37 cursor proteins through Mia40 and the redox centers of Erv1, (Fig. 1A). Sam50 and Sam37 are essential in yeast. Sam50 with a including FAD cofactor to cytochrome c and respiratory chain com- POTRA domain cooperates with Sam35 and Sam37 in the recogni- plex IV, before they reach molecular oxygen as a final acceptor tion, transfer, and release of b-barrel proteins into the lipid bilayer [10,16–19] (Fig. 1B). In addition to the biogenesis of small IMS pro- (Fig. 1A). Additionally, the SAM complex associates with Mdm10, teins, Mia40 functions as a disulfide carrier and chaperone that which is specifically required in the late stage of TOM complex supports the biogenesis of non-canonical substrates, including assembly [5,9,10,12]. The effectiveness of protein sorting is the integral membrane protein Tim22, peripheral membrane- improved by the coupling of TOM and SAM because they are not bound Atp23, and matrix-localized Mrp10 [22–24]. independently acting machineries [13]. Outer mitochondrial mem- brane proteins that are embedded in the OMM with the help of one 2.4. Import of precursor proteins with a cleavable presequence or more a-helical transmembrane segments use TOM complex receptors in a unique way, in which they do not appear to be direc- Over half of all mitochondrial precursor proteins are synthe- ted to the protein-conducting channel of TOM. Instead, these pro- sized with targeting signals at their N-termini, which are called teins are sorted via the mitochondrial import complex, MIM, into presequences, and cleaved inside mitochondria [25]. One charac- the OMM. MIM functions as a heterooligomeric complex that com- teristic feature of presequences is their a-helical structure with prises two subunits, Mim1 and Mim2 [7–9,11] (Fig. 1A). Recently, positively charged and hydrophobic surfaces (Fig. 1C). After cross- the abundant OMM protein, Om45, was shown to be integrated to ing the TOM channel, precursors are directed to the TIM23 com- the OMM from the IMS site with the involvement of the IMM plex [6,26–30]. The TIM23 or presequence pathway is utilized by machinery, TIM23 [14,15]. proteins that are targeted to the matrix and also partially by pro- teins that are localized to the IMM and IMS. Import via the prese- 2.3. Oxidation-driven import into the IMS quence pathway occurs in an IMM electrochemical potential- and (ATP)–dependent manner. The TIM23 The biogenesis of small IMS proteins proceeds via the MIA path- complex is composed of three essential IMM proteins, Tim50, way [10,16–19] (Fig. 1B). Interestingly, small IMS proteins are reg- Tim23, and Tim17. Tim50 serves as a receptor in the initial stages ulated by the ubiquitin-proteasome system prior to their when precursors are passed from TOM to the TIM23 channel. mitochondrial import via MIA [20]. Mitochondrial intermembrane Tim23 and Tim17 have multiple transmembrane helices and form space assembly substrates are small soluble proteins that share a a core channel part of the complex (Fig. 1C). The electrochemical characteristic feature of cysteine residues, typically arranged in potential of the IMM plays an important role in protein transloca-

CX3C and CX9C motifs. An example of MIA substrates with a tion through TIM23 by exerting an electrophoretic force on the CX3C motif are small Tim proteins, whereas many other proteins, positively charged presequence. Additionally, conformational rear- including components involved in the biogenesis of cytochrome c rangements of these components in response to electrochemical oxidase, Cox17 and Cox19, contain a CX9C motif. The formation potential provide the means to sense and regulate the gating of of disulfide bonds between the cysteine residues upon import into the TIM23 channel [6,26–31]. the mitochondria stabilizes the folded structure of these proteins TIM23 associates with the presequence translocase-associated and prevents their leakage from mitochondria. motor (PAM) to guide presequence precursors into the matrix The MIA import pathway is responsible for the catalytic forma- [6,26–30]. mtHsp70 (Ssc1) is a central component of PAM that tion of disulfide bonds within substrates, thereby coupling the binds precursor proteins and utilizes ATP, thereby contributing to import of precursor proteins with their oxidative folding inside their movement inside the organelle (Fig. 1C). Tim44 is peripher- the IMS [10,16–19]. The essential components of the MIA machin- ally associated with IMM and mediates mtHsp70 attachment to ery are Mia40 oxidoreductase and the sulfhydryl oxidase Erv1. TIM23. Additionally, PAM consists of an array of co-chaperones Mia40 is anchored in the IMM, exposing the C-terminal domain and helper proteins, including the J-proteins Pam18 and Pam16, into the IMS (Fig. 1B). Serving as a trans-receptor, Mia40 specifi- the nucleotide exchange factor Mge1, and Pam17, which assists cally recognizes and transiently forms disulfide bonds with with the recruitment of Pam18 and Pam16 to TIM23 (Fig. 1C). reduced precursors that emerge from the TOM channel. Specificity In addition to the TIM23-PAM complex, another version, called is ensured by the presence of mitochondrial intermembrane space TIM23SORT, exists that is functionally different from TIM23-PAM sorting signals within IMS proteins, known as MISS/ITS. These [6,27,29] (Fig. 1C). TIM23SORT is capable of the IMM insertion of signals direct the hydrophobicity-driven positioning of a specific presequence proteins if the presequence is followed by the A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495 2487 hydrophobic transmembrane sorting signal. This functional differ- 3. Architecture and function of mitochondrial protein ence is underscored by changes in the architecture of the complex, translocases in evolution in which the PAM complex is substituted by Tim21. Tim21 plays an important role in the productive transfer of precursor proteins Mitochondrial protein import machineries have been inten- from TOM to TIM23 and bridging of TIM23 with respiratory chain sively studied, both at the molecular and mechanistic levels. How- complexes III and IV. Its interaction with TIM23 is supported by the ever, most of our current knowledge stems from studies that were presence of Mgr2 (Fig. 1C). In addition to Tim21 and Mgr2, Tim17 performed on the yeast S. cerevisiae as a model organism. High evo- was also shown to play an important role in the lateral sorting of lutionary conservation of the import components within the membrane precursor proteins [6,26–30]. eukaryotic kingdom is a striking feature of mitochondrial translo- The presequence-containing precursor proteins undergo vari- cases (Fig. 2). In this chapter, we review the human mitochondrial ous maturation steps [25]. The most important and common event translocases and discuss differences between yeast and mamma- for a large majority of these proteins is the removal of a positively lian mitochondrial import systems. charged presequence, frequently just upon its appearance on the matrix side of TIM23 (Fig. 1C). This important step is executed by 3.1. Biogenesis of outer membrane proteins mitochondrial processing peptidase (MPP). Some matrix-localized proteins require further processing by Icp55 or Oct1 to gain stable The human TOM complex consists of TOMM40, TOMM22, forms. The TIM23 complex is also utilized by a subset of prese- TOMM20, TOMM70A, TOMM5, TOMM6, and TOMM7 (Fig. 2A). quence-containing precursors that are targeted to the IMS. These The engagement of TOM receptors with cytoplasmic chaperones precursors contain the classical MPP-cleavable presequence, fol- that mediate the transition of precursor proteins from ribosomes lowed by a transmembrane domain that is cleaved by the inner to mitochondria has been found to differ between species. In yeast, membrane protease (IMP) at the IMS side of IMM. Upon the second interactions between Tom70 and hydrophobic precursors that cleavage, the mature form of the protein is released into the IMS in carry an internal sorting signal are regulated by cytosolic Hsp70. a soluble form [25]. In humans, a multichaperone complex of Hsp90 and Hsp70 deliv- ers precursor proteins to TOMM70A for subsequent import [37] 2.5. Import of proteins with multiple internal signals (Fig. 2A). Additionally, the TOMM34 co-chaperone, which does not have a yeast homologue, has been found to interact with the A separate IMM translocase, called TIM22, is specialized in the Hsp70/Hsp90 complex [38,39] (Fig. 2A). As TOMM34–/– mice sus- biogenesis of hydrophobic proteins of the carrier family and other tain efficient mitochondrial import, the function of TOMM34 as a multispanning IMM proteins, including translocase components mitochondrial precursor chaperone remains to be clarified [40]. Tim23, Tim22, and Tim17 [5,27,32,33]. This group contains multi- Interestingly, TOMM20 was found to interact with the arylhydro- ple hydrophobic segments