Protein transport machineries for precursor translocation across the inner mitochondrial membrane

Biochimica et Biophysica Acta 1793 (2009) 52–59

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r

Review

Protein transport machineries for precursor translocation across the inner mitochondrial membrane Karina Wagner a,b, David U. Mick a,b,c, Peter Rehling c,⁎ a b c

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg, Germany Fakultät für Biologie, Universität Freiburg, D-79104 Freiburg, Germany Abteilung Biochemie II, Universität Göttingen, D-37073 Göttingen, Germany

a r t i c l e

i n f o

Article history: Received 26 February 2008 Received in revised form 20 May 2008 Accepted 22 May 2008 Available online 11 June 2008 Keywords: Mitochondria Protein complex Import Tim protein Inner membrane

a b s t r a c t The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Mitochondria of eukaryotic cells originated from α-proteobacteria, which lost most of the protein encoding genes to the nucleus of their host cell over time (for review see [1]). Consequently, mitochondrial genomes usually encode only a small subset of mostly hydrophobic proteins. In the yeast S. cerevisiae only eight and in human thirteen proteins are synthesized within the mitochondria. Since most of the approximately 1000 mitochondrial proteins are translated in the cytosol, mitochondria require sophisticated machineries that are competent of importing these proteins into the four mitochondrial subcompartments (outer membrane, intermembrane space, inner membrane, and matrix). Moreover, the proteins that are synthesized in the cytosol require targeting signals, which direct them not only to mitochondria but also to their final destination inside the organelle. The mitochondrial ancestors, α-proteobacteria, already possessed machineries for export of proteins from the cytosol into and across the plasma membrane to the periplasmic space [2]. Some of these machineries were maintained in the mitochondria during evolution (i.e. the export machinery for mitochondrial-encoded proteins) whereas others had to evolve for the import of proteins into the organelle.

⁎ Corresponding author. Tel.: +49 551 39 5947; fax: +49 551 39 5979. E-mail address: [email protected] (P. Rehling). 0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.05.026

2. Import pathways into mitochondria — an overview of the players To traverse the outer and inner mitochondrial membranes precursor proteins need the assistance of membrane embedded translocase machineries. These multi-protein complexes usually contain signal receptors and form pores in the membrane through which the precursor proteins are transported. The process of precursor translocation is energy dependent and the translocases help to translate the driving energies into a vectorial movement of the polypeptide chain [3–6]. The TOM complex (translocase of the outer mitochondrial membrane) is the main entrance into mitochondria. It contains receptors that expose domains at the cytosolic surface of the membrane for binding of incoming precursor proteins [2–6]. The receptors pass the precursors on to Tom40, which forms the protein-conducting pore of the complex. Upon exit from the Tom40 channel, proteins use different transport machineries depending on their final destination and topology. Prior to their insertion into the outer membrane β-barrel proteins are transported into the intermembrane space by the TOM complex and subsequently sorted into the outer membrane by the SAM complex (sorting and assembly machinery) (Fig. 1A). In the intermembrane space a small TIM chaperone complex facilitates the transport of the β-barrel precursor proteins from the TOM to the SAM complex [3,5,7]. The Sam50 protein is related to the bacterial Omp85 and forms a channel in the outer membrane that is closely associated with Sam35, which recognizes the β-signal [2–5,8]. The molecular mechanism by which the

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Fig. 1. Import pathways into mitochondria. (A) β-barrel proteins pass the outer membrane via the TOM machinery and are guided by the small Tim chaperones to the SAM machinery. (B) Preproteins containing cysteine motifs (usually n = 3 or 9) are imported via the TOM complex and the MIA machinery. (C) Presequence-containing preproteins require the TOM and TIM23 complexes for transport into or across the inner membrane. Hydrophobic sorting signals direct precursor proteins laterally into the inner membrane Pre, presequence; hydrophobic sorting signal, hatched. (D) Carrier pathway proteins use the TOM and small Tim proteins for transport to the TIM22 translocase, which inserts them into the inner mitochondrial membrane. OM, outer membrane; IM, inner membrane; IMS, intermembrane space.

