Mitochondrial protein import: two membranes, three translocases Nikolaus Pfanner* and Nils Wiedemann

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1 400 Mitochondrial protein import: two membranes, three translocases Nikolaus Pfanner* and Nils Wiedemann Most mitochondrial proteins are synthesised in the cytosol and must be translocated across one or two membranes to reach their functional destination inside mitochondria. Dynamic protein complexes in the outer and inner membranes function as specific machineries that recognise the various kinds of precursor proteins and promote their translocation through protein-conducting channels. At least three major translocase complexes with a high flexibility and versatility are needed to ensure the proper import of precursor proteins into mitochondria. Addresses Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Strasse 7, D Freiburg, Germany * Nikolaus.Pfanner@biochemie.uni-freiburg.de Current Opinion in Cell Biology 2002, 14: /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. Abbreviations ψ membrane potential GIP general import pore MPP mitochondrial processing peptidase mthsp70 mitochondrial heat shock protein 70 TIM translocase of the inner mitochondrial membrane TOM translocase of the outer mitochondrial membrane Introduction Mitochondria make up ~20% of the mass of eukaryotic cells and contain ~1000 different proteins. Only a few mitochondrial proteins are encoded by the organellar genome and synthesised in the innermost compartment, the matrix. Over 98% of mitochondrial proteins are encoded by nuclear genes and synthesised as precursor proteins on cytosolic ribosomes [1 4]. Two major classes of precursor proteins can be distinguished (Figure 1). Cleavable precursor proteins carry amino-terminal amino acid extensions, termed presequences. The presequences function as targeting signals to direct the proteins to mitochondria and across both outer and inner membranes. The presequences are proteolytically removed in the matrix. Many hydrophobic membrane proteins, however, are synthesised without cleavable extensions. These precursor proteins typically contain several targeting signals that are distributed over the entire length of the protein. Main representatives for this second class of precursor proteins are the numerous metabolite carriers of the mitochondrial inner membrane. Besides these two major classes, several special types of precursor proteins exist, in particular for proteins of the outer membrane and intermembrane space. In this review, we discuss the observation that the mitochondrial import mechanisms show a higher complexity and versatility than previously anticipated. Since there is no consensus targeting sequence, the import machinery must recognise hundreds of different targeting signals. Hydrophilic matrix proteins must be translocated across two membranes and the intermembrane space. Hydrophobic inner membrane proteins have to cross the outer membrane without getting inserted into the membrane and to traverse the hydrophilic intermembrane space. How are mitochondria able to fulfil these tasks? Three membrane protein complexes, one located in the outer membrane and two in the inner membrane, are the main machineries for specific recognition and translocation of precursor proteins (Figure 1). The translocase of the outer membrane (TOM) complex, including the general import pore (GIP), transports all precursor proteins. The presequence translocase of the inner membrane (TIM23 complex) translocates cleavable preproteins. The protein insertion complex of the inner membrane (TIM22 complex or the carrier translocase) mediates the insertion of hydrophobic membrane proteins that carry several internal targeting signals. Translocase of the outer membrane At least seven different integral membrane proteins form a large, dynamic complex known as the TOM complex. This complex, comprising receptors and the GIP, represents the entry gate of mitochondria (Figure 2a). Import receptors Three TOM proteins, Tom20, Tom22 and Tom70, function as receptors with different yet overlapping specificities for precursor proteins. Each of the receptors is anchored in the outer membrane by a single transmembrane segment. Tom20 and Tom70, outer membrane proteins of 20 and 70 kda, respectively, carry the membrane anchor close to their amino terminus and expose a carboxy-terminal domain to the cytosol. Tom22 exposes an amino-terminal domain to the mitochondrial surface and also contains a small carboxy-terminal domain located in the intermembrane space. The presequences of cleavable precursor proteins are first recognised by Tom20 and then by Tom22 [1,4]. Mitochondrial presequences are positively charged and have the ability to form amphipathic α helices. It has been proposed that both surfaces of mitochondrial presequences, the positively charged one and the hydrophobic one, are recognised by different import components during the import process [5,6]. Tom20 is thought to recognise the hydrophobic surface and Tom22 the positively charged surface of the amphipathic helix [5]. The first high-resolution structure (NMR) of a mitochondrial import component, the cytosolic domain of the receptor Tom20 in complex with a presequence, beautifully demonstrated that the presequence formed an α helix that was indeed bound by a hydrophobic groove of the receptor [7 ] (Figure 2b).