that are dispersed throughout their carbon receptor-interacting protein (AIP), a cytosolic factor with polypeptide chain and is not proteolytically processed upon their chaperone-like activity [41]. IMM integration. The biogenesis of metabolite carrier precursors Classically, TOMM20 serves as a receptor for mitochondrial pro- requires the assistance of cytosolic chaperones that are engaged tein precursors that harbor an N-terminally localized targeting sig- in the efficient passage of newly synthesized precursors and pre- nal, whereas TOMM70A engages proteins that carry an internal vent their aggregation. Upon recognition by Tom70 and ATP sorting signal [5,6]. Unlike in yeast, the translocation of human hydrolysis, the chaperones are released, and the precursors are precursor proteins from TOMM70A to the membrane channel is handed over to the Tom40 channel to be transferred in a loop for- assisted by TOMM20 and its affinity for the precursor-binding tet- mation (Fig. 1C). Once on the IMS side, the aggregation of hydro- ratricopeptide repeat (TRP) domain of TOMM70A [42] (Fig. 2A). phobic precursors is prevented by binding to small Tim The human TOM complex contains the small TOMM5, TOMM6, chaperone complexes, Tim9-Tim10 or Tim8-Tim13. The hydro- and TOMM7 proteins, which are involved in the assembly and sta- phobic precursors are delivered to TIM22 with help from the bility of the core complex [43] (Fig. 2A). Interestingly, their func- Tim9-Tim10-Tim12 complex that is peripherally bound to tion is not conserved within the eukaryotic kingdom. The yeast TIM22 (Fig. 1C). Tim22, a core subunit of TIM22, forms a pore TOM complex is stabilized by Tom6 and more dynamic or destabi- and promotes precursor insertion into the lipid bilayer in a mem- lized by the presence of Tom7 [9–11]. In humans, the TOMM7 pro- brane potential-dependent fashion. Tim22 associates with Tim54 tein stabilizes TOM [43]. to form the fully functional translocase (Fig. 1C). The receptor role The SAMM50 protein, the main component of the SAM complex, of Tim54 is fulfilled by a large C-terminal domain that is exposed is conserved between yeast and metazoa [44] (Fig. 2A). It forms a to the IMS. Two other proteins, Tim18 and Sdh3, form a module 200 kDa complex in the OMM and participates in the insertion of that is a stoichiometric part of the TIM22 translocase. Interest- b-barrel proteins, such as VDAC and TOMM40. The human counter- ingly, Sdh3 is part of the succinate dehydrogenase complex (com- parts to the additional SAM components are the peripherally plex II) of the respiratory chain, where it builds an analogous OMM-associated Metaxin-2 (yeast Sam35) and Sam37-like subcomplex with Sdh4 (Tim18 homologue). The precise role of Metaxin-1 and Metaxin-3 [45–48]. Given their high sequence the Tim18-Sdh3 module in membrane integration is currently divergence, clear structural similarity cannot be readily inferred unknown. This is yet another example of links between energy- [48] (Fig. 2A). Only a small fraction of Metaxin-1 is present with transducing complexes and protein translocation machineries SAMM50 in the 200 kDa complex, whereas a larger fraction of [27,33]. Metaxin-1 is found in a distinct complex of 600 kDa together with Oxa1, the homologue of bacterial YidC protein conserved Metaxin-2 [47]. between species, is a core component of the mitochondrial protein export machinery (OXA) involved in the co-translational import of 3.2. Mitochondrial intermembrane space assembly mitochondrially-encoded proteins into the IMM [5,34]. Addition- ally, Oxa1 has been shown to participate in the posttranslational The oxidoreductase MIA40 (CHCHD4) and sulfhydryl oxidase transport of multispanning proteins into the IMM, reflecting the ALR (human Erv1, Augmenter of Liver Regeneration, hepatopoie- cooperation of Oxa1 with the TIM22 and TIM23 translocases tin, GFER) are the human orthologues of key constituents of the [35,36] (Fig. 1C). yeast MIA pathway. The main difference between yeast and 2488 A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495

Fig. 2. Similarities and differences between yeast and humans in the architecture and function of mitochondrial translocases. Translocase components that are evolutionarily conserved are represented in gray, with name differences in pink where indicated. Components that are found only in yeast are in blue, and human-specific proteins are represented in pink. In cases in which more than one paralogue of the human protein exist, both names are given (e.g., 17A/17B). The high sequence divergence between human Metaxin proteins and yeast Sam35/37 might result in a difference in structure or function; therefore, these are shown as individual proteins, represented in pink and blue, respectively. OMM, outer mitochondrial membrane; IMS, intermembrane space; IMM, inner mitochondrial membrane; Dw, electrochemical potential of the inner mitochondrial membrane; TOM, translocase of the outer membrane; SAM, sorting and assembly machinery; MIM, insertase of the mitochondrial outer membrane; eÀ, electron; TIM23, presequence translocase; PAM, presequence translocase-associated motor; TIM22, carrier translocase of the inner membrane. human Mia40 concerns the N-terminal part of the protein. segment, human MIA40 was correctly localized to yeast mito- Human MIA40 lacks the N-terminal portion, including the prese- chondria and able to substitute for the essential function of yeast quence and transmembrane segment. This change results in a oxidoreductase in the biogenesis of IMS proteins, including their difference in the topology of these two proteins. Yeast Mia40 is recognition, formation of intermediates, and oxidation in IMM-anchored, whereas human MIA40 is soluble in the IMS cooperation with ALR [51]. Additionally, a novel role for ALR in [49,50] (Fig. 2B). Nonetheless, the C-terminal domains of human the mitochondrial accumulation of human MIA40 has been and yeast Mia40 have a high level of similarity, including the reported [51]. conserved CPC and twin CX9C motifs. Because of the lack of a The in vivo studies that have been performed in mammalian presequence, human MIA40 is imported into the IMS via the cells showed that the oxidation-driven import of IMS-targeted pre- MIA pathway [50]. Similar to other MIA pathway substrates, cursor proteins is much slower than presequence-dependent human MIA40 trapping in the IMS relies on the oxidation of cys- translocation, and its efficiency strongly depends on the accessibil- teine residues in the twin CX9C motifs [49]. This mechanism is ity of fully functional human MIA40. Manipulating the amount of analogous to the import of an artificial presequence and trans- oxidoreductase showed that MIA40 is a ‘‘bottle neck’’ for oxidative membrane domain-lacking Mia40 protein in yeast [50]. Reconsti- folding in the IMS in intact mammalian cells [52]. Interestingly, in tution of the human MIA pathway in yeast cells confirmed the human cells, MIA40 is present in two ubiquitously expressed splic- high functional conservation of MIA40 and ALR function within ing isoforms that differ by a short stretch of N-terminally located the eukaryotic kingdom. Although deprived of the N-terminal residues [49,53]. A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495 2489

The human sulfhydryl oxidase ALR, similar to its yeast counter- yeast, small Tim proteins form three complexes: a soluble Tim9- part, has a FAD prosthetic group. Both oxidation by MIA40 and Tim10 complex, Tim9-Tim10-Tim12 associated with the core interactions with FAD were suggested to determine the mature TIM22 complex, and a soluble Tim8-Tim13 complex. Tim12 is a conformation of ALR and its entrapment in the IMS [54]. Interest- yeast-specific duplication of yeast Tim10 [68–70] that interacts ingly, ALR has two splicing variants, 23 kDa and 15 kDa, that differ with TIM22. In humans (Fig. 2C), this function is fulfilled by the by 80 amino acids at the N-terminus. The cysteine residues and metazoan-specific TIMM10B (similar to yeast Tim9 and human FAD binding domain are conserved and found in both isoforms, TIMM9) [70]. TIMM10B forms a complex with TIMM9 and but the shorter isoform functions extracellularly as a mitogen TIMM10, also referred to as Tim10a, and is present in the complex and intracellularly as an intracrine of cytosolic and nuclear locali- with TIMM22 [69]. TIMM10 and TIMM9 also form a separate com- zation [55–57]. How the partitioning of both isoforms between plex without TIMM10B [69]. TIMM8 and TIMM13 have two para- various compartments is achieved remains unclear. logues in the . TIMM8A is ubiquitously expressed, whereas TIMM8B mRNA was found at elevated levels in the heart, 3.3. Translocases of the inner membrane liver, and skeletal muscle, although its presence at the protein level was not confirmed [71]. TIMM13A is expressed mainly in the fetus, The general architecture of the TIM23 