SAM machinery inserts β-barrel proteins into the outer membrane is still enigmatic. Moreover, the recent finding that assembly of the αhelical Tom22 protein also depends on the SAM complex suggests that the complex fulfills more functions than anticipated [9]. A set of proteins of the intermembrane space requires the MIA machinery (mitochondrial intermembrane space import and assembly) for import and folding. Substrates of the MIA machinery are proteins that contain cysteine-rich motives such as the small Tim chaperones of the intermembrane space (Fig. 1B). Together with the sulfhydryl oxidase Erv1, Mia40 catalyzes the formation of disulfide bonds in its substrates [10–12]. Depending on the precursors' targeting signals, membrane translocation across the inner mitochondrial membrane can be carried out by one of two distinct translocases: the TIM23 or the TIM22 complex (translocase of the inner mitochondrial membrane) (Fig. 1C and D). While both inner membrane translocases differ with respect to their substrates, the Tim22 and Tim23 proteins that form the proteinconducting pores of the TIM complexes display sequence similarity and only a single common ancestor (Tim22) is present in early eukaryotes with so called degenerate mitochondria [13]. 3. Transport pathway of carrier proteins into the inner membrane Although the inner mitochondrial membrane represents a stringent permeability barrier, mitochondria need to constantly exchange metabolites such as ATP, phosphate, carnitine/acylcarnitine, oxoglutarate/malate, and many others with the cytosol [14]. To pass such metabolites across the inner membrane, specialized carrier proteins are necessary that mediate the transport into and out of the mitochondrial matrix. These metabolite carrier proteins are inserted into the inner membrane of mitochondria through a complex transport

route that has been termed the carrier pathway and which involves the TIM22 translocase complex in the inner mitochondrial membrane. 3.1. Substrates of the carrier translocase complex Substrates of the carrier translocase (TIM22 complex) are especially hydrophobic proteins, which possess several transmembrane segments. These proteins are translated in the cytosol as mature-sized proteins and possess internal targeting signals, which are distributed over the length of the precursor (Fig. 1D). Several of such signals are usually present in a single precursor molecule. Most substrates of the carrier translocase belong to the family of metabolite carriers. Beside their functional similarities, members of the carrier protein family share structural features. Carrier proteins are built of three modules of approximately similar length, each of which consists of two transmembrane segments that are connected by a hydrophilic loop (Fig. 2A). Thus, they form six transmembrane α-helices in the lipid phase of the inner membrane [15]. It is currently assumed, that other carrier proteins display a similar structure and that they associate into dimers in the inner mitochondrial membrane [16]. In addition to the carrier proteins, also other polytopic inner membrane proteins are transported along the carrier transport route, among them are Tim23 and Tim22 [17,18]. In contrast to carrier proteins, Tim22 and Tim23 possess only four predicted transmembrane spans. After their insertion into the lipid phase, Tim23 and Tim22 expose their N- and C-termini into the intermembrane space. Little is known about the targeting information in these proteins, however, it appears that for import of Tim23 into mitochondria, transmembrane helices one and four are important. For translocation across the outer membrane a N-terminal segment of Tim23 is critical, to which the non-essential small Tim

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Fig. 2. Carrier proteins follow the carrier pathway to reach the inner membrane. (C) The carrier transport pathway can be dissected into five consecutive stages. (A) Carrier proteins exhibit a domain structure built of three modules. Each module consists of two transmembrane spans connected by a hydrophilic loop. (B) The carrier translocase is built of six different proteins. OM, outer membrane; IM, inner membrane; IMS, intermembrane space; GIP, general import pore (Tom40, Tom5, Tom6, Tom7, and Tom22).