2 Mitochondrial protein import: two membranes, three translocases Pfanner and Wiedemann 401 Figure 1 Two major pathways for mitochondrial protein import. Most mitochondrial proteins are encoded in the nucleus and are synthesised as precursor proteins on cytosolic polysomes. Preproteins (red) with an amino-terminal targeting signal (the presequence, positively charged) are targeted to the mitochondria by receptors (R) of the outer mitochondrial membrane (OM). These preproteins are translocated as unfolded, linear polypeptide chains through the general import pore of the outer mitochondrial membrane and the presequence translocase of the inner mitochondrial membrane (IM). Transport through the presequence translocase is driven by the membrane potential ( ψ) and the ATP-powered import motor mthsp70. The presequences are cleaved off by the mitochondrial processing peptidase (MPP), and the mature proteins can reach their native conformation with the help of matrix chaperones. The translocation of hydrophobic precursor proteins with internal targeting signals (blue) is also mediated by receptors and the GIP. These precursor proteins are then transferred across the intermembrane space (IMS) to the protein insertion complex, where they are integrated into the inner mitochondrial membrane in a membranepotential-dependent step. Cytosol OM IMS IM ATP mthsp70 Preprotein with presequence R Presequence translocase General import pore ψ Precursor protein with internal signals ATP R Protein insertion complex Mature innermembrane protein Matrix MPP Mature matrix protein Current Opinion in Cell Biology Hydrophobic precursor proteins with several internal targeting signals, such as the inner membrane metabolite carriers, are typically recognised by the receptor Tom70, which forms a dimer in the outer membrane [1,4]. Surprisingly, a single precursor protein is not recognised by a single receptor, but the presence of precursor proteins induces an oligomerisation of Tom70 such that several Tom70 molecules bind to one precursor polypeptide [8 ] (see below). Since these hydrophobic precursors contain several internal targeting signals [9,10], each Tom70 monomer is likely to bind to one targeting signal. So why are multiple receptor monomers simultaneously needed to bind one precursor protein, whereas for cleavable preproteins one precursor is recognised by one receptor monomer? The hydrophobic nature of the non-cleavable precursor proteins favours aggregation in a hydrophilic environment such as the cytosol. To prevent this, the precursors are escorted through the cytosol by molecular chaperones and, upon transfer to Tom70, are bound by multiple receptor sites. The receptor Tom70 might therefore possess chaperone-like properties [8 ]. Below, we will discuss that the aqueous intermembrane space also contains chaperone-like molecules for transfer of the hydrophobic precursor proteins from the outer membrane to the inner membrane. The receptors Tom20 and Tom22 can also recognise the carrier proteins, but with lower efficiency than Tom70. Tom20 and Tom22 might be involved in the step-wise transfer of the carrier proteins from Tom70 to the import pore. General import pore (Tom40) The protein-conducting channel across the outer membrane is formed by Tom40 [11]. The channel has an effective diameter of ~20 Å and thus does not allow the passage of folded protein domains [6,11 14]. Both cleavable and non-cleavable precursor proteins use the Tom40 channel; that is, their import pathways converge during passage across the outer membrane and separate again to distinct inner membrane translocases (Figures 1, 2a). The Tom40 channel is therefore termed the GIP. Cleavable preproteins are translocated through the GIP as linear chains, probably in α-helical or extended conformation.

3 402 Membranes and organelles Figure 2 (a) Cytosol OM IMS (b) ~3.5 nm Preprotein with presequence Tom20 hydrophobic Tom20 N Tom40 Tom22 C Tom5 Tom40 (c) Precursor protein with internal signals Tom7 Tom6 ATP Tom20 Tom22 Tom70 N GIP complex ~13 nm ~2 nm Tom22 ~7 nm The preprotein translocase of the outer mitochondrial membrane. (a) The TOM complex contains three main receptors: Tom20, Tom22 and Tom70. The initial receptors Tom70 and Tom20 are relatively loosely associated with the core GIP complex, which includes the pore-forming protein Tom40 (the GIP), the multifunctional receptor Tom22 and three small Tom proteins, Tom7, Tom6 and Tom5. Preproteins with a presequence are initially recognised by Tom20, transferred to Tom22, Tom5 and Tom40, and translocated across the outer membrane (OM). When the presequence emerges on the intermembrane space (IMS) side of the TOM complex, it can bind to the carboxy-terminal domain of Tom22 before the transfer to the inner membrane. Precursor proteins with internal signals are initially bound by multiple molecules of the receptor Tom70. They are transferred in an ATPdependent step to the GIP complex, where they can bind to Tom22 and Tom5 before they are inserted into the GIP. (b) Model of the cytosolic domain of the receptor Tom20 with a bound presequence. Mitochondrial presequences can form amphipathic α helices. The presequence binds with the hydrophobic side to a hydrophobic binding groove of Tom20 (modelled after the NMR