complex and topology of whereas TIMM13B prevails in adults [71]. The functional features its major components are conserved between species [58] (Fig. 2C). of these proteins remain to be studied. Human TIMM23, TIMM50, TIMM17, and TIMM44 have high simi- larity to their yeast counterparts (Fig. 2C). Interestingly, in humans, 4. Regulatory functions of mitochondrial translocases the that encodes TIMM17 occurs in two copies. Both TIMM17A and TIMM17B are ubiquitously expressed in human tis- Several recent studies led to the proposal that the function of sues and able to assemble into the TIM23 complex. However, they the human MIA pathway extends beyond the framework of the are separated into different subpopulations of the complex because yeast system. The MIA pathway in humans promotes the mito- they do not co-precipitate with each other [58,59]. chondrial localization of some non-IMS proteins that may be The function of the import motor in humans is also evolution- conditionally targeted to mitochondria. An example is AP endonu- arily conserved. MAGMAS, an orthologue of Pam16, can comple- clease 1 (APE1), a nuclear protein of the DNA base excision repair ment the growth of Pam16-deficient yeast [60]. Pam18 has two pathway that is localized to the mitochondrial matrix where it orthologues in the human genome, MCJ and DNAJC19 [61,62] participates in mtDNA repair [72]. The localization of APE1 to mito- (Fig. 2C). Both proteins stimulate the ATP-ase activity of chondria requires cysteine residue C65 and redox-assisted folding mtHsp70/Mortalin and interact with the TIM23 complex [60,62]. that is attributed to the MIA pathway where APE1 is able to inter- Although both proteins are ubiquitously expressed in human tis- act with MIA40 [73]. Another example is the p53 protein, a tran- sues, they are not fully interchangeable functionally [59]. MCJ, scription factor and ‘‘guardian of the genome’’ of differentiated but not DNAJC19, was able to restore the viability of cells that is implicated in broad stress-related signaling pathways Pam18-depleted yeast cells [62]. In fact, DNAJC19 was found to pre- that ultimately affect cell cycle arrest, senescence, and apoptosis. dominantly interact with the prohibitin complexes and to regulate Mutations in the TP53 gene are the most frequently reported muta- cardiolipin metabolism (Thomas Langer, personal communication). tions in cancer-related malignancies [74]. Interestingly, growing Mutations in DNAJC19 result in pathology [61,63]. Interestingly, evidence suggests that the levels of p53 and its subcellular locali- MCJ has been recently found to regulate the activity of respiratory zation control metabolism [75]. Oxidative stress leads to the complex I and its engagement in the supercomplexes [64]. accumulation of p53 in mitochondria where it plays a role in main- In yeast, Tim21 interacts with Tim23 through the Mgr2 protein, taining the integrity of the mitochondrial genome (Fig. 3A). The which is an orthologue of human ROMO1. ROMO1 is involved in transport of p53 to the mitochondrial matrix has been shown to the mitochondrial processing of the optic atrophy 1 (OPA1) pro- depend on MIA40 [76]. tein, and its activity has been implicated in the control of mito- Human stem cells reside in hypoxic niches and energetically chondrial dynamics and reactive oxygen species (ROS)-induced rely on anaerobic glucose metabolism [77]. Nonetheless, mito- signaling [65,66]. Fully assembled respiratory chain complexes in chondrial has been reported to be an essential factor humans are not directly connected to the TIM23 complex. None- for sustaining the proliferative potential and survival of stem cells. theless, human TIMM21 was found in the recently identified The chemical inhibition of Erv1/ALR induced apoptosis in human MITRAC (mitochondrial translation regulation assembly interme- embryonic stem cells (hESCs) but not differentiated cells [78]. diate of cytochrome c oxidase) complex, together with assembly Erv1/ALR knockdown decreased stem cell pluripotency and mito- intermediates of respiratory chain complexes [67] (Fig. 2C). The chondrial function because of a loss of membrane potential and fact that TIMM21 is found in complexes with MITRAC and that excessive fragmentation. At the molecular level, this can be MCJ is involved in the control of the respiratory chain emphasizes explained by the fact that a decrease in Erv1/ALR expression corre- tight coupling between protein import machineries and abundant lates with an increase in dynamin-related protein 1 (Drp1) levels IMM complexes devoted to respiration. and consequently a disturbance in mitochondrial fusion-fission Human hydrophobic proteins with multiple internal targeting balance [79]. The role of Erv1/ALR in sustaining proliferative signals are imported into the IMM via the TIM22 translocase. How- growth may not be restricted to its mitochondrial function. Models ever, in contrast to other translocases, TIM22 differs substantially are discussed for the involvement in hepatocyte proliferation and between lower and higher eukaryotes (Fig. 2C). The core complex liver regeneration, which rely on the cytosolic, nuclear, and extra- of yeast TIM22 is composed of Tim22, Tim18, Tim54, and Sdh3 pro- cellular localization of the shorter forms of Erv1/ALR and its role in teins (Fig. 1C). Tim22 and Sdh3 are the only proteins from the activating extracellular and internal signaling pathways [57,80,81]. TIM22 complex that are present in human cells. No evidence to Terminally differentiated cells produce a bulk of energy through date indicates that Sdh3 interacts with the TIM22 translocase in oxidative phosphorylation and the respiratory chain. Conse- humans. TIMM22 itself exhibits substantial conservation, suggest- quently, cell differentiation should trigger mitochondrial biogene- ing that it cooperates with yet unknown partners [68] (Fig. 2C). sis. Defects in this process can dramatically affect a developing On the IMS side, the TIM22 complex interacts with small Tim embryo. Consistent with these observations, abnormalities in the proteins that shuttle precursor proteins from TOM to TIM22. In amount and function of the components of mitochondrial 2490 A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495

Fig. 3. Mitochondrial translocases as regulatory players in tumorigenesis and signaling. (A) Under basal conditions, p53 shuttles between the nucleus and cytosol and is degraded by the ubiquitin-proteasome system. Oxidative stress prevents p53 degradation and leads to its accumulation in the nucleus and import into mitochondria. (B) Intensively proliferating cancer cells overproduce the translocase components MIA40, TIMM50, TIMM17A, and mtHsp70/Mortalin. (C) In cancer, p53 is transported into mitochondria. The increased import of p53 is linked to the overproduction of MIA40 and mtHsp70/Mortalin. (D) The stabilization of HIF1a is used to activate hypoxic signaling. MIA40 controls the stability of HIF1a via an as-yet-unclear mechanism that may involve the biogenesis and activity of respiratory chain complexes. OMM, outer mitochondrial membrane; IMS, intermembrane space; IMM, inner mitochondrial membrane; TOM, translocase of the outer membrane; TIM23, presequence translocase. translocases are associated with impaired development. The the mitochondrial unfolded protein response (mtUPR). This phe- silencing or inhibition of Erv1/ALR in zebrafish led to abnormalities nomenon was proposed to increase the resistance to stress under in liver and heart development, respectively, without affecting pathological conditions [86]. In summary, in addition to canonical apoptosis [78,81]. Defects in TOMM22 protein, identified in the oli- mitochondrial function in energy conversion, the examples dis- ver zebrafish mutant, resulted in hepatocyte apoptosis that under- cussed above support the regulatory role of mitochondrial protein lies the small liver phenotype [82]. TIMM50 mutant flies were translocases at the cellular level. significantly smaller in size, and a detailed analysis of the cells obtained from these mutants showed a reduction of proliferation 5. Mitochondrial protein translocases: implications in human rates [83]. In the developing zebrafish embryo, the incidence of pathologies increased cell death caused by TIMM50 depletion results in neuro- degeneration, reduced motility, and dysmorphic hearts [84]. The Because of the essential role of mitochondria in cells, it is not growth defects observed in flies and zebrafish upon a reduction surprising that impaired mitochondrial biogenesis results in cell of TIMM50 levels indicate the importance of TIM23 during the and tissue pathologies. The goal of this chapter is to review the early stages of development. TIMM23 homozygous knockout mice involvement of specific mitochondrial translocase components in are not viable, whereas TIMM23 heterozygotes exhibited a neuro- human diseases. logical phenotype with a severely reduced lifespan [85]. Interestingly, a recent report showed that the abundance of one 5.1. Cancer of the TIMM17 paralogues, TIMM17A, is reduced under stress conditions [86]. Decreased TIMM17A resulted in the impaired Cancer cells are similar to stem cells since they metabolically biogenesis of mitochondrial proteins that depend on TIM23 and rely on the Warburg effect and utilize glycolysis over triggered a stress-induced signaling pathway that is related to oxidative phosphorylation to produce energy for growth [87]. A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495 2491