protein complex of Tim8 and Tim13 binds [19,20]. Finally, insertion of Tim23 into the inner membrane depends on positive charges in the matrix-exposed loops [21]. 3.2. Subunits and their interactions in the carrier translocase complex The TIM22 complex selectively transports proteins with multiple transmembrane spans into the inner mitochondrial membrane (Fig.1D). The complex can be divided into two parts: a membrane integrated part consisting of Tim54, Tim22, and Tim18 and a peripheral part that is exposed to the intermembrane space and consists of Tim12, Tim10, and Tim9 (Fig. 2B). These six subunits form a complex of approximately 300 kDa that displays a length of about 110 Å [22]. The essential Tim22 protein is the central component of the membrane integral part of the translocase and forms a hydrophilic channel in the TIM22 complex. Electrophysiological studies of isolated Tim22 estimated the channel diameter to be approximately 18 Å [23]. The Tim22 channel is sensitive towards the membrane potential (Δψ) and internal targeting signals [23]. Interestingly, two functionally coupled pores are present in the TIM22 complex [22]. In the absence of a carrier precursor protein the two pores are mainly in a closed state at physiological membrane potential. However, in the open state, when an internal targeting signal and a Δψ of sufficient magnitude are present, one of the pores closes while the second one is activated. The function of the membrane integral proteins Tim54 and Tim18 is less well understood [24–26]. In the absence of Tim18 neither the stability of the TIM22 complex nor the transport of proteins seem to be affected. In contrast to Tim18, loss of Tim54 function leads to an import defect for carrier proteins. Since tim54 mutant mitochondria display reduced levels of Tim22 it was suggested that Tim54 supports the stability of the complex [24]. Recently a second function for Tim54 in the assembly of the Yme1 protease complex in the inner membrane was suggested, however, it is unclear what role it plays in this process [27]. The fact that Tim54 exposes a large domain into the intermembrane space led to the ideas that it could serve as a receptor for the incoming carrier proteins or that

it might represent a docking site for the peripheral subunits Tim9, Tim10, and Tim12 on the complex. Tim9, Tim10, and Tim12 are essential for cell viability and belong to the protein family of small Tim proteins, which localize to the intermembrane space. Other members of this group are Tim8 and Tim13, which are involved in the transport of the Tim23 precursor protein across the outer membrane (see above). All five proteins contain characteristic cysteine motifs for the formation of disulfide bonds and are related to each other based on sequence similarity. As mentioned above Tim9 and Tim10 form a soluble, hexameric chaperone complex that mediates transport of the carrier precursors across the intermembrane space (Fig. 2C). In addition, a pool of Tim9 and Tim10 in association with Tim12 is bound to the TIM22 complex (Fig. 2B). It is currently unclear how the Tim9/Tim10/Tim12 module binds to the membrane integral part of the translocase. However, with regard to the organization of the membrane bound Tim9/Tim10/ Tim12 module one model suggests that the Tim12 replaces a single Tim10 molecule in the docked form of the hexameric complex [28,29]. 3.3. Insertion of carrier proteins into the inner membrane For the transport of proteins along the carrier pathway from the cytosol into the inner membrane three protein translocases need to cooperate — the TOM complex in the outer membrane, a small TIM chaperone complex in the intermembrane space, and the TIM22 complex in the inner membrane (Fig. 1D). The transport pathway of carrier proteins from the cytosol into the inner mitochondrial membrane can be divided into five distinct stages (Fig. 2C). These stages have been defined by stable translocation intermediates along the transport route, most of which have been found by arresting the precursor artificially. The carrier proteins are synthesized on cytosolic ribosomes. To prevent the hydrophobic precursors from aggregation in the cytosol chaperones associate with the unfolded precursor proteins (stage I) [30,31]. At the outer surface of mitochondria carrier precursors in association with their chaperones

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bind to the Tom70 receptor [30,32]. In the absence of ATP this Tom70bound state can be trapped on the surface of mitochondria (stage II) [33,34]. For translocation across the outer membrane ATP hydrolysis is required that leads to the release of the precursors from its chaperones and the receptor (Fig. 2C). Subsequently, Tom70 hands the carrier precursors over to the pore of the translocase that is formed by the β-barrel protein Tom40. The carrier precursors traverse the Tom40 channel in a hairpin loop like conformation [32,35]. Upon exit from the TOM complex the carrier precursors interact with the Tim9/Tim10 chaperone complex in the intermembrane space (stage III). The hexameric Tim9/Tim10 complex is built of three alternating copies of Tim9 and Tim10, which form an α-helical propeller with a central pore [36]. It is unclear where in the Tim9/Tim10 complex the carrier precursors are bound, however, biochemical analyses have shown that the hydrophobic transmembrane segments of the carrier precursors are recognized by the chaperone complex so that they are shielded during transport through the aqueous intermembrane space [37]. After translocation of the carrier precursors through Tom40, subsequent transport steps across the inner mitochondrial membrane are driven by the membrane potential. Accordingly, when the membrane potential is dissipated, e.g. by treatment with ionophors, the precursors accumulate at the translocation stage III of transport in association with the TOM complex and the small Tim proteins [33,38,39]. However, under these conditions a fraction of carrier proteins is also found tethered to the TIM22 complex of the inner membrane in a Δψindependent manner [22]. Insertion of the precursors into the lipid phase of the inner membrane is driven by the Δψ as the sole external energy source [40]. Through modulation of the Δψ in vitro the insertion process has been dissected into two consecutive steps. A low Δψ allows docking of the precursors to the TIM22 translocase, the Δψ suffices to drive the carrier precursors into the channel that is formed by Tim22