structure of [7 ]). (c) Hypothetical model of the GIP complex containing Tom40, Tom22, Tom7, Tom6 and Tom5, and the more loosely associated Tom20 (the encircled area of [a] is shown). The complex contains up to three pore-like structures with ~2 nm internal diameter each. The complex is roughly 13 nm wide and 7 nm high, spanning the outer mitochondrial membrane (modelled after electron micrographic studies [12,20,79]). Current Opinion in Cell Biology The pore diameter of Tom40, however, is large enough to accommodate two α-helical polypeptide segments at the same time. Indeed, the non-cleavable carrier proteins are not translocated as linear chains. Neither the amino nor the carboxyl terminus of these membrane proteins is translocated first a middle portion of the precursor enters the channel in a loop formation [8 ]. Tom40 is associated with the three receptors Tom20, Tom22 and Tom70 as well as three small Tom proteins Tom5, Tom6 and Tom7 to form the TOM complex. The stable core complex (the GIP complex) is mainly formed by Tom40, Tom22 and the three small Tom proteins, while Tom20 and particularly Tom70 are more loosely associated with the GIP complex [6,12,15,16 ] (Figure 2a). This association of Tom proteins reflects their cooperation in import of precursor proteins. Tom20 and Tom70 are the initial receptors that recognise cleavable and non-cleavable precursors, respectively. Tom22 functions as a central receptor that accepts precursor proteins from the two initial receptors and mediates their transfer to the Tom40 channel. The cytosolic domain of Tom22 functions as docking point for the cytosolic domains of Tom20 and Tom70 [17]. Tom5 is involved in the transfer of all types of precursor proteins from Tom22 to Tom40 [18]. For some special precursors for example, some intermembrane space proteins Tom5 can even function as the only and primary receptor [19]. Upon passage through the Tom40 channel, presequence-carrying preproteins can interact with the intermembrane space domain of Tom22. Tom6 and Tom7 do not directly contact the precursor proteins, but are involved in the assembly of the translocase (see below). Electron microscopic analysis of the purified TOM complex revealed the presence of three pore-like structures [12,20] (Figure 2c). Surprisingly, when the receptor Tom20 was removed from the TOM complex, only two pores were observed [20]. Moreover, after removal of the central receptor (Tom22), the complex dissociates into smaller

4 Mitochondrial protein import: two membranes, three translocases Pfanner and Wiedemann 403 Figure 3 Precursor of Tom40 (a) Targeting, Tom20 Tom22 Tom7 Tom5 Early assembly intermediate: intermembrane space side (250 kda) kda (b) Late assembly intermediate: membrane integration (100 kda) (c) Tom22 Tom6 Completion of assembly: the mature TOM complex ( kda) Tom7 Tom7 Tom22 Tom6 Cycling between mature complex and late intermediate Multistep assembly of the TOM complex of the outer mitochondrial membrane. (a) The precursor of Tom40 (synthesised in the cytosol) is targeted to mitochondria mainly by the receptors Tom20 and Tom22. Tom7 facilitates the release of the receptors from the precursor protein that subsequently assembles with Tom5 to form an early assembly intermediate of ~250 kda. This intermediate is exposed to the intermembrane space side of the outer membrane. (b) The 250 kda intermediate complex disassembles in a time-dependent manner to form a late assembly intermediate, a 100 kda membrane-integrated complex. (c) The assembly pathway of Tom40 is completed by incorporation of Tom6, Tom7 and Tom22, to form the mature TOM complex of kda. The mature complex continuously disassembles to form the late assembly intermediate. Both assembly and disassembly are facilitated by Tom7 [27 ]. units, mainly single pores [14,17,20]. However, the receptors do not form pores by themselves. Each pore is formed by Tom40 proteins, while the membrane anchors of the receptors Tom22 and Tom20 are thought to be required for the stable association of several Tom40 dimers to form a large complex with several pores (Figure 2c). The TOM complex represents the universal entry gate for mitochondrial precursor protein destined for each of the four mitochondrial subcompartments [19,21]. Until recently, it was assumed that the precursor of the intermembrane space protein cytochrome c represented an exception (i.e. that apocytochrome c is directly translocated across the lipid phase of the outer membrane without a requirement for the TOM complex) [22]. In a recent study, however, the import of this precursor has been reconstituted and shown to require the TOM complex [23 ]. Interestingly, its import does not require surface-exposed receptor domains and is not competed for by saturating amounts of cleavable preproteins. Apocytochrome c seems to use the TOM complex in a novel manner that does not overlap with the import pathway of cleavable preproteins. Assembly of the TOM complex Each TOM protein itself is encoded by a nuclear gene and synthesised as non-cleavable precursor protein on cytosolic polysomes. This means that the TOM complex must import its own precursors, leading to the potential risk that a minor mistargeting of, for example, a TOM receptor to an incorrect organelle would subsequently