Import of mitochondrial proteins

IMPORT OF MITOCHONDRIAL PROTEINS

Matthias F. Bauer 1 and Sabine Hofmann Instituteof Clinical Chemistry Molecular Diagnosticsand Mitochondrial Genetics and Diabetes ResearchGroup Academic Hospital Munich-Schwabing K61nerPlatz, D-80804 Miinchen, Germany Waiter Neupert Instituteof Physiological Chemistry University of Munich Butenandtstrasse5 81377 Miinchen, Germany

I. Introduction II. The Pathways of Mitochondrial Preprotein Import A. Targeting and Sorting of Preproteins to Mitochondria is Mediated by Specific Signals B. The Translocation System of the Outer Mitochondrial MembranehThe TOM Complex C. The Presequence Translocase of the Inner Membrane--The TIM23 Complex D. The Translocase for Import of Carder Proteins into the Mitochondrial Inner Membrane--The TIM22 Complex E. Mitochondrial Translocases in Mammals III. Mitochondrial Biogenesis and Human Neurodegenerative Diseases A. Dysfunction of Mitochondrial Preprotein Import as a Cause of Progressive Neurodegeneration--Mohr-Tranebjaerg Syndrome B. Defects of Quality Control of Mitochondrial Inner Membrane Proteins--Hereditary Spastic Paraplegia References

I. Introduction

M i t o c h o n d r i a are p r e s e n t i n virtually all eukaryotic cells, a n d they arise by growth a n d division o f p r e e x i s t i n g m i t o c h o n d r i a . T h i s growth occurs by i n s e r t i o n o f newly synthesized c o m p o n e n t s l e a d i n g to the e x p a n s i o n o f 1 A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d b e a d d r e s s e d . INTERNATIONALREVIEWOF NEUROBIOLOGY,VOL. 53

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the four compartments of the mitochondria. Two of these mitochondrial compartments are formed by the outer and the inner membrane, which delimitate two aqueous compartments, the intermembrane space, and the matrix. There has been considerable advance in the field of transport of mitochondrial constituents, in particular of protein components, to these mitochondrial compartments. Almost all mitochondrial proteins are encoded as precursors by the nuclear genome. A major aspect of mitochondrial biogenesis is, therefore, the transfer of nuclear encoded, cytosplasmically synthesized precursor proteins across and into the mitochondrial membranes and their assembly to the supramolecular complexes of the various mitochondrial compartments. The number of different proteins undergoing these processes may amount to roughly 1000. In contrast, only a few protein components are encoded by the mitochondrial DNA (mtDNA). In mammals the mitochondrial genome contains the genes for RNA species [two ribosomal (rRNAs) and 22 transfer (tRNAs)] required for mitochondrial protein biosynthesis and for 13 polypeptides that represent components of the various complexes of oxidative phosphorylation. All of these latter proteins are synthesized on mitochondrial ribosomes and they are inserted from the matrix side into the mitochondrial inner membrane (Stuart and Neupert, 1996). Together with the imported preproteins encoded by nuclear genes, these mitochondrial gene products are assembled into the hetero-oligomeric respiratory chain complexes I, III, and IV, and the ATP synthase. The use of simple model organisms, such as the yeast Saccharomyces cerevisiae and Neurosporacrassa, has helped considerably to investigate the structure and function of a rather large number of components involved in targeting and sorting of nuclear-encoded preproteins to mitochondria. Several pathways that guide mitochondrial preproteins to their sites of function have been characterized and the energetics of the various steps of import have been studied in some detail (Ryan andJensen, 1995; Schatz, 1996; Bauer et al., 2000; Herrmann and Neupert, 2000; Pfanner and Geissler, 2001). As demonstrated by these investigations, uptake of protein components into mitochondria is a multistep process facilitated by the coordinated action of specialized translocation systems, so-called preprotein translocases. These translocases decode the signal sequences of the precursor proteins and mediate translocation, insertion, and intramitochondrial sorting to their correct destination. Hydrophilic precursor proteins destined for the matrix must cross both membranes as well as the intermembrane space before reaching their final location. The precursors of the membrane-integrated components of the outer membrane are bound on the surface and are sorted directly into the lipid bilayer, whereas inner membranes have to cross the outer membrane without getting arrested in it and have to pass through the aqueous intermembrane space. Notably, all subunits of the mitochondrial

IMPORTOF MITOCHONDRIALPROTEINS translocation systems are themselves nuclear encoded, and the precursors of these components have to be sorted and inserted into the membranes by preexisting translocases. Recent research on mitochondrial protein translocation has focused mainly on the molecular nature of the translocation machineries. These translocation systems are of considerably higher complexity and higher versatility than may have been expected. In this chapter we provide an overview on the structural organization and the function of the import systems that mediate protein targeting to mitochondria. We will also discuss how genetic alterations of these systems contribute to the development of neurodegenerative disorders in humans.