Counterintuitively, reports are growing that link mitochondrial 5.2. Genetically inherited mitochondrial syndromes biogenesis pathways to tumor progression. The upregulation of TIMM17A expression has been identified as a diagnostic marker The vast majority of mitochondrial translocase components and poor prognosis factor for cancer [59,88,89] (Fig. 3B). A similar have been found to be essential for life of a simple unicellular scenario is observed for two other mitochondrial protein transloca- organism, the yeast S. cerevisiae. The fact that very little pathogenic tion components, mtHsp70/Mortalin and MIA40. The overexpres- mutations of the core components of these pathways have been sion of human mtHsp70/Mortalin or MIA40 has been associated reported in higher eukaryotes, including humans, is not surprising. with the incidence of various types of human cancer and poor sur- Consistent with the fundamental role played by translocases in vival prognosis [53,90–92] (Fig. 3B). A decrease in mtHsp70/Mort- yeast, such mutations likely lead to early prenatal lethality. The alin levels impaired tumor growth and, importantly, sensitized known diseases caused by alterations in mitochondrial protein cancer cells to chemotherapy and drug-induced senescence translocases are extremely rare. [92,93]. The first mitochondrial disease that was associated with Mutation variants of p53 in malignancies show many molecular impaired function of the mitochondrial import machinery is and functional abnormalities, some of which are mitochondria- human deafness dystonia syndrome, which leads to hearing loss, related. First, p53 has been found to stimulate aerobic respiration; mental retardation, and blindness [100]. This recessive, X-chromo- therefore, p53 mutations in glycolytic cancer tissues may serve to some-linked condition was found to be caused by mutations that sustain non-aerobic metabolism [94]. Second, p53 plays a funda- result in the loss of deafness dystonia protein 1 (DDP1), subse- mental role in initiating programmed cell death, in which the quently identified as the small mitochondrial IMS chaperone release of cytochrome c from mitochondria is a critical step. The TIMM8A [100]. An analysis of patient samples revealed mutations avoidance of apoptosis, a feature that cancer cells can acquire via in the CX3C motif of TIMM8A [101,102], which are likely responsi- p53 mutations, is a well-known hallmark of cancer [87]. Interest- ble for its MIA pathway-mediated oxidative folding and retention ingly, mitochondrial translocase components, such as mtHsp70/ in the IMS. Because of the improper folding, the mutant protein Mortalin, MIA40, and TIMM50, have a connection with p53. can no longer assemble into a complex with TIMM13 and under- mtHsp70/Mortalin interacts with both wildtype and mutant p53 goes rapid turnover [102,103]. The dramatic clinical phenotype and sequesters its function in stress-mediated cellular signaling that results from TIMM8A mutations underscores the significance (Fig. 3C). This feature is exploited by cancer cells. Through the of the oxidative folding of IMS proteins, which further impacts excessive production of mtHsp70/Mortalin, cancer cells can escape downstream mitochondrial protein biogenesis. p53-mediated apoptosis [95]. A recent study reported that the An homozygous R194H mutation in sulfhydryl oxidase Erv1/ mitochondrial localization of p53 can be modulated by MIA40 ALR was identified in patients diagnosed with infantile autosomal expression and is respiration-driven [76] (Fig. 3C). p53 does not recessive myopathy (ARM) [104]. This condition is phenotypically look like a classical substrate protein for the MIA pathway [6], characterized by congenital cataract, muscle hypotonia, hearing but its interaction with MIA40 depends on the CPC motif of oxido- loss, and developmental delay [104]. The R194H mutation affects reductase and the cysteine residue at position 135 in p53 [76]. the stability of Erv1/ALR and enhances its degradation [105].At Another interesting correlation has been found between TIMM50 the cellular level, the Erv1/ALR mutation leads to the inefficient and p53. The levels of TIMM50 were found to be elevated in cancer import of MIA pathway substrates, including components of the cells that harbor specific mutations in the TP53 oncogene [96] respiratory chain. A decrease in the activity of respiratory com- (Fig. 3B). TIMM50 downregulation makes cells more vulnerable plexes is consequently observed, in addition to abnormal mito- to death stimuli, a scenario that p53 mutant cancer cells can over- chondrial structure and accelerated mtDNA degeneration [104]. come through TIMM50 overexpression [84,96]. A tempting specu- Using a yeast model with a reconstituted human MIA pathway, lation is that the increased levels of protein translocation the molecular basis of the disease was better defined. A patholog- components in cancer cells can prevent p53-mediated stress ical mutation in the sulfhydryl oxidase Erv1/ALR affects the mito- responses that are typical for healthy cells by binding, oxidizing, chondrial accumulation of human MIA40, resulting in the and sequestering p53 to mitochondria. Going one step further, pre- impaired biogenesis of other MIA substrates [51]. venting an increase in MIA40, TIMM50, or mtHsp70/Mortalin Patients who suffer from Pelizaeus-Merzbacher disease (PMD), activity may be a possible step toward more effective cancer in addition to rodent models of PMD, carry mutations in the PLP therapies. gene. This gene encodes the proteolipid protein (PLP) and its shorter The role of MIA40 in cancer is not limited to an interplay with isoform DM20, the two most abundant membrane proteins in mye- p53. MIA40 has been proposed to promote the hypoxic response lin [106]. The consequences of disease-related PLP gene duplication, that is required for metabolic adaptation and tumor angiogenesis which are phenotypically more severe than null mutations, include because of HIF1a stabilization (Fig. 3D). The mechanism by which the accumulation of PLP in mitochondria. At the molecular level, MIA40 influences HIF1a stability is unclear. At least three mito- PMD is characterized by metabolic abnormalities as a possible con- chondria-dependent mechanisms of HIF1a stabilization have been sequence of overexpressed PLP localization to mitochondria described: (i) ROS generation by complex III of the respiratory [107,108]. Interestingly, overexpressed PLP that carried cysteine- chain [97],(ii) elevated oxygen consumption by complex IV to-alanine mutations in the N-terminally located CX3C and CX9C [53,98], and (iii) increased concentration of the complex II sub- motifs was no longer targeted to mitochondria, but its ability to strate succinate [99]. Whether MIA40 stabilizes HIF1a indirectly localize to the plasma membrane remained unchanged [108]. These by modulating the biogenesis and activity of the respiratory chain observations suggest the possible involvement of Mia40 [109] in or more directly through an as-yet-unidentified mechanism the mitochondrial import of PLP in PMD. Further studies are needed requires further studies (Fig. 3D). to better understand the disease-linked localization of PLP in mito- In conclusion, several components of protein translocases are chondria, involvement of Mia40, and mechanisms by which this frequently upregulated in cancer. In addition to direct involvement abnormal PLP behavior alters physiology. in the transport of proteins with known roles in cancer, such as p53, the increased activity of single protein translocation may 5.3. Role of mitochondrial protein translocases in neurodegeneration likely result in an imbalance in mitochondrial protein biogenesis and homeostasis and thus less directly modulate the cellular Frequently, neurodegenerative diseases have a late onset, with responses to stimuli and stress. sporadic deterioration of various types of neuronal cells. The 2492 A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495 symptoms are mirrored by rare familial cases that are determined amyloid precursor protein (APP) [121]. Interestingly, studies that genetically. Over one-third of the genetic mutations associated mimicked AD pathology by chronically exposing human cells to with the inherited predisposition to neurodegeneration are func- Ab peptide reported blockage of the TOM channel for the import tionally linked to mitochondria [110,111]. Furthermore, the symp- of mitochondria-targeted proteins [122]. Indeed, Ab peptide enters toms of many, or perhaps all, types of neurodegeneration include mitochondria through the TOM channel, and its import is indepen- various modes of mitochondrial dysfunction. Here, we focus on dent of mitochondrial membrane potential [123]. Studies on the the aspects of these devastating pathologies that are directly or submitochondrial localization of Ab peptide using in vitro import indirectly related to mitochondrial protein translocases at the experiments and AD patient biopsies revealed that Ab peptide molecular level. resides within cristae, the folds of the IMM, and is cleared from mitochondria by the redox-regulated peptidase PreP [123]. 