cause the mistargeting of more receptors and many other mitochondrial proteins. This scenario can be excluded since none of the TOM proteins is able to import its own precursor, but the import of TOM precursors typically requires several other TOM proteins [24 26, 27,28]. The best-studied assembly pathway is that of the essential channel protein Tom40, which involves virtually all other TOM proteins (Figure 3). The precursor of Tom40 requires the surface receptors, in particular Tom20 and Tom22, for targeting to mitochondria [26,27,29]. Unexpectedly, the precursor is then translocated through the TOM machinery to the intermembrane space side of the outer membrane, associates with Tom5 and forms an early assembly intermediate of ~250 kda [27 ]. Subsequently this complex is converted to a late assembly intermediate of smaller size (100 kda) that is integrated into the outer membrane. The precursor of Tom40 is thus inserted into the lipid phase of the outer membrane from the trans side; that is, the intermembrane space side. Tom6 and Tom22 are finally needed to promote assembly of the 100 kda complexes to the large mature TOM complex of kda. While Tom6 functions as an assembly factor for the TOM complex, Tom7 has in part an antagonistic function [15,27,30]. Tom7 favours the dissociation of TOM proteins at several stages of the assembly pathway. Tom7 promotes the release of the Tom40 precursor from Tom20 and Tom22 and, most remarkably, supports a dissociation of the mature TOM complex to the late-assembly 100 kda intermediate (Figure 3). This dynamic behaviour of the TOM complex explains the observation that the mature

5 404 Membranes and organelles Figure 4 Figure 4 legend (a) IMS IM ATP N Tim23 Tim44 mthsp70 Mge1 Preprotein with presequence Tim17 ψ The mitochondrial presequence translocase and the processing peptidase. (a) The presequence translocase consists of an innermembrane-integrated pore complex (the Tim23 complex) and the peripherally attached import motor at the matrix side. Tim23 and Tim17 form a stable complex in the inner membrane (IM). The amino terminus of Tim23 in the intermembrane space (IMS) is important for recognising the presequences as they emerge from the TOM complex, whereas the carboxy-terminal membrane-integrated domain of Tim23 forms a channel for preproteins. This channel is activated by the membrane potential ( ψ), which also exerts a pulling force on the positively charged presequence. The import motor consists of mthsp70, the membrane-associated protein Tim44, and the homodimeric co-chaperone mitochondrial GrpE (Mge1, a nucleotide exchange factor). The motor is driven by hydrolysis of ATP. The heterodimeric MPP cleaves the presequences. (b) The MPP binds the presequences in an extended conformation in a large central cavity between the α and β subunits (the encircled area of MPP of [a] is shown; modelled after the structure of yeast MPP [54 ]). MPPβ binds the conserved hydrophobic ( hydro ) sidechain of the first amino acid residue of the mature protein in a hydrophobic pocket that includes a phenylalanine (Phe77). The conserved, positively charged arginine residue in the 2 position is bound by negatively charged residues (Glu160 and Asp164) on MPPβ. This brings the scissile peptide bond (arrow) close to the essential active-site zinc ion. The preprotein with the presequence is shown in red and blue. Amino acid residues in green belong to MPPβ (the numbering starts with residue 1 of the preprotein of Saccharomyces cerevisiae MPPβ). Matrix MPPα β is a continuous cycling between the mature TOM complex and the late assembly intermediate, driven by the action of Tom7 and Tom6 (Figure 3). In this way, new subunits can associate with the 100 kda intermediate complex and subsequently be incorporated into the mature TOM complex. (b) Zn 2 Phe77 hydro MPPβ Glu160 Asp164 A r X g Mature matrix protein N Current Opinion in Cell Biology Presequence translocase and import motor The Tim23 channel for cleavable preproteins Upon passage through the TOM complex, the presequences are bound by Tim23, which exposes a domain to the intermembrane space [3,4,32,33]. The membrane domain of Tim23, consisting of four predicted transmembrane helices, forms a channel across the inner membrane (Figure 4a). Channel formation by Tim23 was demonstrated directly by reconstitution of the purified protein into liposomes [34 ]. The channel formed by Tim23 alone shows the typical characteristics of the presequencesensitive channel observed in mitochondrial inner membranes [35] (i.e. has a high affinity for presequences, cation-selectivity and multiple conductance states), demonstrating that Tim23 is a major component of the presequence translocase [34 ]. Although the actual channel is formed by the membrane domain, the intermembrane space domain of Tim23 is also needed for its high cation selectivity that correlates with the high affinity for positively charged presequences. complex apparently can exchange its subunits. In fact, radiolabelling experiments indicate that each individual TOM protein can be incorporated into pre-existing TOM complexes (i.e. can replace the corresponding old subunit) [27,31]. The mechanism behind this surprising property A second inner membrane protein, Tim17, shows a weak yet significant similarity to the membrane domain of Tim23 [3,4]. Tim23 is essential for cell viability, and Tim17, which forms a complex with Tim23, is, alone, also essential for cell viability and involved in protein import [36 38]. The exact

