- Email: [email protected]
How mitochondria import hydrophilic and hydrophobic proteins
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22 Lamaze, C. et al. (2001) Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell 7, 661–671 23 Nichols, B.J. et al. (2001) Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153, 529–542 24 Sabhararanjak, S. et al. (2002) GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423 25 Puri, V. et al. (2001) Clathrin-dependent and independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J. Cell Biol. 154, 535–547 26 Nichols, B.J. A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex. Nat. Cell Biol. (in press) 27 Nichols, B.J. and Lippincott-Schwartz, J. (2001) Endocytosis without clathrin coats. Trends Cell Biol. 11, 406–412 28 Simons, K. and Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39 29 Gomez-Mouton, C. et al. (2001) Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization. Proc. Natl. Acad. Sci. U. S. A. 98, 9642–9647 30 Millan, J. et al. (2002) Lipid rafts mediate biosynthetic transport to the T lymphocyte uropod subdomain and are necessary for uropod integrity and function. Blood 99, 978–984 31 Lin, S.X. et al. (2002) Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (glu) microtubules and kinesin. Mol. Biol. Cell 13, 96–109 32 Pelkmans, L. et al. (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step
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vesicular-transport pathway to the ER. Nat. Cell Biol. 3, 473–483 Parton, R.G. (2001) Cell biology. Life without caveolae. Science 293, 2404–2405 Drab, M. et al. (2001) Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293, 2449–2452 Schubert, W. et al. (2001) Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J. Biol. Chem. 276, 48619–48622 Thomsen, P. et al. (2002) Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell 13, 238–250 Le, P.U. et al. (2002) Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J. Biol. Chem. 277, 3371–3379 van der Goot, F.G. and Harder, T. (2001) Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack. Semin. Immunol. 13, 89–97 Norkin, L.C. et al. (2001) Association of caveolin with Chlamydia trachomatis inclusions at early and late stages of infection. Exp. Cell Res. 266, 229–238 Bavari, S. et al. (2002) Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 195, 593–602 Nguyen, D.H. and Taub, D. (2002) CXCR4 function requires membrane cholesterol: implications for HIV infection. J. Immunol. 168, 4121–4126 Falguieres, T. et al. (2001) Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes. Mol. Biol. Cell 12, 2453–2468
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43 Gatfield, J. and Pieters, J. (2000) Essential role for cholesterol in entry of mycobacteria into macrophages. Science 288, 1647–1650 44 Fratti, R.A. et al. (2001) Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J. Cell Biol. 154, 631–644 45 Duclos, S. and Desjardins, M. (2000) Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell. Microbiol. 2, 365–377 46 Garin, J. et al. (2001) The phagosome proteome: insight into phagosome functions. J. Cell Biol. 152, 165–180 47 Nagai, H. et al. (2002) A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295, 679–682 48 Bishop, N. et al. (2002) Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein ubiquitin conjugates. J. Cell Biol. 157, 91–101
F. Gisou van der Goot Dept of Genetics and Microbiology, CMU, 1 rue Michel Servet, University of Geneva, CH-1211 Geneva 4, Switzerland. e-mail: [email protected] Jean Gruenberg Dept of Biochemistry, 30 quai Ernest Ansermet, University of Geneva, CH-1211 Geneva 4, Switzerland.
How mitochondria import hydrophilic and hydrophobic proteins Agnieszka Chacinska, Nikolaus Pfanner and Chris Meisinger Most mitochondrial proteins are nuclear encoded and have to be transported into the organelle after synthesis on cytosolic ribosomes. Three multimeric protein complexes have been identified that import precursor proteins destined for the mitochondria: the TOM complex in the outer membrane and two TIM complexes in the inner membrane. Recent work has provided a detailed view of the different mechanisms operating during the import of the two major classes of mitochondrial proteins – hydrophilic proteins with cleavable presequences and hydrophobic proteins with multiple internal signals.
Mitochondria contain about 1000 different proteins (estimates range from 600–2000), of which only a few are synthesized in the http://tcb.trends.com
organelle directly. This implicates a requirement for a highly organized protein import system that ensures proper recognition, transport and sorting of the cytosolically synthesized proteins destined to mitochondrial subcompartments [1–3]. The signals that direct these proteins to mitochondria can be classified into two major categories (Fig. 1). The most common class contains mitochondrial precursor proteins with cleavable, N-terminal extensions, or ‘presequences’, of 20–50 amino acid residues (range between 10–80 residues), which can form amphipathic helices bearing positively charged amino acid residues on one side and a hydrophobic non-charged surface on the other side. Most of these proteins are destined for the mitochondrial matrix. The second major class of
mitochondrial precursor proteins includes many proteins of the mitochondrial inner membrane, such as the metabolite carriers. These hydrophobic precursors contain internal signal segments that are distributed throughout the entire length of the protein. Three different multimeric protein complexes localized in the two mitochondrial membranes have been identified so far (Fig. 1; Box 1). The translocase of the outer membrane (TOM complex) consists of at least seven different subunits and can be divided into a very stable core complex of 400 kDa [general import pore (GIP)] and two loosely associated receptors (Tom20, Tom70). While the presequence-carrying preproteins bind mainly to Tom20, the carrier preproteins bind preferentially to Tom70, although there can also be
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Fig. 1. The two main pathways of protein translocation into mitochondria. First, preproteins with an aminoterminal presequence (blue) and precursor proteins with internal targeting signals (green) are recognized by receptors (R). In the next step, the proteins are translocated through the general import pore (GIP) across the outer membrane. After crossing the intermembrane space (IMS), the preproteins carrying a presequence are translocated across the inner membrane (IM) by the presequence translocase (TIM23 complex). This requires the membrane potential ∆ψ and the ATP-dependent activity of the mitochondrial heat-shock protein, mtHsp70. The presequences are cleaved off by the mitochondrial processing peptidase (MPP) and the mature protein is released into the matrix. Precursor proteins with internal signals are transferred across the intermembrane space to the inner membrane protein insertion complex (TIM22 complex) where they are inserted into the membrane in a ∆ψ-dependent fashion.