Ih The Palhways of Mitochondrial Preprotein Import Nuclear-encoded mitochondrial proteins are synthesized on ribosomes in the cytosol as precursor proteins (or preproteins) that are directed to the submitochondrial compartments by means of specific mitochondrial targeting signals. Newly synthesized mitochondrial preproteins in the cytosol are believed to be maintained in a translocafion competent state by specific binding proteins. A number of cytosolic components were reported to interact with nascent polypeptide chains, i.e., even before they are released from the ribosome to mediate stabilization and (partial) folding (for review, see Hart, 1996). In particular, cytosolic Hsp70s, members of the heat shock protein family of 70 kDa as well as binding factors specific for presequences appear to be involved in these processes. Moreover, proteins in the cytosol may exert a more specific function, namely guiding preproteins to the surface of mitochondria, in a similar way as has been discovered for secretory proteins in bacteria and eukaryotes ("targeting function") (Rapoport et al., 1996; Schatz and Dobberstein, 1996). At the outer surface of the mitochondrial outer membrane, specific receptors are exposed that recognize and bind the precursors prior to their translocafion. The transfer across the membranes is then mediated by the distinct import systems embedded in the outer and the inner membranes (Fig. 1). Upon translocafion of a precursor into the mitochondrial matrix, these machineries interact dynamically, thereby bringing the two membranes into close proximity. In eukaryotes, three distinct preprotein import systems have been described (Ryan and Jensen, 1995; Schatz, 1996; Bauer et al., 2000; Herrmann and Neupert, 2000; Pfanner and Geissler, 2001). The TOM complex ("translocase of the outer mitochondrial membrane") mediates the initial recognition of preproteins, their transfer through the outer membrane, and the insertion of resident outer membrane

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FIG. 1. Mitochondrial preprotein import and export pathways. Nuclear-encoded preproteins are imported and distributed into the four ntitochondrial compartments along distinct pathways (schematically depicted as arrows). Cytosolic preproteins are recognized by specialized import receptors of the TOM complex of the outer membrane, and then, depending on their final destination, sorted into the outer membrane (OM) and the intermembrane space (IMS), or are handed over to the TIM translocases of the inner membrane (IM). Preproteins carrying a presequence (matrix-targeting signal) are imported via the TIM23 complex into the inner membrane or the matrix space. Hydrophobic proteins are shuttled by the help of soluble chaperones across the intermembrane space to the TIM22 complex, which mediates their insertion into the inner membrane. Mitochondrial protein components encoded by the mtDNA are exported into the inner membrane via the OXA translocase and by the help of Pntl. p r o t e i n s (Fig. 1). T h i s c o m p l e x is m o s t likely u s e d b y all n u c l e a r - e n c o d e d p r e cursors. T h e T O M c o m p l e x c o n t a i n s specific h y d r o p h i l i c r e c e p t o r s r e c o g n i z i n g newly s y n t h e s i z e d p r e c u r s o r s in t h e cytosol ( K i e b l e r et al., 1990; Pfaller et al., 1988). T h e b o u n d p r e c u r s o r p r o t e i n s a r e t h e n t r a n s f e r r e d to a p r o t e i n c o n d u c t i n g c h a n n e l , also r e f e r r e d to as t h e " g e n e r a l i m p o r t / i n s e r t i o n p o r e " ( G I P ) , w h i c h t r a n s l o c a t e s p r e p r o t e i n s across t h e o u t e r m e m b r a n e i n t o t h e i n t e r m e m b r a n e s p a c e (Pfaller et al., 1988; Hill et al., 1998; K u n k e l e et al., 1998a). P r e p r o t e i n s cross t h e m e m b r a n e s in a n u n f o l d e d c o n f o r m a t i o n , and folded domains of preproteins present on the surface of mitochondria a r e u n f o l d e d d u r i n g this t r a n s l o c a t i o n process. F u r t h e r m o v e m e n t o f t h e t r a n s l o c a t i o n i n t e r m e d i a t e s i n t o a n d across t h e i n n e r m e m b r a n e is m e d i a t e d by two d i s t i n c t t r a n s l o c a s e s in t h e i n n e r m e m b r a n e , t h e T I M 2 3 a n d t h e T I M 2 2 c o m p l e x (Fig. 1). B o t h T I M c o m p l e x e s c o o p e r a t e with t h e T O M c o m p l e x u p o n t r a n s f e r o f a p r e p r o t e i n i n t o a n d across t h e m i t o c h o n d r i a l