6 Mitochondrial protein import: two membranes, three translocases Pfanner and Wiedemann 405 function of Tim17 is unknown, however; but it may be involved in channel formation or protein sorting to the inner membrane. Surprisingly, it has been reported that the intermembrane space domain of Tim23 extends into the outer membrane, suggesting that Tim23 spans across both mitochondrial membranes [39 ]. This observation could provide an explanation for how mitochondrial outer- and innermembrane translocases get into close contact. However, no contact of Tim23 to the TOM machinery was found [39 ]. Further studies will be needed to clarify a possible relationship between Tim23 and the outer membrane machinery. Two driving forces for protein import Translocation of precursor proteins across the inner membrane requires two energy sources: the electrical membrane potential ( ψ) across this membrane; and the hydrolysis of ATP in the matrix (Figure 4a). ψ is strictly required for the translocation of the presequences into or across the inner membrane. ψ (which is negative on the matrix side) operates by two mechanisms: it exerts an electrophoretic effect on the positively charged presequences [40] and activates the channel protein Tim23 [32,34 ]. ATP powers the preprotein import motor. This motor consists of the matrix heat shock protein 70 (mthsp70), its co-chaperone (nucleotide exchange factor) Mge1, and the peripheral membrane protein Tim44, which associates with the matrix side of the import channel [1 4]. Although molecular chaperones of the Hsp70 class, which consist of an ATPase domain and a polypeptide-binding domain, are typically soluble proteins, a fraction of mthsp70 is transiently converted into an inner-membrane-bound protein by interaction with Tim44 [41,42]. Since Tim44 is loosely associated with the Tim23 Tim17 import channel complex, mthsp70 is positioned at the outlet of the import channel and can directly interact with presequence and mature segments of preproteins emerging on the matrix side. mthsp70 is therefore essential, in an ATP-dependent manner, for the completion of preprotein translocation into the matrix [43]. Although this requirement for mthsp70 and ATP is undisputed, the exact molecular mechanism of mthsp70 function in protein import is still the subject of an ongoing debate. The two controversial views can be summarised as follows. On the one hand, mitochondria and mthsp70 are thought to play a passive role in protein import [44]. mthsp70 recognises unfolded polypeptide segments and thus preprotein segments emerging in the matrix are bound by mthsp70 and their backsliding in the import channels is prevented. By binding of more and more mthsp70 molecules, the preprotein in transit is thus trapped in the matrix (the trapping model). On the other hand, mthsp70 is thought to form an active import motor in cooperation with Tim44 and Mge1 and thereby pull the polypeptide chain into the matrix [45 48]. Tim44-associated mthsp70 is thought to undergo ATP-dependent conformational changes that generate an active pulling force on the preprotein (the motor model). Since mthsp70 is needed to promote the unfolding of folded preprotein domains that are still located on the cytosolic side, both views try to explain protein unfolding by different means. In the passive trapping model, folded domains undergo global spontaneous unfolding, allowing the movement of further preprotein segments into mitochondria that are subsequently trapped by mthsp70. Passive trapping by mthsp70 thus stabilises the unfolded states of preproteins. In the motor model, a pulling force generated on the polypeptide chain will facilitate a labilisation of the folded domain and thus promote its unfolding. Different views exist regarding which parts of mthsp70 bind to Tim44. It was reported [42] that the peptide-binding domain of mthsp70 interacts with Tim44 as well as the preprotein; thus, a pulling mechanism would be unlikely, favouring a passive trapping model. A recent study [41], however, indicates that the ATPase domain of mthsp70 binds to Tim44, while the peptide-binding domain plays an important regulatory role in this interaction. This suggests a more complex mode of interaction between mthsp70 and Tim44, in agreement with a motor model. As in most cases involving controversial discussions, both models contain important aspects of the actual mechanism. Indeed, recent studies suggest that both mechanisms, passive trapping and active pulling, cooperate to import and unfold mitochondrial preproteins [46,48,49 ]. Protein unfolding by mitochondria is faster than spontaneous unfolding in solution; thus it is clear that mitochondria actively unfold proteins [47,50]. Using a model protein, Huang et al. [51] showed that mitochondria change the unfolding pathway of a folded domain by unravelling it from its amino terminus that is, by first unravelling the portion that directly follows the presequence thereby demonstrating catalysis of protein unfolding by mitochondria. Simple trapping of preprotein