overlapping interactions. After binding to these receptors, the precursor proteins are transferred to the GIP, where translocation into and through the outer membrane occurs. Both classes of precursor proteins are transported across the outer membrane via the TOM complex but diverge afterwards for transport through or into the inner membrane. Matrix-destined preproteins with cleavable signal sequences are transported through the inner membrane via the TIM23 complex (TIM, translocase of the inner membrane), which consists of Tim23, Tim17 and Tim44. Mitochondrial Hsp70 (mtHsp70), which is positioned in close proximity to the TIM23 import channel, drives the translocation of these precursors into the mitochondrial matrix in an ATP-dependent manner, with the help of the nucleotide-exchange factor Mge1. The metabolite carrier precursor proteins are guided through the aqueous intermembrane space by soluble oligomeric http://tcb.trends.com
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Box 1. The protein translocases of mitochondria TOM The TOM complex (translocase of the outer membrane) contains three receptor proteins and the general import pore (GIP). The receptor Tom20 (molecular mass of 20 kDa) mainly recognizes presequences, whereas the receptor Tom70 recognizes hydrophobic precursor proteins with internal signals. Major components of the GIP complex are the channel-forming protein, Tom40, tightly associated with the organizer protein, Tom22. Tom22 also plays a role as a central receptor. Additionally, the GIP complex contains three small proteins: Tom5, Tom6 and Tom7. Tom5 links the receptors to the GIP and is involved in the transfer of preproteins into the channel. Tom6 and Tom7 play antagonistic roles: Tom6 is involved in the assembly, whereas Tom7 favors the dissociation, of the TOM complex. TIM23 The TIM23 complex (presequence translocase) contains Tim23, Tim17 and Tim44. Tim23 is an integral membrane protein that forms a channel for preproteins containing a cleavable
complexes of small Tim proteins. These prevent the aggregation of this hydrophobic class of precursor proteins as they pass on their way from the TOM complex to the TIM22 complex, where they are inserted into the inner membrane. Recent studies have provided functional insights into the translocation processes. Mitochondria do not use one common mechanism for importing all precursor proteins but have evolved import machineries with high flexibility and versatility for different types of protein. Transport of proteins carrying cleavable presequences
The mitochondrial precursors carrying cleavable presequences are first recognized by receptor proteins exposed to the cytosolic side of the mitochondrial outer membrane (Fig. 2a). What drives the import through the outer membrane? An active energyconsuming step has not been detected so far. One explanation is the so called ‘acid chain hypothesis’: the positively charged amino acids are bound at negatively charged patches along the transport pathway from the Tom receptors to the Tom22 trans binding site, until they reach the N-terminal Tim23 region facing the intermembrane space [4]. This model, however, does not explain the involvement of hydrophobic interactions, as described recently [5,6]. It is clear that Tom20 is the first receptor to recognize and bind to presequence-carrying preproteins. In addition, there is also no doubt that the
presequence. The role of the second integral membrane protein, Tim17, is unknown (a function as a channel has been proposed). The peripherally attached Tim44 protein serves as an anchor for the ATP-dependent import motor formed by the mitochondrial Hsp70 and the co-chaperone Mge1. TIM22 The TIM22 complex (inner membrane insertion complex) comprises the membrane proteins Tim22, Tim54, Tim18 and Tim12, as well as two soluble oligomeric complexes of the intermembrane space. The membraneintegrated Tim22 forms a translocation channel, while the role of Tim54 and Tim18 is unknown. The Tim9–Tim10 complex of the intermembrane space protects hydrophobic precursor proteins with internal signals from aggregation and guides them to the TIM22 channel through association with Tim12. Another soluble complex in the intermembrane space, Tim8–Tim13, is most likely involved in the import of some special proteins (such as the precursor of Tim23).
presequences can form amphipathic helices, with a hydrophobic surface on one side and a positively charged surface at the opposite side. The question remained, however, as to what kind of interaction (ionic, hydrophobic, or both) is responsible for this first contact and whether the helical character of the presequence is required for targeting. Abe et al. [7] provided the answer with a highresolution NMR structure of a Tom20 core fragment containing a bound presequence peptide. The presequence is bound in an α-helical conformation and, moreover, it is bound through only three hydrophobic presequence residues in a hydrophobic groove within the Tom20 domain. Charged and hydrophilic residues are exposed to the solvent but seem not to play a role in binding. The contribution of hydrophobic forces to the transport through the outer membrane [5–7] led to a revised model of the mechanism, termed ‘the binding chain hypothesis’. In this model, the preproteins are transported through a guidance system of multiple interaction sites containing every type of noncovalent binding force [6]. After passage through the outer membrane, the preprotein reaches the TIM23 complex of the inner membrane (Fig. 2a, b). The presequences can bind to a Tim23 domain exposed to the intermembrane space. At this stage, it is not yet clear whether further structural requirements in the intermembrane space are needed for proper recognition and insertion into the translocase. It was recently reported that the extreme
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Fig. 2. Import of preproteins containing an amino-terminal presequence into mitochondria. (a) The presequence forms an amphipathic helix, with positively charged amino acid residues on one side (green) and a hydrophobic segment on the other side (red). The presequence is initially recognized by the receptors Tom20 and Tom22 of the outer membrane (OM). After passing through the general import pore (GIP), the presequence can interact with the trans binding site of Tom22 and the intermembrane space domain of Tim23 (blue). (b) The presequence is translocated through the TIM23 channel, driven by the electrophoretic force of the membrane potential ∆ψ. (c) The preprotein binds to the ATP-driven import motor containing mitochondrial heat-shock protein 70 (mtHsp70), its membrane anchor Tim44 and the co-chaperone Mge1. The force generated by the mtHsp70 motor unravels the precursor protein and leads to its translocation into the matrix. (d) The heterodimeric mitochondrial processing peptidase (MPP) binds to the presequence that is now in an extended conformation and removes it from the mature mitochondrial protein.