5.3.1. Parkinson’s disease Whether the pathology observed in AD can be modulated by regu- To sustain cellular homeostasis in the context of such dynamic lating the uptake and stability of Ab peptide inside mitochondria is organelles as mitochondria, maintaining their continuous biogene- an important question for future studies. sis and removing damaged pools are equally important. A failure of mitochondria quality control at the organellar level has been 5.3.3. Huntington’s disease reported to be a predisposing factor to Parkinson’s disease (PD) This neurodegenerative disease is genetically inherited and [111,112]. A subpopulation of patients who suffer from the familial caused by the expansion of polyglutamine repeats in the hunting- form of PD carry loss-of-function mutations in the PTEN-induced tin protein. This fatal disease is additionally linked to mitochon- putative kinase-1 (PINK1) and PARK2 . PARK2 encodes drial dysfunction [110,111]. The list of mitochondrial E3-ubiquitin ligase, which is involved in mitochondrial turnover abnormalities found in neurons in HD patients and mouse models [113]. is complex. Examples include impaired glucose metabolism and a In the case of mitochondrial dysfunction with the hallmark defective respiratory chain [110,111]. The levels of Erv1/ALR pro- decrease in electrochemical IMM potential, the PINK1-Parkin path- tein have recently been reported to be substantially decreased in way is responsible for marking damaged mitochondria for degra- mouse heterozygous or homozygous huntingtin mutant striatal dation by selective autophagy [112]. Under physiological cells, with either unchanged or slightly reduced MIA40 levels conditions, PINK1 is synthesized in the cytosol as a 63 kDa precur- [124]. Consequently, the substrates of the MIA pathway and com- sor that, upon relocating to mitochondria, is processed into the ponents of respiratory chain complex I, NDUFB7 and NDUFS5, were mature 52 kDa form and subsequently degraded. Thus, Parkin is significantly altered, indicating problems with complex I assembly not recruited to healthy mitochondria. Under stress conditions that and activity and reduced oxidative phosphorylation [124]. are experimentally mimicked by treatment with uncouplers to dis- sipate IMM potential, the import of PINK1 differs such that the 5.3.4. Amyotrophic lateral sclerosis unprocessed form is released to the OMM and recruits cytosolically Amyotrophic lateral sclerosis (ALS) is a devastating disease that located Parkin instead of being targeted to degradation. The role of affects motor neurons [111,125,126]. Its genetically inherited cases the TOM complex or its single components in the import of PINK1 are frequently associated with mutations in Sod1, an into mitochondria is commonly known. PINK1 was found in com- responsible for ROS scavenging [125,126]. The disease-causing fac- plex with TOM or its components [114–117]. However, still tor is not the inability to neutralize ROS but rather the formation of unclear are the TOM components on which the import pathway toxic protein Sod1 aggregates. Although most Sod1 is found in the of PINK1 relies in healthy and damaged mitochondria. Importantly, cytosol, a small pool is localized to the IMS of mitochondria. Thus, an unbiased approach identified TOMM7 as a functionally relevant in the context of Sod1 the mitochondrial dysfunction observed in factor that is involved in anchoring and stabilizing PINK1 kinase on ALS is not surprising [125,126]. To be retained in the IMS, Sod1 damaged mitochondria [118]. Interestingly, the impaired mito- requires Ccs1, a copper chaperone that is also involved in the oxi- chondrial import through the TOM complex was shown to play a dation of Sod1. In yeast, the localization of Ccs1 depends on the pivotal role in activating PINK1-Parkin-dependent mitochondrial MIA pathway [127,128]. In human cells, the situation is less clear, clearance [116]. Consistent with this observation, TOMM40 (i.e., but two possible scenarios have been proposed. The first scenario the core component of the TOM complex) was found to be down- implies a role for MIA40, and the second scenario suggests the sole regulated in midbrain samples from PD patients and mouse models involvement of Ccs1 in the import of Ccs1 to the IMS [129,130]. of PD [119]. Furthermore, TOM components, similar to many other Interestingly, Kawamata and Manfredi found that the mechanisms OMM proteins, are the ubiquitination targets of Parkin upon its that drive the IMS localization of mutant versions of Sod1 in recruitment to mitochondria [120]. Remaining questions are human cells differ from wildtype Sod1 [129]. In yeast, studies on whether TOM activity is a decisive step in activating the mitochon- Sod1 with conserved amino acid substitutions that cause ALS in drial clearance pathway and the extent to which it contributes to humans supported and extended the view of unique pathways PD. for pathogenic variants of Sod1 [131]. Disease-related forms of Sod1 that fail to undergo oxidation are localized to the IMS 5.3.2. Alzheimer’s disease [131]. Interestingly, the import of these Sod1 variants and reduced Alzheimer’s disease (AD) is the most frequent age-related disor- wildtype Sod1 did not depend on Ccs1. Instead, this process relied der, characterized by the gradual accumulation of neuronal abnor- on the unconventional action of Mia40 that did not result in disul- malities that result in progressive dementia in patients. Growing fide bond formation [131]. The lack of MICOS, the mitochondrial evidence indicates that mitochondrial defects contribute to the contact site and cristae-organizing system that affects the architec- neuronal cell pathologies observed in patients and experimental ture of mitochondria, was found to increase the mitochondrial lev- models of AD [111,121]. These defects include increased ROS pro- els of wildtype oxidized Sod1. Intriguingly, the depletion of one duction, the fragmentation of mitochondria, and the decreased MICOS component, Mic60 (Fcj1/Mitofilin), led to the accumulation activity of canonical mitochondrial processes, such as oxidative of reduced aberrant forms of Sod1 [131]. Thus, Mia40 and MICOS phosphorylation [121]. In addition to other typical features, such are novel factors that affect the biogenesis of pathogenic variants as the accumulation of hyperphosphorylated Tau protein and dis- of Sod1 in simple eukaryotic cells. Future studies will assess turbed calcium homeostasis, AD is accompanied by an accumula- whether these proteins are also important in the pathology of tion of amyloid b (Ab) peptide, a proteolytic product of the ALS. Independent of which pathway the conformationally altered A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495 2493