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(stage IV) [22]. A full membrane potential and an internal targeting signal in the precursor activate the translocase and lead to the subsequent lateral release of the polypeptide chain into the membrane. The third module of the carrier has been shown to be critical for membrane insertion by the TIM22 complex since it contains an internal targeting signal in the N-terminal portion of the fifth transmembrane segment [22,23,41]. In fact, insertion of the other two modules depends on the presence of the third module [42–45]. Finally, the last stage (stage V) in the carrier pathway is the maturation of the metabolite carrier into its functional dimeric state [31,33,44]. 4. Transport of presequence proteins across the inner mitochondrial membrane 4.1. Substrates of the presequence pathway Substrates for the presequence translocase (TIM23 complex) are mostly proteins that contain N-terminal targeting signals that are termed presequences. In most cases the presequence of a precursor is cleaved off in the mitochondrial matrix by the mitochondrial processing peptidase MPP after import (Fig. 3A) (for review see [46]). Usually presequences have a length of 10 to 80 amino acids and form an amphipathic α-helix with a positive net charge [47–49]. The presequence typically directs the mitochondrial precursor into the matrix. However, the presence of a hydrophobic sorting signal adjacent to the presequence can stall translocation across the inner membrane and direct the precursor laterally into the membrane (Fig. 3A). This process is commonly referred to as inner membrane sorting. If such a sorted precursor undergoes a second processing on the intermembrane space side of the inner membrane, a mature soluble protein can be released into the intermembrane space (Fig. 3A) [50,51]. The inner membrane

Fig. 3. The presequence translocase is a versatile multi-protein-complex. (A) Depending on their destination within the mitochondria preproteins possess different targeting signals. Cleavage of the precursor can occur after the presequence or the hydrophobic sorting signal (blue box). (B) The preproteins follow different import pathways. After crossing the outer membrane via the TOM complex the TIM23 translocase can either translocate soluble preproteins into the matrix with the help of the PAM complex, or sort proteins into the inner membrane. Association with respiratory chain supercomplexes (III/IV) can promote membrane potential dependent translocation of sorted precursors. The TIM23 translocase exists in two different states, either in the Tim21-containing, sorting competent form, TIM23SORT (C), or in the Tim21-free, PAM-bound form, TIM23Motor (D). OM, outer membrane; IM, inner membrane; IMS, intermembrane space; GIP, general import pore.