N-terminal region of Tim23 is even anchored in the outer membrane, but is not in contact with Tom proteins [8]. Whether this contact of inner and outer membrane plays a role in import has to be studied further. How does the preprotein then cross the inner membrane? Truscott et al. [9] identified Tim23 as the pore-forming subunit of the presequence translocase by reconstitution of the purified protein in planar lipid bilayers. Electrophysiological measurements revealed a cation-selective channel with multiple conductance states and an affinity for presequences. This type of channel activity was also observed in purified inner membrane vesicles. The Tim23 membrane domain alone is sufficient to form a channel; however, the domain of Tim23 exposed to the intermembrane space is required for the selectivity for cations and presequences. A direct function for Tim17, the second http://tcb.trends.com
main component of the presequence translocase, has not yet been elucidated. The translocation of the precursor proteins carrying cleavable presequences through the inner membrane requires two forces – the membrane potential ∆ψ, which exerts an electrophoretic force onto the positively charged amino acids, and ATP, hydrolyzed by the motor complex of mtHsp70, Tim44 and Mge1. These two strong energy sources are thought to be responsible for the unidirectionality of the translocation reaction as binding of the presequence to the individual components along the binding chain is of rather low affinity [3]. Two roles have been attributed to the electrochemical potential of the inner membrane. First, it activates the TIM23 complex [9,10]. Second, the electrical field is strictly required for inserting the positively charged presequence into the TIM23
channel and driving its translocation to the negatively charged matrix side, apparently by an electrophoretic effect. Also, Huang et al. [11] recently reported a novel function for ∆ψ: the electrophoretic effect produced by ∆ψ can promote an active unfolding of the precursor protein. The requirement for this function of ∆ψ depends on the length of the precursor and its folding state, such that pulling at the presequence facilitates the labilization of a domain during entry into the GIP (Fig. 2a, b). It is commonly accepted that the mtHsp70 and ATP hydrolysis are crucial for the translocation process (Fig. 2c). However, the exact mechanism and function of mtHsp70 in active unfolding of precursors is the subject of an ongoing debate. Two models have been considered: a passive role for mtHsp70 in the trapping of the emerging precursor, and an active role for the mtHsp70 motor in generating a pulling force. Recent studies show that both mechanisms are not exclusive [12,13]. Most likely, diverse mechanisms, including trapping by mtHsp70 as well as active unfolding driven either by mtHsp70 or ∆Ψ, are utilized in order to translocate the presequence-containing precursors. The choice of energy source depends on the intrinsic features of the precursors,
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Fig. 3. Import of hydrophobic precursor proteins containing internal signals into the inner membrane of mitochondria. (a) The precursor protein binds through multiple internal segments to several Tom70 molecules. (b) The precursor is translocated through the general import pore (GIP) of the outer membrane (OM) in a loop formation and interacts with the Tim9–Tim10 complex in the intermembrane space (IMS). (c) The Tim9–Tim10 complex guides the precursor across the IMS to the TIM22 translocase. Insertion of the precursor protein into the inner membrane requires a membrane potential (∆ψ). After lateral release into the inner membrane, the protein assembles into its functional mature form.
their lengths, their folding state and the stage of the translocation process. When the presequence reaches the matrix, it is cleaved off by the mitochondrial processing peptidase (MPP), which consists of two homologous subunits, α and β (Fig. 2d). Cleavage occurs typically at a position where the penultimate residue (–2) is an arginine and the next residue (+1) contains an aromatic side chain. The highresolution crystal structures of active MPP and a cleavage-deficient mutant of MPP in complex with synthetic processing substrates have brought new insights into the mechanisms of MPP action and the structural requirements of presequences [14]. A large central cavity between the two homologous subunits forms the active site and also contains a Zn2+ ion. The positively charged amino acid (–2) of the presequence is in contact with glutamate and aspartate residues of βMPP, and the aromatic residue at +1 interacts with a phenylalanine of βMPP. The active site is similar to that of thermolysin, suggesting that their catalytic mechanisms might be comparable. The most interesting result, however, was that the presequence is not bound to the active site as an α-helix but binds in an extended conformation. This means that presequences must undergo a conformational change on their way from receptor binding at the TOM complex to processing in the matrix. http://tcb.trends.com
Taken together, these new results extend our view of how all mitochondrial presequence-containing proteins are transported via the same import pathway. Although their targeting signals do not show any primary structure homology, the presequences do share common structural features. Mitochondrial targeting is therefore mediated by distinct structural properties of the presequences. These are: (i) the presence of positively charged residues required for electrostatic interactions with components of the import machineries and also for responding to electrophoretic forces of the membrane potential; (ii) the formation of an amphipathic α-helix containing a positively charged surface and an opposite, hydrophobic surface, which also interacts with components of the import binding chain; and (iii) the ability to change conformation between α-helical and extended forms. Transport of proteins bearing internal targeting signals
Members of the second major class of mitochondrial precursor proteins (Fig. 3), which are synthesized without a cleavable presequence, contain distinct hydrophobic segments and currently unclassified internal targeting sequences. After translation, these proteins are guided through the cytosol by chaperones, such as
cytosolic Hsp70, and handed over to the main import receptor Tom70. A detailed study of the binding of carrier proteins to Tom70 and their subsequent insertion into the translocation pore revealed that this pathway differs from the presequence pathway as early as the level of the outer membrane [15]. As shown for the ADP/ATP carrier, several internal segments of the precursor protein interact with Tom70 and promote a homo-oligomerization of the receptor. Thus, several Tom70 molecules cooperate to bind to one precursor protein. By simultaneous binding to multiple sites of these hydrophobic precursor proteins, Tom70 apparently prevents their aggregation and might exert a chaperonelike function (Fig. 3a). Insertion into the general import pore also requires the cooperative action of the multiple targeting sequences. While the presequence-carrying proteins are translocated as a linear chain through the pore, the carrier proteins are inserted and transported through the outer membrane, with a leading internal loop segment (Fig. 3b). At the intermembrane space side, the carrier proteins are bound by the Tim9–Tim10 system, a complex most likely consisting of three Tim9 and three Tim10 molecules. This complex guides the precursor proteins through the aqueous intermembrane space to the protein insertion complex of the inner membrane (TIM22 complex). Curran et al. [16] reported that the Tim9–Tim10 complex binds to the carrier proteins at their hydrophobic regions and could therefore prevent their