Sod1 forms take to reach the IMS, they were shown to inhibit the [13] Qiu, J. et al. (2013) Coupling of mitochondrial import and export translocases import of proteins into spinal cord mitochondria [132]. by receptor-mediated supercomplex formation. Cell 154 (3), 596–608. [14] Song, J., Tamura, Y., Yoshihisa, T. and Endo, T. (2014) A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 6. Concluding remarks complex. EMBO Rep., http://dx.doi.org/10.1002/embr.201338142. [15] Wenz, L.S. et al. (2014) The presequence pathway is involved in protein sorting to the mitochondrial outer membrane. EMBO Rep., http://dx.doi.org/ Undeniably, mitochondria are essential for life. Protein 10.1002/embr.201338144. translocases are fundamental players in the effective biogenesis [16] Koehler, C.M. and Tienson, H.L. (2009) Redox regulation of protein folding in of mitochondria. Interestingly, growing evidence indicates the the mitochondrial intermembrane space. Biochim. Biophys. Acta 1793 (1), 139–145. involvement of mitochondrial translocase components in human [17] Sideris, D.P. and Tokatlidis, K. (2010) Oxidative protein folding in the pathologies and cancer. A tempting speculation is that the mito- mitochondrial intermembrane space. Antioxid. Redox Signal. 13 (8), chondrial transport components that have been found to be upreg- 1189–1204. [18] Herrmann, J.M. and Riemer, J. (2012) Mitochondrial disulfide relay: redox- ulated in cancer may become new targets for drugs, which can regulated protein import into the intermembrane space. J. Biol. Chem. 287 potentiate anti-cancer therapies in the future. Recent studies (7), 4426–4433. greatly expanded the view on the role of the MIA pathway in the [19] Stojanovski, D., Bragoszewski, P. and Chacinska, A. (2012) The MIA pathway: a tight bond between protein transport and oxidative folding in cell by identifying its function in hypoxic signaling and the condi- mitochondria. Biochim. Biophys. Acta 7 (1823), 1142–1150. tional stress-regulated mitochondrial transport of proteins, which [20] Bragoszewski, P., Gornicka, A., Sztolsztener, M.E. and Chacinska, A. (2013) The have important canonical functions beyond mitochondria. An aris- ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins. Mol. Cell. Biol. 33 (11), 2136–2148. ing theme involves alterations in mitochondrial translocases and [21] Pfanner, N. et al. (2014) Uniform nomenclature for the mitochondrial contact protein import in various types of neurodegeneration. Disturbed site and cristae organizing system. J. Cell Biol. 204 (7), 1083–1086. protein homeostasis is a common hallmark of neurodegenerative [22] Weckbecker, D., Longen, S., Riemer, J. and Herrmann, J.M. (2012) Atp23 biogenesis reveals a chaperone-like folding activity of Mia40 in the IMS of pathologies, thus it is tempting to speculate that the reduced effi- mitochondria. EMBO J. 31 (22), 4348–4358. ciency of mitochondrial protein import, which is deleterious to cel- [23] Wrobel, L., Trojanowska, A., Sztolsztener, M.E. and Chacinska, A. (2013) lular proteostasis, may be an important process that contributes to Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the these pathologies. Future research should provide insights into the inner membrane of mitochondria. Mol. Biol. Cell 24 (5), 543–554. [24] Longen, S., Woellhaf, M.W., Petrungaro, C., Riemer, J. and Herrmann, J.M. links and networks that involve dynamic mitochondrial protein (2014) The disulfide relay of the intermembrane space oxidizes the transport and biogenesis processes that are responsible for the ribosomal subunit mrp10 on its transit into the mitochondrial matrix. Dev. health of the cell and entire organism. Cell 28 (1), 30–42. [25] Mossmann, D., Meisinger, C. and Vogtle, F.N. (2012) Processing of mitochondrial presequences. Biochim. Biophys. Acta 9–10 (1819), Acknowledgements 1098–1106. [26] Endo, T. and Yamano, K. (2009) Multiple pathways for mitochondrial protein traffic. Biol. Chem. 390 (8), 723–730. Research in the AC laboratory is supported by the Foundation [27] Wagner, K., Mick, D.U. and Rehling, P. (2009) Protein transport machineries for Polish Science–Welcome Programme co-financed by the EU for precursor translocation across the inner mitochondrial membrane. within the European Regional Development Fund, EMBO Installa- Biochim. Biophys. Acta 1793 (1), 52–59. [28] Mokranjac, D. and Neupert, W. (2010) The many faces of the mitochondrial tion grant, National Science Centre (NCN; grant 2011/02/B/NZ2/ TIM23 complex. Biochim. Biophys. Acta 1797 (6–7), 1045–1054. 01402) and FishMed within the EU Seventh Framework Pro- [29] van der Laan, M., Hutu, D.P. and Rehling, P. (2010) On the mechanism of gramme (no. 316125). AMS is supported by FishMed. MES is sup- preprotein import by the mitochondrial presequence translocase. Biochim. Biophys. Acta 6 (1803), 732–739. ported by Welcome. MW is a recipient of the NCN grant 2012/ [30] Marom, M., Azem, A. and Mokranjac, D. (2011) Understanding the molecular 05/B/NZ3/00781. EH is an ARC FL Postdoctoral Research Fellow. mechanism of protein translocation across the mitochondrial inner membrane: still a long way to go. Biochim. Biophys. Acta 3 (1808), 990–1001. References [31] Malhotra, K., Sathappa, M., Landin, J.S., Johnson, A.E. and Alder, N.N. (2013) Structural changes in the mitochondrial Tim23 channel are coupled to the proton-motive force. Nat. Struct. Mol. Biol. 20 (8), 965–972. [1] Dolezal, P., Likic, V., Tachezy, J. and Lithgow, T. (2006) Evolution of the [32] Baker, M.J., Frazier, A.E., Gulbis, J.M. and Ryan, M.T. (2007) Mitochondrial molecular machines for protein import into mitochondria. Science 313 protein-import machinery: correlating structure with function. Trends Cell (5785), 314–318. Biol. 17 (9), 456–464. [2] Sickmann, A. et al. (2003) The proteome of Saccharomyces cerevisiae [33] Kulawiak, B., Hopker, J., Gebert, M., Guiard, B., Wiedemann, N. and Gebert, N. mitochondria. Proc. Natl. Acad. Sci. USA 100 (23), 13207–13212. (2013) The mitochondrial protein import machinery has multiple [3] Pagliarini, D.J. et al. (2008) A mitochondrial protein compendium elucidates connections to the respiratory chain. Biochim. Biophys. Acta 5 (1827), complex I disease biology. Cell 134 (1), 112–123. 612–626. [4] Nunnari, J. and Suomalainen, A. (2012) Mitochondria: in sickness and in [34] Bonnefoy, N., Fiumera, H.L., Dujardin, G. and Fox, T.D. (2009) Roles of Oxa1- health. Cell 148 (6), 1145–1159. related inner-membrane translocases in assembly of respiratory chain [5] Neupert, W. and Herrmann, J.M. (2007) Translocation of proteins into complexes. Biochim. Biophys. Acta 1793 (1), 60–70. mitochondria. Annu. Rev. Biochem. 76, 723–749. [35] Bohnert, M., Rehling, P., Guiard, B., Herrmann, J.M., Pfanner, N. and van der [6] Chacinska, A., Koehler, C.M., Milenkovic, D., Lithgow, T. and Pfanner, N. (2009) Laan, M. (2010) Cooperation of stop-transfer and conservative sorting Importing mitochondrial proteins: machineries and mechanisms. Cell 138 mechanisms in mitochondrial protein transport. Curr. Biol. 20 (13), (4), 628–644. 1227–1232. [7] Schmidt, O., Pfanner, N. and Meisinger, C. (2010) Mitochondrial protein [36] Hildenbeutel, M., Theis, M., Geier, M., Haferkamp, I., Neuhaus, H.E., import: from proteomics to functional mechanisms. Nat. Rev. Mol. Cell Biol. Herrmann, J.M. and Ott, M. (2012) The membrane insertase Oxa1 is 11 (9), 655–667. required for efficient import of carrier proteins into mitochondria. J. Mol. [8] Harbauer, A.B., Zahedi, R.P., Sickmann, A., Pfanner, N. and Meisinger, C. (2014) Biol. 423 (4), 590–599. The protein import machinery of mitochondria-a regulatory hub in [37] Young, J.C., Hoogenraad, N.J. and Hartl, F.U. (2003) Molecular chaperones metabolism, stress, and disease. Cell Metab. 19 (3), 357–372. Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor [9] Dukanovic, J. and Rapaport, D. (2011) Multiple pathways in the integration of Tom70. Cell 112 (1), 41–50. proteins into the mitochondrial outer membrane. Biochim. Biophys. Acta 3 [38] Chewawiwat, N., Yano, M., Terada, K., Hoogenraad, N.J. and Mori, M. (1999) (1808), 971–980. Characterization of the novel mitochondrial protein import component, [10] Endo, T., Yamano, K. and Kawano, S. (1808) Structural insight into the Tom34, in mammalian cells. J. Biochem. 125 (4), 721–727. mitochondrial protein import system. Biochim. Biophys. Acta 3 (2011), 955– [39] Faou, P. and Hoogenraad, N.J. (2012) Tom34: a cytosolic cochaperone of the 970. Hsp90/Hsp70 protein complex involved in mitochondrial protein import. [11] Becker, T., Bottinger, L. and Pfanner, N. (2012) Mitochondrial protein import: Biochim. Biophys. Acta 2 (1823), 348–357. from transport pathways to an integrated network. Trends Biochem. Sci. 37 [40] Terada, K. et al. (2003) Expression of Tom34 splicing isoforms in mouse testis (3), 85–91. and knockout of Tom34 in mice. J. Biochem. 133 (5), 625–631. [12] Kutik, S., Guiard, B., Meyer, H.E., Wiedemann, N. and Pfanner, N. (2007) [41] Yano, M., Terada, K. and Mori, M. (2003) AIP is a mitochondrial import Cooperation of translocase complexes in mitochondrial protein import. J. Cell mediator that binds to both import receptor Tom20 and preproteins. J. Cell Biol. 179 (4), 585–591. Biol. 163 (1), 45–56. 2494 A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495