segments by mthsp70 without an efficient mthsp70 Tim44 interaction was found to be enough to import loosely folded preproteins, while preproteins with folded domains also required an efficient mthsp70 Tim44 reaction cycle, suggesting that pulling is needed [48,49 ]. Huang et al. [52 ] reported a further twist on the mechanism of protein unfolding. They used a preprotein consisting of a folded domain and a short presequence that was unable to reach into the matrix when the mature protein was folded. The presequence was pulled into the matrix by the membrane potential and as a result, unfolding of the mature protein was promoted. The pulling force generated by ψ is therefore essential to unfold this preprotein. When the presequence is made longer, mthsp70 drove the unfolding [52 ]. Since the electrophoretic effect of ψ on the positively charged presequence doubtless generates a pulling force, the results of Huang et al. [52 ] provide further evidence for an active motor function of the mitochondrial protein import and unfolding machinery.

7 406 Membranes and organelles Figure 5 Stage I: Carrier precursor protein bound by cytosolic chaperone Stage II: Receptor complex ATP Cytosol Tom70 Outer membrane GIP Intermembrane space Inner membrane Stage III: Intermembrane space intermediate ψ 9 10 Tim Tim54 Matrix Stage IV: Membrane insertion Stage V: Assembled dimeric carrier protein Current Opinion in Cell Biology Import stages of hydrophobic membrane proteins to the mitochondrial inner membrane. Stage I: Cytosolic chaperones bind to the precursor of a carrier protein (used here as an example of a membrane protein) and keep it in an import-competent state. Stage II: The precursor protein is bound by several Tom70 receptor molecules. The precursor is then released in an ATP-dependent step and transferred to the GIP. Stage III: The precursor protein is translocated in a loop formation through the GIP and binds to the Tim9 Tim10 complex (via hydrophobic regions of the precursor). Stage IV: Membrane integration of the carrier precursor by the protein insertion complex (Tim22 complex) of the inner membrane depends on the membrane potential ( ψ). Stage V: The carrier protein assembles to the mature homodimer in the inner membrane. Mitochondrial processing peptidase The heterodimeric mitochondrial processing peptidase (MPP) proteolytically removes the amino-terminal presequences (Figure 4a,b). The cleavage site typically contains a positively charged residue at the 2 position (i.e. the penultimate residue of the presequence) and a bulky hydrophobic residue as the first residue of the mature protein [53]. Moreover, the function of MPP depends on zinc ions. The determination of the crystal structure of yeast MPP in complex with processing substrates revealed important insights into its function [54 ]. For example, the active site of MPP is located in a large central cavity between the homologous subunits α and β. Surprisingly, the bound presequence is not in a helical conformation (as was found for the Tom20 presequence complex), but is in an extended conformation. An activesite Zn 2 ion is found close to the cleavage site. Two negatively charged residues of MPPβ are near the positively charged residue at position 2 of the presequence, and a phenylalanine of MPPβ interacts with the bulky hydrophobic residue (Figure 4b). The structure of MPP thus beautifully explains the various observations on cleavage specificity that have been made for more than a decade. The binding chain hypothesis Mitochondrial presequences do not share an obvious primary structure similarity. However, they possess three important physical characteristics that explain their multiple roles in protein import: first, presequences are positively charged; second, they have the ability to form amphipathic α helices with a basic side and a hydrophobic side; and third, presequences have a conformational flexibility, allowing exchange between helical and extended conformations. On their journey into mitochondria, presequences sequentially interact with numerous import components (Figures 1, 2a and 4a), starting with the receptors Tom20 and Tom22, followed by interaction with Tom5, the channel Tom40 and the intermembrane space domain of Tom22. Upon transfer through the intermembrane space, presequences interact with Tim23 and are driven through the import channel of the inner membrane via an

8 Mitochondrial protein import: two membranes, three translocases Pfanner and Wiedemann 407 electrophoretic effect of ψ. mthsp70 binds the presequences and presents them, possibly in an extended conformation, to the matrix processing peptidase. Although it had been assumed originally that the positive charges of presequences are the major critical element for the recognition by the various import components (known as the acid chain hypothesis) [55,56], it is now clear that each of the three characteristics of presequences is important for the import process. Electrostatic interactions between presequence and negatively charged patches of import components were shown to be important in Tom22 and MPP. The basic character of presequences is also essential for responding to the membrane potential. Hydrophobic-type interactions are involved in the interaction with Tom20, Tom40 [5 7,57] and mthsp70. Surprisingly, the two high-resolution structures of presequences in complex with an import component (Tom20 or MPP) showed that presequences dramatically change their conformation during their import process (α-helical versus extended). It is thus possible that presequences adopt distinct conformations during interaction with each individual import component. The affinities determined for the interaction with each individual component are relatively low, such that presequences can, in principle, move back and forth; however, the chain of binding proteins provides a sequence of binding sites that guides presequences into the mitochondrial matrix (the binding chain hypothesis) [6]. The two energy inputs, ψ and mthsp70-atp, then promote unidirectional translocation of the presequences with the attached mature proteins into mitochondria. The protein insertion complex (carrier translocase) The mitochondrial inner membrane contains a second translocase for importing protein: the protein insertion complex, Tim22 complex or carrier translocase. This functions independently of the presequence translocase, yet is also essential for cell viability. Numerous inner membrane proteins synthesised without presequences act as substrates, such as the metabolite carriers of the inner membrane. These proteins contain several hydrophobic segments and internal targeting signals. Their import pathway can be divided into five stages (Figure 5) [58,59]. The hydrophobic proteins are guided through the cytosol by molecular chaperones, most likely cytosolic Hsp70s (stage I). Tom70 functions as the major receptor. As outlined in the section on import receptors, the multiple targeting signals in a carrier precursor are bound by several Tom70 molecules simultaneously, suggesting a cooperative action of the targeting signals (stage II) [8 ]. With the help of the additional receptors and Tom5, the hydrophobic proteins are transferred to the import channel (GIP) [18]. While presequence-containing preproteins are translocated as linear polypeptide chains, typically with the amino terminus first, the hydrophobic carrier proteins use the outer membrane pore in a different manner and are threaded through the channel via internal loop regions first [8 ]. Upon emergence on the intermembrane space side, carrier segments are bound by a specialised machinery, the Tim9 Tim10 complex. This hetero-oligomeric complex, which probably consists of three Tim9 and three Tim10 molecules, might possess chaperone-like properties [60,61 ]. It preferentially binds hydrophobic segments of the carrier proteins [61 ] and guides the precursor proteins through the aqueous intermembrane space (stage III). The Tim9 Tim10 complex associates with the protein insertion complex of the inner membrane. This complex carries a peripheral membrane protein, Tim12, on its intermembrane space side. Tim12 is homologous to Tim9 and Tim10 [62 65] and probably accepts the Tim9 Tim10 complex together with the bound precursor protein [66]. The actual insertion of the carrier protein into the inner membrane is mediated by a complex of three integral membrane proteins, Tim22, Tim54 and Tim18 (stage IV) [67 70]. The functions of Tim54 and Tim18 are unknown. Tim54 exposes a large domain to the intermembrane space and may possess receptor-like properties. Recent evidence showed a critical role for Tim22, which was found to be the only transmembrane protein of the protein insertion complex essential for cell viability [71 ]. This protein, with four predicted transmembrane segments and a weak, yet significant, homology to Tim23 and Tim17 of the presequence translocase, has been found to form a cationselective channel. The Tim22 channel specifically responds to internal targeting signals of carrier proteins, but not to presequences, indicating that Tim22 forms the core of the membranes insertion machinery [71 ]. The membrane potential represents the only external energy source to promote insertion of carrier proteins into the inner membrane in contrast to the presequence translocase, where both ψ and ATP are used to drive translocation [58,72]. The properties of purified and reconstituted Tim22 revealed an important clue about the energetics of translocation. The opening of Tim22 is favoured by an increase in the membrane potential, and the presence of a carrier targeting signal leads to activation of channel gating. When both a high ψ and a specific targeting signal are present, the frequency of opening and closing of the Tim22 channel is stimulated more than 100-fold [71 ]. This rapid gating of the channel probably resembles the situation during the active insertion of polypeptide loops into the translocase. The Tim22 channel has two flexible pore sizes, ~11 Å and ~18 Å [71 ]. The larger pore size is just enough for insertion of a tightly packed polypeptide loop; the smaller pore size, however, can only accommodate one polypeptide segment and may be important during the lateral release of polypeptides into the lipid phase of the membrane. During this process, half of a loop is still in the translocase, while the other half is already membrane-inserted. Thus the flexible pore size of Tim22 will help to maintain the electrochemical barrier across the inner membrane. The multiple targeting signals in carrier proteins are not only important for interaction with the receptor Tom70,