Research Update
aggregation, suggesting a chaperone-like function for this intermembrane space complex too. The precursors are then transferred from the Tim9–Tim10 complex to Tim12, a peripheral membrane protein associated with the protein insertion complex (Fig. 3c; Box 1). Further components of the complex are the integral inner membrane proteins Tim22, Tim54 and Tim18. Kovermann et al. [17] have identified Tim22 as the channel-forming subunit. The channel is cation-selective and activated by internal targeting signals but not by presequence peptides, thereby supporting its function as the central pore-forming subunit of the inner membrane insertion complex. The only known energy source required for insertion of inner membrane proteins by the TIM22 complex is the membrane potential ∆ψ, whereas presequencecarrying preproteins require both ∆ψ and ATP. Opening of the Tim22 channel is promoted by an increase of the membrane potential. When a high membrane potential was applied together with internal targeting signal peptides, a 100-fold increase in the gating frequency of the channel was observed – that is, a rapid opening and closing, possibly reflecting active conformational changes of the channel during the insertion process. Tim22 shows subconductance states and a variable pore size. When the channel is fully open, the pore size of ~18 Å should allow for the insertion of two tightly packed α-helices, supporting the view that proteins insert in a loop conformation. A smaller pore size of ~11 Å at a subconductance state only allows for the presence of a single α-helix. This could reflect the lateral release of the preprotein into the membrane. The detailed function of the two other components of the TIM22 translocase, Tim54 and Tim18, is unknown so far. Concluding remarks
The two main classes of mitochondrial precursor proteins use different import mechanisms. The only common features are that they utilize the GIP (Tom40) as a common pore for translocation through the outer membrane and that both require a membrane potential as an active force for transport through or into the inner membrane. Both pathways have adapted specifically to the different characteristics of each type of targeting signal. While the presequence-carrying proteins are directed by a single N-terminal signal region and transported in a linear fashion http://tcb.trends.com
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along a chain of binding sites, the hydrophobic inner membrane proteins contain multiple internal signals and are translocated in loop formations. Besides these two major classes of import pathways, recent work has revealed the existence of other, specialized mechanisms. For example, the Tom and Tim proteins themselves are all nuclear encoded and have to be imported and assembled into their functional complexes. The best-studied case is the biogenesis of Tom40 which assembles into the TOM complex through a complicated pathway involving multiple intermediate stages in which all other Tom proteins are involved [18,19]. For the import of Tim23, Paschen et al. [20] found that, under conditions of limited membrane potential, another soluble TIM complex in the intermembrane space, the Tim8–Tim13 complex, is required. Moreover, a crossingover of some precursor proteins between the two main import pathways has been observed [21]. The identification of export machineries for transport of mitochondrially encoded proteins from the matrix into the inner membrane has opened a new field of translocation research and will be an important topic in the future [22,23]. It is likely that the great variety of mitochondrial precursor proteins and the high versatility of the translocase machineries will hold many more surprises. Acknowledgements
We thank Ann Frazier for critical comments on the manuscript. The work of the authors’ laboratory was supported by the Deutsche Forschungsgemeinschaft, SFB 388 and the Fonds der Chemischen Industrie/BMBF. A.C. is a recipient of a postdoctoral FEBS fellowship. References 1 Koehler, C.M. et al. (1999) How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem. Sci. 24, 428–432 2 Bauer, M.F. et al. (2000) Protein translocation into mitochondria: the role of TIM complexes. Trends Cell Biol. 10, 25–31 3 Pfanner, N., and Geissler, A. (2001) Versatility of the mitochondrial protein import machinery. Nat. Rev. Mol. Cell Biol. 2, 339–349 4 Schatz, G. (1997) Just follow the acid chain. Nature 388, 121–122 5 Brix, J. et al. (1997) Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J. Biol. Chem. 272, 20730–20735 6 Meisinger, C. et al. (2001) Protein import channel of the outer mitochondrial membrane: a highly stable Tom40–Tom22 core structure differentially interacts with preproteins, small Tom proteins, and import receptors. Mol. Cell. Biol. 21, 2337–2348
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7 Abe, Y. et al. (2000) Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell 100, 551–560 8 Donzeau, M. et al. (2000) Tim23 links the inner and outer mitochondrial membranes. Cell 101, 401–412 9 Truscott, K.N. et al. (2001) A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nat. Struct. Biol. 8, 1074–1082 10 Bauer, M.F. et al. (1996) Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria. Cell 87, 33–41 11 Huang, S. et al. (2002) Protein unfolding by the mitochondrial membrane potential. Nat. Struct. Biol. 9, 301–307 12 Voisine, C. et al. (1999) The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70. Cell 97, 565–574 13 Geissler, A. et al. (2001) Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates translocation and reduces the membrane potential dependence of loosely folded preproteins. Mol. Cell. Biol. 21, 7097–7104 14 Taylor, A.B. et al. (2001) Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences. Structure 9, 615–625 15 Wiedemann, N. et al. (2001) The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria. EMBO J. 20, 951–960 16 Curran, S.P. et al. (2002) The Tim9p–Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier. EMBO J. 21, 942–953 17 Kovermann, P. et al. (2002) Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signalgated channel. Mol. Cell 9, 363–373 18 Rapaport, D. and Neupert, W. (1999) Biogenesis of Tom40, core component of the TOM complex of mitochondria. J. Cell Biol. 146, 321–331 19 Model, K. et al. (2001) Multistep assembly of the protein import channel of the mitochondrial outer membrane. Nat. Struct. Biol. 8, 361–370 20 Paschen, S.A. et al. (2000) The role of the TIM8–13 complex in the import of Tim23 into mitochondria. EMBO J. 19, 6392–6400 21 Kurz, M. et al. (1999) Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway. Mol. Biol. Cell 10, 2461–2474 22 Hell, K. et al. (2001) Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA. EMBO J. 20, 1281–1288 23 Saracco, S.A. and Fox, T.D. (2002) Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane. Mol. Biol. Cell 13, 1122–1131