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peptidase complex (IMP) mediates this processing event by cleavage of the precursor behind the stop-transfer signal [52,53]. Thus, in contrast to the TIM22 complex, the presequence translocase (TIM23 complex) delivers its substrate proteins to three different mitochondrial compartments: the matrix, the inner membrane, and the intermembrane space. Therefore, it must be able to handle proteins, which vary strongly in length, topology, and structure. Consequently, the transport machinery for presequence-carrying proteins needs to be able to adapt to the specific requirements of each individual precursor for translocation. The membrane integral presequence translocase mediates the initial transport steps of the precursor proteins with presequences across the inner mitochondrial membrane. The membrane potential across the inner membrane drives the membrane translocation of the presequences. The force exerted by the Δψ acts in an electrophoretic manner on the positively charged presequence [54]. Since the Δψ is a spatially restricted force in proximity to the inner membrane it is insufficient to drive full transport of precursors into the matrix. However, protein sorting into the inner mitochondrial membrane can be driven by the Δψ alone [6,55–58] (Fig. 3B). Full translocation of the precursor into the matrix, however, requires an additional driving force that is generated by the PAM complex (presequence translocase-associated motor). The PAM complex is a multi-subunit protein complex exposed to the mitochondrial matrix. It associates to the TIM23 complex in a dynamic manner and is essential for matrix translocation (Fig. 3B). 4.2. Components and organization of the TIM23 complex The large variety of substrates that are imported into the different mitochondrial subcompartments requires a remarkably dynamic multi-subunit transport machinery. Three subunits build the membrane integral TIM23 complex that can associate to the matrix-exposed presequence translocase-associated motor complex (PAM). The core components of the TIM23 complex, that are present in all forms of the translocase, are Tim23, Tim17, and Tim50 (Fig. 3C and D). All three proteins are membrane integrated and perform essential functions: formation and regulation of the protein-conducting channel for transport of precursor proteins. Tim23, as well as Tim17, are related to the Tim22 channel of the carrier translocase. The Tim23 protein forms the protein-conducting channel of the presequence translocase complex [59]. Tim50 is an essential inner membrane protein that binds to the Nterminal intermembrane space domain of Tim23 [60–63]. In the inactive state of the Tim23 channel, when no precursor is transported, the intermembrane space domain of Tim50 keeps the Tim23 channel in a closed state [64]. This regulation is critical to maintain the electrochemical gradient across the inner mitochondrial membrane by preventing an uncontrolled flux of ions. Despite the fact that Tim17 and Tim23 form the core of the translocase and associate into a 90 kDa subcomplex, the function of Tim17 is not well understood [65,66]. The analysis of tim17 mutants showed that the protein is not only essential for import of proteins into the matrix but also plays an active role in inner membrane sorting [67]. Moreover, recent electrophysiological analyses of the Tim23 channel in tim17 mutants suggested a function of Tim17 in channel gating [68]. In addition to Tim17, Tim23, and Tim50 a fourth integral membrane protein is found as a subunit of the presequence translocase — Tim21. In yeast Tim21 is not required for growth under all conditions but critical for growth under conditions that require an increased mitochondrial activity [67]. The Tim21 protein exposes a ∼16 kDa domain (Tim21IMS), which is built of two α-helices flanked by an eight-stranded β-sheet, into the intermembrane space [69]. The Tim21IMS-domain is able to bind to the intermembrane space domain of the receptor Tom22 (Tom22IMS) through ionic interactions between positively charged amino acids on Tim21IMS and negatively charged segments in Tom22IMS [67,69,70]. The intermembrane space domain of Tom22 serves as a trans binding site for presequences of precursor

proteins in transit that have passed through the TOM complex [71– 75]. Subsequently, the interaction of Tim21 with the intermembrane space domain of Tom22 leads to the release of the presequence from the Tom22IMS domain. Consequently, the presequence can be passed over from the TOM complex to the presequence translocase in the inner mitochondrial membrane [67,70]. The presequence translocase mediates the initial transport steps of the presequence across the inner membrane. For translocation of preproteins that are sorted into the inner mitochondrial membrane, the active TIM23 complex can associate with respiratory chain complexes via the Tim21 protein (Fig. 3B) [76–78]. Although Tim21 seems not to be the only component that is responsible for this interaction, its importance for protein sorting becomes evident under conditions of a reduced membrane potential [77,78]. 4.3. The presequence translocase-associated motor complex — PAM When the presequence has entered the mitochondrial matrix, the TIM23 translocase can associate with the PAM complex to drive translocation of the precursor into the matrix (Fig. 3D). Corresponding to the Tim21 containing translocase, which is termed TIM23SORT, the TIM23 complex in association with the PAM complex is termed TIM23MOTOR. The central component of the PAM complex is the essential mtHsp70 (Ssc1 in yeast), an ATP-dependent chaperone that binds to the incoming unfolded polypeptide chain [79]. Cycles of ATP-dependent binding and subsequent release of the precursor by mtHsp70 promote the forward movement of the polypeptide into the matrix. The ATPase cycle of mtHsp70 is strongly regulated and therefore the PAM complex consists of five additional subunits that are involved in positioning and regulation of mtHsp70. The second essential component of the PAM complex is the membrane integral co-chaperone Pam18, a J-domain protein that stimulates the ATPase activity of mtHsp70 [80–82]. Nucleotide exchange of ADP against ATP in mtHsp70 is stimulated by the soluble nucleotide exchange factor Mge1 [83]. The PAM complex consists of two additional essential components — Pam16 and Tim44 (Fig. 3D). The membrane-associated J-like protein Pam16 is unable to activate mtHsp70 but rather regulates the activity of Pam18 in the ATPase cycle of mtHsp70 [84–87]. Pam16 and Pam18 interact with each other through their matrix-exposed domains. In the X-ray structure of the complex it becomes evident that Pam16 maintains Pam18 in an inactive conformation [88]. Tim44 is a membrane-associated protein that tethers mtHsp70 to the translocation channel [3,7,89]. The last PAM component is the recently identified Pam17 protein, which is involved in the organization of the PAM complex and promotes the association of Pam16/Pam18 with the TIM23 translocase [90]. The molecular functions of the Pam proteins during the protein transport process and the dynamics of their physical interactions are still enigmatic. Recent analyses have shown that the association of the PAM complex with the presequence translocase is mediated through a complex network of independent protein–protein interactions: The intermembrane space domain of Pam18 interacts with Tim17 [67,89], Pam16 promotes the association of Pam18 with the TIM23 complex [85,86,89], Pam17 interacts with Tim23 [91], and Tim44 plays a role in PAM association with the translocon [89]. Accordingly, the PAM complex connects physically to the TIM23 complex through at least four contact sites. These multiple interactions are especially important with regard to the dynamics of the presequence translocase during protein translocation, since association of the PAM complex is thought to induce dissociation of Tim21 from the complex and vice versa. Moreover, a novel mitochondrial protein termed Tam41/Mmp37 was recently identified. It affects the association of the TIM23 complex with the PAM complex. However, it is still an open question how Tam41/Mmp37 acts on the TIM23 translocase [92,93]. Thus, based on this finding it is tempting to speculate that additional factors might exist in the mitochondria that affect TIM23 complex dynamics.