9 408 Membranes and organelles but apparently also cooperate during translocation across the outer membrane, interaction with the Tim9 Tim10 complex and insertion into the inner membrane [8,61,73]. Mitochondria thus use two completely different mechanisms for import of precursor proteins. Hydrophilic preproteins with presequence are directed by the aminoterminal signal sequence along the chain of binding sites in a linear mode, while hydrophobic precursor proteins with internal signals are transferred in loop formation where the multiple targeting signals cooperate at several stages of the import process. This latter mechanism is probably important to prevent aggregation and misfolding of the hydrophobic proteins during passage through membrane translocases and the aqueous intermembrane space. Besides these two major pathways, several variations of import mechanisms exist. Special mechanisms have been found in particular for components of the import machineries themselves. Like the Tom proteins, also all Tim proteins are encoded by nuclear genes and synthesised on cytosolic ribosomes. For example, the precursor of Tim54 is recognised by Tom70, but then crosses over to the presequence pathway and is inserted into the inner membrane by the presequence translocase. The precursor of Tim22 uses Tom20 as receptor like presequence-containing proteins, but then crosses over to the carrier import pathway [19]. The intermembrane space contains a second complex of tiny TIM proteins, the Tim8 Tim13 complex (Figure 5). This complex is not important for import of carrier proteins, but plays a role in translocation of the precursor of Tim23 across the outer membrane [74,75 ]. While the Tim9 Tim10 complex binds to hydrophobic segments of precursor proteins [61 ], the Tim8 Tim13 complex binds to the hydrophilic amino terminus of the Tim23 precursor and helps to trap the precursor in the intermembrane space [74,75 ]. Conclusions and perspectives Mitochondria must import many different precursor proteins. The mitochondrial outer membrane contains the dynamic TOM complex with several receptors and the general import pore. The TOM machinery is able to handle all different kinds of preproteins and mediate their translocation into and across the outer membrane. The inner membrane, which has to maintain an electrochemical proton gradient, has at least two distinct translocases for precursor proteins: a specific presequence translocase for cleavable preproteins, and a protein insertion complex for hydrophobic membrane proteins with multiple internal targeting signals. While previous studies tried to find the common mechanism for import of all mitochondrial precursor proteins, it is becoming more and more evident that mitochondria have evolved a multitude of mechanisms for recognition and translocation of the various kinds of precursor proteins. Future studies will be directed towards a molecular understanding of the recognition of hundreds of targeting signals by receptors and other import components. How do the import channels permit the passage of many different polypeptide chains while the membrane barriers for small molecules are maintained? The sorting of precursor proteins into the outer or inner membranes, as well as the mechanisms of assembly of the translocases, are major questions. How are the energy inputs converted into import-driving activities? Finally, the relationship between the protein import translocases and the recently discovered machineries for export of some proteins from the matrix into the inner membrane [76 78] will attract a lot of attention in the future. Acknowledgements Work of the authors laboratory was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 388 Freiburg and the Fonds der Chemischen Industrie/BMBF. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Schatz G, Dobberstein B: Common principles of protein translocation across membranes. Science 1996, 271: Bauer MF, Hofmann S, Neupert W, Brunner M: Protein translocation into mitochondria: the role of TIM complexes. Trends Cell Biol 2000, 10: Jensen RE, Johnson AE: Opening the door to mitochondrial protein import. Nat Struct Biol 2001, 8: Pfanner N, Geissler A: Versatility of the mitochondrial protein import machinery. Nat Rev Mol Cell Biol 2001, 2: Brix J, Dietmeier K, Pfanner N: Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. 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EMBO J 2001, 20: The import of the ADP/ATP carrier is directed by internal targeting signals. Wiedemann et al. report that several targeting signals function simultaneously in import of the precursor protein such that several Tom70 molecules are recruited to one carrier molecule. The targeting signals cooperate also in the transfer of the precursor through the general import pore of the outer membrane. In contrast to presequence-containing preproteins that are translocated as linear polypeptide chain, the carrier precursor crosses the outer membrane in a loop formation. 9. Brix J, Rüdiger S, Bukau B, Schneider-Mergener J, Pfanner N: Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein. 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