Agnieszka Chacinska Nikolaus Pfanner* Chris Meisinger Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany. *e-mail: nikolaus.pfanner@biochemie. uni-freiburg.de
22 Lamaze, C. et al. (2001) Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol. Cell 7, 661–671 23 Nichols, B.J. et al. (2001) Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153, 529–542 24 Sabhararanjak, S. et al. (2002) GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423 25 Puri, V. et al. (2001) Clathrin-dependent and independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. J. Cell Biol. 154, 535–547 26 Nichols, B.J. A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex. Nat. Cell Biol. (in press) 27 Nichols, B.J. and Lippincott-Schwartz, J. (2001) Endocytosis without clathrin coats. Trends Cell Biol. 11, 406–412 28 Simons, K. and Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39 29 Gomez-Mouton, C. et al. (2001) Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization. Proc. Natl. Acad. Sci. U. S. A. 98, 9642–9647 30 Millan, J. et al. (2002) Lipid rafts mediate biosynthetic transport to the T lymphocyte uropod subdomain and are necessary for uropod integrity and function. Blood 99, 978–984 31 Lin, S.X. et al. (2002) Export from pericentriolar endocytic recycling compartment to cell surface depends on stable, detyrosinated (glu) microtubules and kinesin. Mol. Biol. Cell 13, 96–109 32 Pelkmans, L. et al. (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step
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vesicular-transport pathway to the ER. Nat. Cell Biol. 3, 473–483 Parton, R.G. (2001) Cell biology. Life without caveolae. Science 293, 2404–2405 Drab, M. et al. (2001) Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293, 2449–2452 Schubert, W. et al. (2001) Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J. Biol. Chem. 276, 48619–48622 Thomsen, P. et al. (2002) Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol. Biol. Cell 13, 238–250 Le, P.U. et al. (2002) Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J. Biol. Chem. 277, 3371–3379 van der Goot, F.G. and Harder, T. (2001) Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack. Semin. Immunol. 13, 89–97 Norkin, L.C. et al. (2001) Association of caveolin with Chlamydia trachomatis inclusions at early and late stages of infection. Exp. Cell Res. 266, 229–238 Bavari, S. et al. (2002) Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 195, 593–602 Nguyen, D.H. and Taub, D. (2002) CXCR4 function requires membrane cholesterol: implications for HIV infection. J. Immunol. 168, 4121–4126 Falguieres, T. et al. (2001) Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes. Mol. Biol. Cell 12, 2453–2468
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43 Gatfield, J. and Pieters, J. (2000) Essential role for cholesterol in entry of mycobacteria into macrophages. Science 288, 1647–1650 44 Fratti, R.A. et al. (2001) Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J. Cell Biol. 154, 631–644 45 Duclos, S. and Desjardins, M. (2000) Subversion of a young phagosome: the survival strategies of intracellular pathogens. Cell. Microbiol. 2, 365–377 46 Garin, J. et al. (2001) The phagosome proteome: insight into phagosome functions. J. Cell Biol. 152, 165–180 47 Nagai, H. et al. (2002) A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295, 679–682 48 Bishop, N. et al. (2002) Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein ubiquitin conjugates. J. Cell Biol. 157, 91–101
F. Gisou van der Goot Dept of Genetics and Microbiology, CMU, 1 rue Michel Servet, University of Geneva, CH-1211 Geneva 4, Switzerland. e-mail: [email protected] Jean Gruenberg Dept of Biochemistry, 30 quai Ernest Ansermet, University of Geneva, CH-1211 Geneva 4, Switzerland.
How mitochondria import hydrophilic and hydrophobic proteins Agnieszka Chacinska, Nikolaus Pfanner and Chris Meisinger Most mitochondrial proteins are nuclear encoded and have to be transported into the organelle after synthesis on cytosolic ribosomes. Three multimeric protein complexes have been identified that import precursor proteins destined for the mitochondria: the TOM complex in the outer membrane and two TIM complexes in the inner membrane. Recent work has provided a detailed view of the different mechanisms operating during the import of the two major classes of mitochondrial proteins – hydrophilic proteins with cleavable presequences and hydrophobic proteins with multiple internal signals.
Mitochondria contain about 1000 different proteins (estimates range from 600–2000), of which only a few are synthesized in the http://tcb.trends.com
organelle directly. This implicates a requirement for a highly organized protein import system that ensures proper recognition, transport and sorting of the cytosolically synthesized proteins destined to mitochondrial subcompartments [1–3]. The signals that direct these proteins to mitochondria can be classified into two major categories (Fig. 1). The most common class contains mitochondrial precursor proteins with cleavable, N-terminal extensions, or ‘presequences’, of 20–50 amino acid residues (range between 10–80 residues), which can form amphipathic helices bearing positively charged amino acid residues on one side and a hydrophobic non-charged surface on the other side. Most of these proteins are destined for the mitochondrial matrix. The second major class of
mitochondrial precursor proteins includes many proteins of the mitochondrial inner membrane, such as the metabolite carriers. These hydrophobic precursors contain internal signal segments that are distributed throughout the entire length of the protein. Three different multimeric protein complexes localized in the two mitochondrial membranes have been identified so far (Fig. 1; Box 1). The translocase of the outer membrane (TOM complex) consists of at least seven different subunits and can be divided into a very stable core complex of 400 kDa [general import pore (GIP)] and two loosely associated receptors (Tom20, Tom70). While the presequence-carrying preproteins bind mainly to Tom20, the carrier preproteins bind preferentially to Tom70, although there can also be
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(02)02310-3
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Fig. 1. The two main pathways of protein translocation into mitochondria. First, preproteins with an aminoterminal presequence (blue) and precursor proteins with internal targeting signals (green) are recognized by receptors (R). In the next step, the proteins are translocated through the general import pore (GIP) across the outer membrane. After crossing the intermembrane space (IMS), the preproteins carrying a presequence are translocated across the inner membrane (IM) by the presequence translocase (TIM23 complex). This requires the membrane potential ∆ψ and the ATP-dependent activity of the mitochondrial heat-shock protein, mtHsp70. The presequences are cleaved off by the mitochondrial processing peptidase (MPP) and the mature protein is released into the matrix. Precursor proteins with internal signals are transferred across the intermembrane space to the inner membrane protein insertion complex (TIM22 complex) where they are inserted into the membrane in a ∆ψ-dependent fashion.