[42] Fan, A.C., Kozlov, G., Hoegl, A., Marcellus, R.C., Wong, M.J., Gehring, K. and [69] Muhlenbein, N., Hofmann, S., Rothbauer, U. and Bauer, M.F. (2004) Young, J.C. (2011) Interaction between the human mitochondrial import Organization and function of the small Tim complexes acting along the receptors Tom20 and Tom70 in vitro suggests a chaperone displacement import pathway of metabolite carriers into mammalian mitochondria. J. Biol. mechanism. J. Biol. Chem. 286 (37), 32208–32219. Chem. 279 (14), 13540–13546. [43] Kato, H. and Mihara, K. (2008) Identification of Tom5 and Tom6 in the [70] Gentle, I.E. et al. (2007) Conserved motifs reveal details of ancestry and preprotein translocase complex of human mitochondrial outer membrane. structure in the small TIM chaperones of the mitochondrial intermembrane Biochem. Biophys. Res. Commun. 369 (3), 958–963. space. Mol. Biol. Evol. 24 (5), 1149–1160. [44] Gentle, I., Gabriel, K., Beech, P., Waller, R. and Lithgow, T. (2004) The Omp85 [71] Jin, H., Kendall, E., Freeman, T.C., Roberts, R.G. and Vetrie, D.L. (1999) The family of proteins is essential for outer membrane biogenesis in human family of Deafness/Dystonia peptide (DDP) related mitochondrial mitochondria and bacteria. J. Cell Biol. 164 (1), 19–24. import proteins. Genomics 61 (3), 259–267. [45] Armstrong, L.C., Komiya, T., Bergman, B.E., Mihara, K. and Bornstein, P. (1997) [72] Szczesny, B., Tann, A.W., Longley, M.J., Copeland, W.C. and Mitra, S. (2008) Metaxin is a component of a preprotein import complex in the outer Long patch base excision repair in mammalian mitochondrial genomes. J. membrane of the mammalian . J. Biol. Chem. 272 (10), 6510– Biol. Chem. 283 (39), 26349–26356. 6518. [73] Vascotto, C. et al. (2011) Knock-in reconstitution studies reveal an [46] Armstrong, L.C., Saenz, A.J. and Bornstein, P. (1999) Metaxin 1 interacts with unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking metaxin 2, a novel related protein associated with the mammalian and function. Mol. Biol. Cell 22 (20), 3887–3901. mitochondrial outer membrane. J. Cell. Biochem. 74 (1), 11–22. [74] Vogelstein, B., Lane, D. and Levine, A.J. (2000) Surfing the p53 network. [47] Kozjak-Pavlovic, V., Ross, K., Benlasfer, N., Kimmig, S., Karlas, A. and Rudel, T. Nature 408 (6810), 307–310. (2007) Conserved roles of Sam50 and metaxins in VDAC biogenesis. EMBO [75] Vousden, K.H. and Ryan, K.M. (2009) P53 and metabolism. Nat. Rev. Cancer 9 Rep. 8 (6), 576–582. (10), 691–700. [48] Qu, Y. et al. (2012) Mitochondrial sorting and assembly machinery subunit [76] Zhuang, J., Wang, P.Y., Huang, X., Chen, X., Kang, J.G. and Hwang, P.M. (2013) Sam37 in Candida albicans: insight into the roles of mitochondria in fitness, Mitochondrial disulfide relay mediates translocation of p53 and partitions its cell wall integrity, and virulence. Eukaryot. Cell 11 (4), 532–544. subcellular activity. Proc. Natl. Acad. Sci. USA 110 (43), 17356–17361. [49] Hofmann, S., Rothbauer, U., Muhlenbein, N., Baiker, K., Hell, K. and Bauer, M.F. [77] Agathocleous, M. and Harris, W.A. (2013) Metabolism in physiological cell (2005) Functional and mutational characterization of human MIA40 acting proliferation and differentiation. Trends Cell Biol. 23 (10), 484–492. during import into the mitochondrial intermembrane space. J. Mol. Biol. 353 [78] Dabir, D.V. et al. (2013) A small molecule inhibitor of redox-regulated protein (3), 517–528. translocation into mitochondria. Dev. Cell 25 (1), 81–92. [50] Chacinska, A., Guiard, B., Muller, J.M., Schulze-Specking, A., Gabriel, K., Kutik, [79] Todd, L.R., Damin, M.N., Gomathinayagam, R., Horn, S.R., Means, A.R. and S. and Pfanner, N. (2008) Mitochondrial biogenesis, switching the sorting Sankar, U. (2010) Growth factor erv1-like modulates Drp1 to preserve pathway of the intermembrane space receptor Mia40. J. Biol. Chem. 283 (44), mitochondrial dynamics and function in mouse embryonic stem cells. Mol. 29723–29729. Biol. Cell 21 (7), 1225–1236. [51] Sztolsztener, M.E., Brewinska, A., Guiard, B. and Chacinska, A. (2013) [80] Teng, E.C., Todd, L.R., Ribar, T.J., Lento, W., Dimascio, L., Means, A.R. and Disulfide bond formation: sulfhydryl oxidase ALR controls mitochondrial Sankar, U. (2011) Gfer inhibits Jab1-mediated degradation of p27kip1 to biogenesis of human MIA40. Traffic 14 (3), 309–320. restrict proliferation of hematopoietic stem cells. Mol. Biol. Cell 22 (8), 1312– [52] Fischer, M. et al. (2013) Protein import and oxidative folding in the 1320. mitochondrial intermembrane space of intact mammalian cells. Mol. Biol. [81] Li, Y. et al. (2012) Augmenter of liver regeneration (alr) promotes liver Cell 24 (14), 2160–2170. outgrowth during zebrafish hepatogenesis. PLoS One 7 (1), e30835. [53] Yang, J. et al. (2012) Human CHCHD4 mitochondrial proteins regulate [82] Curado, S., Ober, E.A., Walsh, S., Cortes-Hernandez, P., Verkade, H., Koehler, cellular oxygen consumption rate and metabolism and provide a critical C.M. and Stainier, D.Y. (2010) The mitochondrial import gene is role in hypoxia signaling and tumor progression. J. Clin. Invest. 122 (2), specifically required for hepatocyte survival and provides a liver regeneration 600–611. model. Dis. Model. Mech. 3 (7–8), 486–495. [54] Kallergi, E. et al. (2012) Targeting and maturation of Erv1/ALR in the [83] Sugiyama, S., Moritoh, S., Furukawa, Y., Mizuno, T., Lim, Y.M., Tsuda, L. and mitochondrial intermembrane space. ACS Chem. Biol. 7 (4), 707–714. Nishida, Y. (2007) Involvement of the mitochondrial protein translocator [55] Wang, G. et al. (1999) Identification and characterization of receptor for component tim50 in growth, cell proliferation and the modulation of mammalian hepatopoietin that is homologous to yeast ERV1. J. Biol. Chem. respiration in Drosophila. Genetics 176 (2), 927–936. 274 (17), 11469–11472. [84] Guo, Y., Cheong, N., Zhang, Z., De Rose, R., Deng, Y., Farber, S.A., Fernandes- [56] Li, Y. et al. (2000) Stimulation of the mitogen-activated protein kinase Alnemri, T. and Alnemri, E.S. (2004) Tim50, a component of the cascade and tyrosine phosphorylation of the epidermal growth factor mitochondrial translocator, regulates mitochondrial integrity and cell receptor by hepatopoietin. J. Biol. Chem. 275 (48), 37443–37447. death. J. Biol. Chem. 279 (23), 24813–24825. [57] Lu, C. et al. (2002) Intracrine hepatopoietin potentiates AP-1 activity through [85] Ahting, U. et al. (2009) Neurological phenotype and reduced lifespan in JAB1 independent of MAPK pathway. FASEB J. 16 (1), 90–92. heterozygous Tim23 knockout mice, the first mouse model of defective [58] Bauer, M.F. et al. (1999) Genetic and structural characterization of the human mitochondrial import. Biochim. Biophys. Acta 1787 (5), 371–376. mitochondrial inner membrane translocase. J. Mol. Biol. 289 (1), 69–82. [86] Rainbolt, T.K., Atanassova, N., Genereux, J.C. and Wiseman, R.L. (2013) Stress- [59] Sinha, D., Srivastava, S., Krishna, L. and D’Silva, P. (2014) Unraveling the regulated translational attenuation adapts mitochondrial protein import intricate organization of Mammalian mitochondrial presequence through Tim17A degradation. Cell Metab. 18 (6), 908–919. translocases: existence of multiple translocases for maintenance of [87] Hanahan, D. and Weinberg, R.A. (2011) Hallmarks of cancer: the next mitochondrial function. Mol. Cell. Biol. 34 (10), 1757–1775. generation. Cell 144 (5), 646–674. [60] Sinha, D., Joshi, N., Chittoor, B., Samji, P. and D’Silva, P. (2010) Role of Magmas [88] Kannangai, R., Vivekanandan, P., Martinez-Murillo, F., Choti, M. and in protein transport and human mitochondria biogenesis. Hum. Mol. Genet. Torbenson, M. (2007) Fibrolamellar carcinomas show overexpression of 19 (7), 1248–1262. genes in the RAS, MAPK, PIK3, and xenobiotic degradation pathways. Hum. [61] Davey, K.M. et al. (2006) Mutation of DNAJC19, a human homologue of yeast Pathol. 38 (4), 639–644. inner mitochondrial membrane co-chaperones, causes DCMA syndrome, a [89] Xu, X. et al. (2010) Quantitative proteomics study of breast cancer cell lines novel autosomal recessive Barth syndrome-like condition. J. Med. Genet. 43 isolated from a single patient: discovery of TIMM17A as a marker for breast (5), 385–393. cancer. Proteomics 10 (7), 1374–1390. [62] Schusdziarra, C., Blamowska, M., Azem, A. and Hell, K. (2013) Methylation- [90] Dundas, S.R., Lawrie, L.C., Rooney, P.H. and Murray, G.I. (2005) Mortalin is controlled J-protein MCJ acts in the import of proteins into human over-expressed by colorectal adenocarcinomas and correlates with poor mitochondria. Hum. Mol. Genet. 22 (7), 1348–1357. survival. J. Pathol. 205 (1), 74–81. [63] Ojala, T., Polinati, P., Manninen, T., Hiippala, A., Rajantie, J., Karikoski, R., [91] Wadhwa, R., Takano, S., Kaur, K., Deocaris, C.C., Pereira-Smith, O.M., Reddel, Suomalainen, A. and Tyni, T. (2012) New mutation of mitochondrial DNAJC19 R.R. and Kaul, S.C. (2006) Upregulation of mortalin/mthsp70/Grp75 causing dilated and noncompaction cardiomyopathy, anemia, ataxia, and contributes to human carcinogenesis. Int. J. Cancer 118 (12), 2973–2980. male genital anomalies. Pediatr. Res. 72 (4), 432–437. [92] Deocaris, C.C., Widodo, N., Shrestha, B.G., Kaur, K., Ohtaka, M., Yamasaki, K., [64] Hatle, K.M. et al. (2013) MCJ/DnaJC15, an endogenous mitochondrial Kaul, S.C. and Wadhwa, R. (2007) Mortalin sensitizes human cancer cells to repressor of the respiratory chain that controls metabolic alterations. Mol. MKT-077-induced senescence. Cancer Lett. 252 (2), 259–269. Cell. Biol. 33 (11), 2302–2314. [93] Yang, L., Li, H., Jiang, Y., Zuo, J. and Liu, W. (2013) Inhibition of mortalin [65] Chung, Y.M., Lee, S.B., Kim, H.J., Park, S.H., Kim, J.J., Chung, J.S. and Yoo, Y.D. expression reverses cisplatin resistance and attenuates growth of ovarian (2008) Replicative senescence induced by Romo1-derived reactive oxygen cancer cells. Cancer Lett. 336 (1), 213–221. species. J. Biol. Chem. 283 (48), 33763–33771. [94] Matoba, S. et al. (2006) P53 regulates mitochondrial respiration. Science 312 [66] Norton, M. et al. (2014) ROMO1 is an essential redox-dependent regulator of (5780), 1650–1653. mitochondrial dynamics. Sci. Signal. 