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The respiratory chain complexes are abundant mitochondrial inner membrane protein complexes and present in excess over the protein translocase complexes. The recruitment of complexes III and IV to the TIM23 complex has been found to promote membrane potential dependent protein sorting into the inner membrane [76,77]. However, the respiratory chain complexes are not essential for protein translocation, since mitochondria that lack mitochondrial DNA and therefore are respiratory deficient are still able to maintain a membrane potential that suffices to allow protein transport. Nevertheless, beside their interaction with the presequence translocase complex, subunits of the PAM complex have also been found in association with the respiratory chain complexes III and IV. Recent data shows that Pam16 and Pam18 interact with the respiratory chain complexes in a Tim21-independent manner while Tim44 and mtHsp70 are not associated [78]. In agreement with this observation a matrix-destined precursor in transit through the TIM23 complex can be co-isolated with respiratory chain complexes III and IV [78]. Based on the observation that Pam16 and Pam18 are found associated with the respiratory chain but not Tim44 and mtHsp70 it was suggested that the PAM complex does not assemble to the TIM23 complex as a single building block but rather in a step-wise manner. A recently suggested model is, that a respiratory-chain-bound Pam16/Pam18 module connects to the TIM23 complex to replace Tim21. Upon release of the Pam16/18 module from the respiratory chain Tim44 and mtHsp70 are recruited to form the complete PAM module. Thus, matrix proteins utilize the respiratory chain complexes associated TIM23SORT form at their initial stages of protein translocation. Subsequently, a switch to the respiratory-chain-bound Pam16/Pam18 containing translocase occurs for subsequent membrane potential dependent translocation steps. However, through the presence of the Pam16/Pam18 module, the translocase is already primed for the subsequent recruitment of mtHsp70 at this stage. While this model is in line with the current data, further analysis will be required to critically test it.

matrix translocation Tim21 dissociates from the TIM23 core while the PAM complex is recruited [67]. In contrast, a recent study by Tamura et al. [93] suggested that at steady state a form of the TIM23 translocase exists that contains Tim21 and the PAM complex. This finding suggests that additional forms of the TIM23 complex might exist during protein translocation. However, the fact that the reconstituted Tim21 containing and motor-free form of the translocase is able to insert proteins into a membrane demonstrates that this is the minimal form of the translocase that can mediate motor-independent protein translocation and membrane insertion of a precursor protein [55]. Interestingly, at early stages of matrix protein translocation the respiratory chain complexes III and IV are bound to the presequence translocase in a Tim21 independent manner [78]. However, it is still unclear if at a later stage of transport a TIM23 complex in association with PAM and the respiratory chain exists. When the unfolded polypeptide enters the matrix it is bound by mtHsp70. Hydrolysis of ATP by mtHsp70 that is stimulated by Pam18 leads to closure of the substratebinding domain upon the unfolded polypeptide chain [95]. Two alternative models have been suggested to explain how the interaction between mtHsp70 and the precursor protein leads to movement of the polypeptide chain into the matrix. In the “Brownian ratchet” model the association of mtHsp70 to the precursor prevents its backsliding. Several mtHsp70 molecules bind in a hand-over-hand fashion to the unfolded precursor in order to lead to a directional movement. The “pulling model” suggests that mtHsp70 performs a power stroke on the precursor, which leads to forward movement of the polypeptide chain towards the matrix. Evidence in support of both models exists and it is still open if these models are mutually exclusive or if both mechanisms have a role in the protein transport [61,84,96–99]. Given the complexity of the PAM complex and the large number of different precursors that are transported with its help, it appears that we are just beginning to understand the dynamics of the presequence translocase and PAM complex and how they mediate precursor translocation.