overlapping interactions. After binding to these receptors, the precursor proteins are transferred to the GIP, where translocation into and through the outer membrane occurs. Both classes of precursor proteins are transported across the outer membrane via the TOM complex but diverge afterwards for transport through or into the inner membrane. Matrix-destined preproteins with cleavable signal sequences are transported through the inner membrane via the TIM23 complex (TIM, translocase of the inner membrane), which consists of Tim23, Tim17 and Tim44. Mitochondrial Hsp70 (mtHsp70), which is positioned in close proximity to the TIM23 import channel, drives the translocation of these precursors into the mitochondrial matrix in an ATP-dependent manner, with the help of the nucleotide-exchange factor Mge1. The metabolite carrier precursor proteins are guided through the aqueous intermembrane space by soluble oligomeric http://tcb.trends.com
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Box 1. The protein translocases of mitochondria TOM The TOM complex (translocase of the outer membrane) contains three receptor proteins and the general import pore (GIP). The receptor Tom20 (molecular mass of 20 kDa) mainly recognizes presequences, whereas the receptor Tom70 recognizes hydrophobic precursor proteins with internal signals. Major components of the GIP complex are the channel-forming protein, Tom40, tightly associated with the organizer protein, Tom22. Tom22 also plays a role as a central receptor. Additionally, the GIP complex contains three small proteins: Tom5, Tom6 and Tom7. Tom5 links the receptors to the GIP and is involved in the transfer of preproteins into the channel. Tom6 and Tom7 play antagonistic roles: Tom6 is involved in the assembly, whereas Tom7 favors the dissociation, of the TOM complex. TIM23 The TIM23 complex (presequence translocase) contains Tim23, Tim17 and Tim44. Tim23 is an integral membrane protein that forms a channel for preproteins containing a cleavable
complexes of small Tim proteins. These prevent the aggregation of this hydrophobic class of precursor proteins as they pass on their way from the TOM complex to the TIM22 complex, where they are inserted into the inner membrane. Recent studies have provided functional insights into the translocation processes. Mitochondria do not use one common mechanism for importing all precursor proteins but have evolved import machineries with high flexibility and versatility for different types of protein. Transport of proteins carrying cleavable presequences
The mitochondrial precursors carrying cleavable presequences are first recognized by receptor proteins exposed to the cytosolic side of the mitochondrial outer membrane (Fig. 2a). What drives the import through the outer membrane? An active energyconsuming step has not been detected so far. One explanation is the so called ‘acid chain hypothesis’: the positively charged amino acids are bound at negatively charged patches along the transport pathway from the Tom receptors to the Tom22 trans binding site, until they reach the N-terminal Tim23 region facing the intermembrane space [4]. This model, however, does not explain the involvement of hydrophobic interactions, as described recently [5,6]. It is clear that Tom20 is the first receptor to recognize and bind to presequence-carrying preproteins. In addition, there is also no doubt that the
presequence. The role of the second integral membrane protein, Tim17, is unknown (a function as a channel has been proposed). The peripherally attached Tim44 protein serves as an anchor for the ATP-dependent import motor formed by the mitochondrial Hsp70 and the co-chaperone Mge1. TIM22 The TIM22 complex (inner membrane insertion complex) comprises the membrane proteins Tim22, Tim54, Tim18 and Tim12, as well as two soluble oligomeric complexes of the intermembrane space. The membraneintegrated Tim22 forms a translocation channel, while the role of Tim54 and Tim18 is unknown. The Tim9–Tim10 complex of the intermembrane space protects hydrophobic precursor proteins with internal signals from aggregation and guides them to the TIM22 channel through association with Tim12. Another soluble complex in the intermembrane space, Tim8–Tim13, is most likely involved in the import of some special proteins (such as the precursor of Tim23).
presequences can form amphipathic helices, with a hydrophobic surface on one side and a positively charged surface at the opposite side. The question remained, however, as to what kind of interaction (ionic, hydrophobic, or both) is responsible for this first contact and whether the helical character of the presequence is required for targeting. Abe et al. [7] provided the answer with a highresolution NMR structure of a Tom20 core fragment containing a bound presequence peptide. The presequence is bound in an α-helical conformation and, moreover, it is bound through only three hydrophobic presequence residues in a hydrophobic groove within the Tom20 domain. Charged and hydrophilic residues are exposed to the solvent but seem not to play a role in binding. The contribution of hydrophobic forces to the transport through the outer membrane [5–7] led to a revised model of the mechanism, termed ‘the binding chain hypothesis’. In this model, the preproteins are transported through a guidance system of multiple interaction sites containing every type of noncovalent binding force [6]. After passage through the outer membrane, the preprotein reaches the TIM23 complex of the inner membrane (Fig. 2a, b). The presequences can bind to a Tim23 domain exposed to the intermembrane space. At this stage, it is not yet clear whether further structural requirements in the intermembrane space are needed for proper recognition and insertion into the translocase. It was recently reported that the extreme
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Fig. 2. Import of preproteins containing an amino-terminal presequence into mitochondria. (a) The presequence forms an amphipathic helix, with positively charged amino acid residues on one side (green) and a hydrophobic segment on the other side (red). The presequence is initially recognized by the receptors Tom20 and Tom22 of the outer membrane (OM). After passing through the general import pore (GIP), the presequence can interact with the trans binding site of Tom22 and the intermembrane space domain of Tim23 (blue). (b) The presequence is translocated through the TIM23 channel, driven by the electrophoretic force of the membrane potential ∆ψ. (c) The preprotein binds to the ATP-driven import motor containing mitochondrial heat-shock protein 70 (mtHsp70), its membrane anchor Tim44 and the co-chaperone Mge1. The force generated by the mtHsp70 motor unravels the precursor protein and leads to its translocation into the matrix. (d) The heterodimeric mitochondrial processing peptidase (MPP) binds to the presequence that is now in an extended conformation and removes it from the mature mitochondrial protein.