7 (310), ra10. [95] Lu, W.J., Lee, N.P., Kaul, S.C., Lan, F., Poon, R.T., Wadhwa, R. and Luk, J.M. [67] Mick, D.U. et al. (2012) MITRAC links mitochondrial protein translocation to (2011) Mortalin-p53 interaction in cancer cells is stress dependent and respiratory-chain assembly and translational regulation. Cell 151 (7), constitutes a selective target for cancer therapy. Cell Death Differ. 18 (6), 1528–1541. 1046–1056. [68] Bauer, M.F., Rothbauer, U., Muhlenbein, N., Smith, R.J., Gerbitz, K., Neupert, [96] Sankala, H., Vaughan, C., Wang, J., Deb, S. and Graves, P.R. (2011) W., Brunner, M. and Hofmann, S. (1999) The mitochondrial TIM22 preprotein Upregulation of the mitochondrial transport protein, Tim50, by mutant p53 translocase is highly conserved throughout the eukaryotic kingdom. FEBS contributes to cell growth and chemoresistance. Arch. Biochem. Biophys. 512 Lett. 464 (1–2), 41–47. (1), 52–60. A.M. Sokol et al. / FEBS Letters 588 (2014) 2484–2495 2495

[97] Chandel, N.S., McClintock, D.S., Feliciano, C.E., Wood, T.M., Melendez, J.A., [115] Lazarou, M., Jin, S.M., Kane, L.A. and Youle, R.J. (2012) Role of PINK1 binding Rodriguez, A.M. and Schumacker, P.T. (2000) Reactive oxygen species to the TOM complex and alternate intracellular membranes in recruitment generated at mitochondrial complex III stabilize hypoxia-inducible factor- and activation of the E3 ligase Parkin. Dev. Cell 22 (2), 320–333. 1alpha during hypoxia: a mechanism of O2 sensing. J. Biol. Chem. 275 (33), [116] Bertolin, G. et al. (2013) The TOMM machinery is a molecular switch in 25130–25138. PINK1 and PARK2/PARKIN-dependent mitochondrial clearance. Autophagy 9 [98] Hagen, T., Taylor, C.T., Lam, F. and Moncada, S. (2003) Redistribution of (11), 1801–1817. intracellular oxygen in hypoxia by nitric oxide: effect on HIF1alpha. Science [117] Kato, H., Lu, Q., Rapaport, D. and Kozjak-Pavlovic, V. (2013) Tom70 is 302 (5652), 1975–1978. essential for PINK1 import into mitochondria. PLoS One 8 (3), e58435. [99] Sciacovelli, M. et al. (2013) The mitochondrial chaperone TRAP1 promotes [118] Hasson, S.A. et al. (2013) High-content genome-wide RNAi screens identify neoplastic growth by inhibiting succinate dehydrogenase. Cell Metab. 17 (6), regulators of parkin upstream of mitophagy. Nature 504 (7479), 291–295. 988–999. [119] Bender, A. et al. (2013) TOM40 mediates mitochondrial dysfunction induced [100] Koehler, C.M., Leuenberger, D., Merchant, S., Renold, A., Junne, T. and Schatz, by alpha-synuclein accumulation in Parkinson’s disease. PLoS One 8 (4), G. (1999) Human deafness dystonia syndrome is a mitochondrial disease. e62277. Proc. Natl. Acad. Sci. USA 96 (5), 2141–2146. [120] Sarraf, S.A., Raman, M., Guarani-Pereira, V., Sowa, M.E., Huttlin, E.L., Gygi, S.P. [101] Tranebjaerg, L., Hamel, B.C., Gabreels, F.J., Renier, W.O. and Van Ghelue, M. and Harper, J.W. (2013) Landscape of the PARKIN-dependent ubiquitylome in (2000) A de novo missense mutation in a critical domain of the X-linked DDP response to mitochondrial depolarization. Nature 496 (7445), 372–376. gene causes the typical deafness-dystonia-optic atrophy syndrome. Eur. J. [121] DuBoff, B., Feany, M. and Gotz, J. (2013) Why size matters – balancing Hum. Genet. 8 (6), 464–467. mitochondrial dynamics in Alzheimer’s disease. Trends Neurosci. 36 (6), [102] Roesch, K., Curran, S.P., Tranebjaerg, L. and Koehler, C.M. (2002) Human 325–335. deafness dystonia syndrome is caused by a defect in assembly of the DDP1/ [122] Sirk, D., Zhu, Z., Wadia, J.S., Shulyakova, N., Phan, N., Fong, J. and Mills, L.R. TIMM8a-TIMM13 complex. Hum. Mol. Genet. 11 (5), 477–486. (2007) Chronic exposure to sub-lethal beta-amyloid (Abeta) inhibits the [103] Hofmann, S., Rothbauer, U., Muhlenbein, N., Neupert, W., Gerbitz, K.D., import of nuclear-encoded proteins to mitochondria in differentiated PC12 Brunner, M. and Bauer, M.F. (2002) The C66W mutation in the deafness cells. J. Neurochem. 103 (5), 1989–2003. dystonia peptide 1 (DDP1) affects the formation of functional DDP1.TIM13 [123] Hansson Petersen, C.A. et al. (2008) The amyloid beta-peptide is imported complexes in the mitochondrial intermembrane space. J. Biol. Chem. 277 into mitochondria via the TOM import machinery and localized to (26), 23287–23293. mitochondrial cristae. Proc. Natl. Acad. Sci. USA 105 (35), 13145–13150. [104] Di Fonzo, A. et al. (2009) The mitochondrial disulfide relay system protein [124] Napoli, E., Wong, S., Hung, C., Ross-Inta, C., Bomdica, P. and Giulivi, C. (2013) GFER is mutated in autosomal-recessive myopathy with cataract and Defective mitochondrial disulfide relay system, altered mitochondrial combined respiratory-chain deficiency. Am. J. Hum. Genet. 84 (5), 594–604. morphology and function in Huntington’s disease. Hum. Mol. Genet. 22 (5), [105] Daithankar, V.N., Schaefer, S.A., Dong, M., Bahnson, B.J. and Thorpe, C. (2010) 989–1004. Structure of the human sulfhydryl oxidase augmenter of liver regeneration [125] Valentine, J.S., Doucette, P.A. and Zittin Potter, S. (2005) Copper-zinc and characterization of a human mutation causing an autosomal recessive superoxide dismutase and amyotrophic lateral sclerosis. Annu. Rev. myopathy. Biochemistry 49 (31), 6737–6745. Biochem. 74, 563–593. [106] Yool, D.A., Edgar, J.M., Montague, P. and Malcolm, S. (2000) The proteolipid [126] Carri, M.T. and Cozzolino, M. (2011) SOD1 and mitochondria in ALS: a protein gene and myelin disorders in man and animal models. Hum. Mol. dangerous liaison. J. Bioenerg. Biomembr. 43 (6), 593–599. Genet. 9 (6), 987–992. [127] Gross, D.P., Burgard, C.A., Reddehase, S., Leitch, J.M., Culotta, V.C. and Hell, K. [107] Huttemann, M., Zhang, Z., Mullins, C., Bessert, D., Lee, I., Nave, K.A., Appikatla, (2011) Mitochondrial Ccs1 contains a structural disulfide bond crucial for the S. and Skoff, R.P. (2009) Different proteolipid protein mutants exhibit unique import of this unconventional substrate by the disulfide relay system. Mol. metabolic defects. ASN Neuro 1 (3), e00014. Biol. Cell 22 (20), 3758–3767. [108] Appikatla, S., Bessert, D., Lee, I., Huttemann, M., Mullins, C., Somayajulu-Nitu, [128] Kloppel, C., Suzuki, Y., Kojer, K., Petrungaro, C., Longen, S., Fiedler, S., Keller, S. M., Yao, F. and Skoff, R.P. (2014) Insertion of proteolipid protein into and Riemer, J. (2011) Mia40-dependent oxidation of cysteines in domain I of oligodendrocyte mitochondria regulates extracellular pH and adenosine Ccs1 controls its distribution between mitochondria and the cytosol. Mol. triphosphate. Glia 62 (3), 356–373. Biol. Cell 22 (20), 3749–3757. [109] Chacinska, A. et al. (2004) Essential role of Mia40 in import and assembly of [129] Kawamata, H. and Manfredi, G. (2008) Different regulation of wild-type and mitochondrial intermembrane space proteins. EMBO J. 23 (19), 3735–3746. mutant Cu, Zn superoxide dismutase localization in mammalian [110] Lin, M.T. and Beal, M.F. (2006) Mitochondrial dysfunction and oxidative mitochondria. Hum. Mol. Genet. 17 (21), 3303–3317. stress in neurodegenerative diseases. Nature 443 (7113), 787–795. [130] Suzuki, Y., Ali, M., Fischer, M. and Riemer, J. (2013) Human copper chaperone [111] Schon, E.A. and Przedborski, S. (2011) Mitochondria: the next for superoxide dismutase 1 mediates its own oxidation-dependent import (neurode)generation. Neuron 70 (6), 1033–1053. into mitochondria. Nat. Commun. 4, 2430. [112] Narendra, D., Walker, J.E. and Youle, R. (2012) Mitochondrial quality control [131] Varabyova, A., Topf, U., Kwiatkowska, P., Wrobel, L., Kaus-Drobek, M. and mediated by PINK1 and Parkin: links to parkinsonism. Cold Spring Harb. Chacinska, A. (2013) Mia40 and MINOS act in parallel with Ccs1 in the Perspect. Biol. 4 (11), a011338. biogenesis of mitochondrial Sod1. FEBS J. 280 (20), 4943–4959. [113] McLelland, G.L., Soubannier, V., Chen, C.X., McBride, H.M. and Fon, E.A. (2014) [132] Vande Velde, C., Miller, T.M., Cashman, N.R. and Cleveland, D.W. (2008) Parkin and PINK1 function in a vesicular trafficking pathway regulating Selective association of misfolded ALS-linked mutant SOD1 with the mitochondrial quality control. EMBO J. 33 (4), 282–295. cytoplasmic face of mitochondria. Proc. Natl. Acad. Sci. USA 105 (10), [114] Becker, D., Richter, J., Tocilescu, M.A., Przedborski, S. and Voos, W. (2012) 4022–4027. Pink1 kinase and its membrane potential (Deltapsi)-dependent cleavage product both localize to outer mitochondrial membrane by unique targeting mode. J. Biol. Chem. 287 (27), 22969–22987.