4.4. A model for precursor translocation by the presequence translocase

5. Concluding remarks

The import of presequence-carrying preproteins from the cytosol to their final destination in the mitochondria requires an initial translocation through the TOM complex. Therefore, TOM receptors bind to the presequence and guide the precursor to the Tom40 channel. On the intermembrane space side of the outer membrane the C-terminal intermembrane space domain of Tom22 (Tom22IMS) binds the presequence that emerges from the Tom40 channel. Subsequently, the Tim21 containing form of the presequence translocase contacts Tom22IMS with the Tim21IMS domain connecting the two translocases of the outer and inner mitochondrial membranes [67,70]. Thereby, the unfolded preprotein is handed over from the TOM to the TIM23 complex. At this stage Tim50 keeps the Tim23 channel in a closed state in order to maintain the electrochemical gradient across the membrane [64]. Interaction of the presequence with Tim23 triggers the opening of the channel so that the protein can enter the translocation pore [64,94]. Depending on the destination and requirements of the preprotein in transit it was suggested that the TIM23 complex associates with different protein complexes for the subsequent transport steps: For protein sorting into the inner membrane the Tim21 containing, motor-free form of the TIM23 complex (TIM23SORT) recruits the respiratory chain supercomplexes consisting of complexes III and IV [76,77]. This association occurs after release of Tim21 from the TOM complex since the TOM complex does not co-isolate together with the respiratory chain and the Tim21 bound presequence translocase [77]. In a reconstituted system, van der Laan et al. [55] demonstrated that the purified motor-free form of the TIM23 complex is able to transport a precursor with a hydrophobic sorting signal into a lipid bilayer. This study demonstrated that a minimal complex composed of Tim17, Tim23, Tim50, and Tim21 is able to transport a precursor across the inner membrane and into the lipid phase. For

Recent research on protein transport across the inner mitochondrial membrane has led to the discovery of an unexpected number of novel components. It is likely that we have now identified most of the core components of the TIM and PAM machineries. However, regulatory factors, which are not essential for protein import but critical for the fine-tuning of the system, might still await their discovery. Novel approaches will be required to identify such factors. Moreover, a molecular understanding of the function of many of the new components is still lacking. Thus, the field of mitochondrial protein transport will need to focus on mechanistic aspects of the translocation process in the future. Acknowledgements We would like to thank Dr. M. van der Laan, A. Chacinska, and C. Meisinger for their helpful discussion and comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft. References [1] M. van der Giezen, J. Tovar, Degenerate mitochondria, EMBO reports 6 (2005) 525–530. [2] P. Dolezal, V. Likic, J. Tachezy, T. Lithgow, Evolution of the molecular machines for protein import into mitochondria, Science 313 (2006) 314–318. [3] W. Neupert, J.M. Herrmann, Translocation of proteins into mitochondria, Annu. Rev. Cell Dev. Biol. 76 (2007) 723–749. [4] T. Endo, H. Yamamoto, M. Esaki, Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles, J. Cell Sci. 116 (2003) 3259–3267. [5] S. Kutik, B. Guiard, H.E. Meyer, N. Wiedemann, N. Pfanner, Cooperation of translocase complexes in mitochondrial protein import, J. Cell Biol. 179 (2007) 585–591. [6] P. Rehling, K. Brandner, N. Pfanner, Mitochondrial import and the twin-pore translocase, Nat. Rev. Mol. Cell. Biol. 5 (2004) 519–530.

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