N-terminal region of Tim23 is even anchored in the outer membrane, but is not in contact with Tom proteins [8]. Whether this contact of inner and outer membrane plays a role in import has to be studied further. How does the preprotein then cross the inner membrane? Truscott et al. [9] identified Tim23 as the pore-forming subunit of the presequence translocase by reconstitution of the purified protein in planar lipid bilayers. Electrophysiological measurements revealed a cation-selective channel with multiple conductance states and an affinity for presequences. This type of channel activity was also observed in purified inner membrane vesicles. The Tim23 membrane domain alone is sufficient to form a channel; however, the domain of Tim23 exposed to the intermembrane space is required for the selectivity for cations and presequences. A direct function for Tim17, the second http://tcb.trends.com
main component of the presequence translocase, has not yet been elucidated. The translocation of the precursor proteins carrying cleavable presequences through the inner membrane requires two forces – the membrane potential ∆ψ, which exerts an electrophoretic force onto the positively charged amino acids, and ATP, hydrolyzed by the motor complex of mtHsp70, Tim44 and Mge1. These two strong energy sources are thought to be responsible for the unidirectionality of the translocation reaction as binding of the presequence to the individual components along the binding chain is of rather low affinity [3]. Two roles have been attributed to the electrochemical potential of the inner membrane. First, it activates the TIM23 complex [9,10]. Second, the electrical field is strictly required for inserting the positively charged presequence into the TIM23
channel and driving its translocation to the negatively charged matrix side, apparently by an electrophoretic effect. Also, Huang et al. [11] recently reported a novel function for ∆ψ: the electrophoretic effect produced by ∆ψ can promote an active unfolding of the precursor protein. The requirement for this function of ∆ψ depends on the length of the precursor and its folding state, such that pulling at the presequence facilitates the labilization of a domain during entry into the GIP (Fig. 2a, b). It is commonly accepted that the mtHsp70 and ATP hydrolysis are crucial for the translocation process (Fig. 2c). However, the exact mechanism and function of mtHsp70 in active unfolding of precursors is the subject of an ongoing debate. Two models have been considered: a passive role for mtHsp70 in the trapping of the emerging precursor, and an active role for the mtHsp70 motor in generating a pulling force. Recent studies show that both mechanisms are not exclusive [12,13]. Most likely, diverse mechanisms, including trapping by mtHsp70 as well as active unfolding driven either by mtHsp70 or ∆Ψ, are utilized in order to translocate the presequence-containing precursors. The choice of energy source depends on the intrinsic features of the precursors,
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Fig. 3. Import of hydrophobic precursor proteins containing internal signals into the inner membrane of mitochondria. (a) The precursor protein binds through multiple internal segments to several Tom70 molecules. (b) The precursor is translocated through the general import pore (GIP) of the outer membrane (OM) in a loop formation and interacts with the Tim9–Tim10 complex in the intermembrane space (IMS). (c) The Tim9–Tim10 complex guides the precursor across the IMS to the TIM22 translocase. Insertion of the precursor protein into the inner membrane requires a membrane potential (∆ψ). After lateral release into the inner membrane, the protein assembles into its functional mature form.
their lengths, their folding state and the stage of the translocation process. When the presequence reaches the matrix, it is cleaved off by the mitochondrial processing peptidase (MPP), which consists of two homologous subunits, α and β (Fig. 2d). Cleavage occurs typically at a position where the penultimate residue (–2) is an arginine and the next residue (+1) contains an aromatic side chain. The highresolution crystal structures of active MPP and a cleavage-deficient mutant of MPP in complex with synthetic processing substrates have brought new insights into the mechanisms of MPP action and the structural requirements of presequences [14]. A large central cavity between the two homologous subunits forms the active site and also contains a Zn2+ ion. The positively charged amino acid (–2) of the presequence is in contact with glutamate and aspartate residues of βMPP, and the aromatic residue at +1 interacts with a phenylalanine of βMPP. The active site is similar to that of thermolysin, suggesting that their catalytic mechanisms might be comparable. The most interesting result, however, was that the presequence is not bound to the active site as an α-helix but binds in an extended conformation. This means that presequences must undergo a conformational change on their way from receptor binding at the TOM complex to processing in the matrix. http://tcb.trends.com
Taken together, these new results extend our view of how all mitochondrial presequence-containing proteins are transported via the same import pathway. Although their targeting signals do not show any primary structure homology, the presequences do share common structural features. Mitochondrial targeting is therefore mediated by distinct structural properties of the presequences. These are: (i) the presence of positively charged residues required for electrostatic interactions with components of the import machineries and also for responding to electrophoretic forces of the membrane potential; (ii) the formation of an amphipathic α-helix containing a positively charged surface and an opposite, hydrophobic surface, which also interacts with components of the import binding chain; and (iii) the ability to change conformation between α-helical and extended forms. Transport of proteins bearing internal targeting signals
Members of the second major class of mitochondrial precursor proteins (Fig. 3), which are synthesized without a cleavable presequence, contain distinct hydrophobic segments and currently unclassified internal targeting sequences. After translation, these proteins are guided through the cytosol by chaperones, such as
cytosolic Hsp70, and handed over to the main import receptor Tom70. A detailed study of the binding of carrier proteins to Tom70 and their subsequent insertion into the translocation pore revealed that this pathway differs from the presequence pathway as early as the level of the outer membrane [15]. As shown for the ADP/ATP carrier, several internal segments of the precursor protein interact with Tom70 and promote a homo-oligomerization of the receptor. Thus, several Tom70 molecules cooperate to bind to one precursor protein. By simultaneous binding to multiple sites of these hydrophobic precursor proteins, Tom70 apparently prevents their aggregation and might exert a chaperonelike function (Fig. 3a). Insertion into the general import pore also requires the cooperative action of the multiple targeting sequences. While the presequence-carrying proteins are translocated as a linear chain through the pore, the carrier proteins are inserted and transported through the outer membrane, with a leading internal loop segment (Fig. 3b). At the intermembrane space side, the carrier proteins are bound by the Tim9–Tim10 system, a complex most likely consisting of three Tim9 and three Tim10 molecules. This complex guides the precursor proteins through the aqueous intermembrane space to the protein insertion complex of the inner membrane (TIM22 complex). Curran et al. [16] reported that the Tim9–Tim10 complex binds to the carrier proteins at their hydrophobic regions and could therefore prevent their
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aggregation, suggesting a chaperone-like function for this intermembrane space complex too. The precursors are then transferred from the Tim9–Tim10 complex to Tim12, a peripheral membrane protein associated with the protein insertion complex (Fig. 3c; Box 1). Further components of the complex are the integral inner membrane proteins Tim22, Tim54 and Tim18. Kovermann et al. [17] have identified Tim22 as the channel-forming subunit. The channel is cation-selective and activated by internal targeting signals but not by presequence peptides, thereby supporting its function as the central pore-forming subunit of the inner membrane insertion complex. The only known energy source required for insertion of inner membrane proteins by the TIM22 complex is the membrane potential ∆ψ, whereas presequencecarrying preproteins require both ∆ψ and ATP. Opening of the Tim22 channel is promoted by an increase of the membrane potential. When a high membrane potential was applied together with internal targeting signal peptides, a 100-fold increase in the gating frequency of the channel was observed – that is, a rapid opening and closing, possibly reflecting active conformational changes of the channel during the insertion process. Tim22 shows subconductance states and a variable pore size. When the channel is fully open, the pore size of ~18 Å should allow for the insertion of two tightly packed α-helices, supporting the view that proteins insert in a loop conformation. A smaller pore size of ~11 Å at a subconductance state only allows for the presence of a single α-helix. This could reflect the lateral release of the preprotein into the membrane. The detailed function of the two other components of the TIM22 translocase, Tim54 and Tim18, is unknown so far. Concluding remarks
The two main classes of mitochondrial precursor proteins use different import mechanisms. The only common features are that they utilize the GIP (Tom40) as a common pore for translocation through the outer membrane and that both require a membrane potential as an active force for transport through or into the inner membrane. Both pathways have adapted specifically to the different characteristics of each type of targeting signal. While the presequence-carrying proteins are directed by a single N-terminal signal region and transported in a linear fashion http://tcb.trends.com
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along a chain of binding sites, the hydrophobic inner membrane proteins contain multiple internal signals and are translocated in loop formations. Besides these two major classes of import pathways, recent work has revealed the existence of other, specialized mechanisms. For example, the Tom and Tim proteins themselves are all nuclear encoded and have to be imported and assembled into their functional complexes. The best-studied case is the biogenesis of Tom40 which assembles into the TOM complex through a complicated pathway involving multiple intermediate stages in which all other Tom proteins are involved [18,19]. For the import of Tim23, Paschen et al. [20] found that, under conditions of limited membrane potential, another soluble TIM complex in the intermembrane space, the Tim8–Tim13 complex, is required. Moreover, a crossingover of some precursor proteins between the two main import pathways has been observed [21]. The identification of export machineries for transport of mitochondrially encoded proteins from the matrix into the inner membrane has opened a new field of translocation research and will be an important topic in the future [22,23]. It is likely that the great variety of mitochondrial precursor proteins and the high versatility of the translocase machineries will hold many more surprises. Acknowledgements
We thank Ann Frazier for critical comments on the manuscript. The work of the authors’ laboratory was supported by the Deutsche Forschungsgemeinschaft, SFB 388 and the Fonds der Chemischen Industrie/BMBF. A.C. is a recipient of a postdoctoral FEBS fellowship. References 1 Koehler, C.M. et al. (1999) How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem. Sci. 24, 428–432 2 Bauer, M.F. et al. (2000) Protein translocation into mitochondria: the role of TIM complexes. Trends Cell Biol. 10, 25–31 3 Pfanner, N., and Geissler, A. (2001) Versatility of the mitochondrial protein import machinery. Nat. Rev. Mol. Cell Biol. 2, 339–349 4 Schatz, G. (1997) Just follow the acid chain. Nature 388, 121–122 5 Brix, J. et al. (1997) Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J. Biol. Chem. 272, 20730–20735 6 Meisinger, C. et al. (2001) Protein import channel of the outer mitochondrial membrane: a highly stable Tom40–Tom22 core structure differentially interacts with preproteins, small Tom proteins, and import receptors. Mol. Cell. Biol. 21, 2337–2348
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7 Abe, Y. et al. (2000) Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell 100, 551–560 8 Donzeau, M. et al. (2000) Tim23 links the inner and outer mitochondrial membranes. Cell 101, 401–412 9 Truscott, K.N. et al. (2001) A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nat. Struct. Biol. 8, 1074–1082 10 Bauer, M.F. et al. (1996) Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria. Cell 87, 33–41 11 Huang, S. et al. (2002) Protein unfolding by the mitochondrial membrane potential. Nat. Struct. Biol. 9, 301–307 12 Voisine, C. et al. (1999) The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70. Cell 97, 565–574 13 Geissler, A. et al. (2001) Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates translocation and reduces the membrane potential dependence of loosely folded preproteins. Mol. Cell. Biol. 21, 7097–7104 14 Taylor, A.B. et al. (2001) Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences. Structure 9, 615–625 15 Wiedemann, N. et al. (2001) The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria. EMBO J. 20, 951–960 16 Curran, S.P. et al. (2002) The Tim9p–Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier. EMBO J. 21, 942–953 17 Kovermann, P. et al. (2002) Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signalgated channel. Mol. Cell 9, 363–373 18 Rapaport, D. and Neupert, W. (1999) Biogenesis of Tom40, core component of the TOM complex of mitochondria. J. Cell Biol. 146, 321–331 19 Model, K. et al. (2001) Multistep assembly of the protein import channel of the mitochondrial outer membrane. Nat. Struct. Biol. 8, 361–370 20 Paschen, S.A. et al. (2000) The role of the TIM8–13 complex in the import of Tim23 into mitochondria. EMBO J. 19, 6392–6400 21 Kurz, M. et al. (1999) Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway. Mol. Biol. Cell 10, 2461–2474 22 Hell, K. et al. (2001) Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA. EMBO J. 20, 1281–1288 23 Saracco, S.A. and Fox, T.D. (2002) Cox18p is required for export of the mitochondrially encoded Saccharomyces cerevisiae Cox2p C-tail and interacts with Pnt1p and Mss2p in the inner membrane. Mol. Biol. Cell 13, 1122–1131
Agnieszka Chacinska Nikolaus Pfanner* Chris Meisinger Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany. *e-mail: nikolaus.pfanner@biochemie. uni-freiburg.de