IMPORT OF MITOCHONDRIAL PROTEINS

inner membrane thereby forming so-called translocation contact sites. The two different TIM complexes differ in their specificity for preprotein substrates and direct preproteins to different destinations (Ryan and Jensen, 1995; Sirrenberg et al., 1996; Kerscher et al., 1997; Koehler et al., 1998a,b; Sirrenberg et al., 1998). The transfer of preproteins via the TIM23 complex across the inner membrane strictly requires both an electrochemical potential (A~) across the inner membrane and ATP in the matrix as energy sources. The TIM22mediated insertion of hydrophobic proteins into the inner membrane depends on the presence of a A ~ but does not require ATP (Ryan andJensen, 1995; Schatz, 1996; Bauer et al., 2000; Herrmann and Neupert, 2000; Pfanner and Geissler, 2001). At least one further translocase, called OXA, exists in the inner membrane (Fig. 1). This translocase contains the Oxal protein and mediates insertion of distinct classes of preprotein substrates from the matrix side into the inner membrane. These substrates include mitochondrially encoded subunits of the respiratory chain complexes and certain nuclear-encoded inner membrane proteins that are first imported into the matrix space via the TIM23 complex and from there into the inner membrane (Hell et al., 1998). This insertion pathway also requires, at least in many cases, a membrane potential across the inner membrane, and it resembles the Secindependent, ApH-dependent insertion of polytopic proteins into the bacterial plasma membrane (Herrmann et al., 1997). Recently, a second export component, Pntl, has been identified in a genetic approach screening for yeast mutants defective for the export of mitochondrially encoded proteins (Fig. 1) (He and Fox, 1999). Pntl is involved in the export of the C-terminus of subunit 2 of the cytochrome c oxidase (Cox2). Its precise role in export, however, has not been determined, and there is experimental evidence that Pntl and Oxalp exhibit overlapping functions in yeast. It has become clear that a variety of additional steps exist that act on precursors during the import and allow them to reach their final destinations. In particular, molecular chaperones support folding of precursors, and facilitate their assembly into functional complexes or sorting to the correct compartment. In addition, maturation steps can occur during import, which include covalent and noncovalent modifications.

A. TARGETING AND SORTING OF PREPROTEINS TO MITOCHONDRIA IS MEDIATED BY SPECIFIC SIGNALS

All proteins of an eukaryotic cell with the exception of the few mitochondrially encoded ones are translated on cytosolic polysomes and are

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eventually sorted to the various different cellular compartments. The information necessary for targeting of preproteins to mitochondria is contained in relatively minor portions of the nascent or newly completed polypepfide chains. These portions were designated as signal or targeting sequences. Most targeting signals are contained within N-terminal extensions, the majority of which are cleaved upon import into the mitochondria. As they direct preproteins, at least partially, into the matrix space they are referred to as "matrix targeting signals." Many preproteins, on the other hand, do not carry N-terminal presequences but internal targeting signals. Additional signals exist in preproteins that mediate sorting and insertion into the mitochondrial membrane. 1. Matrix-Targeting Signals (Presequences) The majority of precursor proteins carry the targeting sequence in an N-terminal extension of about 10-80 amino acid residues. In particular, most soluble matrix proteins carry such presequences, which are proteolytically cleaved off upon reaching their final destination. When fused to a cytosolic protein presequences could be shown to specifically direct "passenger" proteins across both membranes into the matrix (Hurt et al., 1984; Horwich et al., 1985). In addition, N-terminal presequences or presequence-like signals are also sufficient to target many preproteins to the other three compartments, the outer membrane, the intermembrane space, and the inner membrane (see below). The presequences of different precursors do not share sequence similarities. They share, however, distinct structural features. They contain abundant positively charged, and quite frequently hydrophobic and hydroxylated, amino acid residues (von Heijne et al., 1989). As a rule, they are predicted to form amphipathic a-helices presenting a positively charged face on one side and a hydrophobic face on the opposite side of the helix (Roise et al., 1986; Roise and Schatz, 1988). The potential to form a polar a-helical structure is thought to prevail in a hydrophobic environment and is considered an essential prerequisite for the function of the presequences (Gavel et al., 1988). It has been proposed that this helical structure is responsible for the initial interaction with the lipid bilayer of the outer membrane mainly on the basis of experiments with artificial lipid vesicles (de Kroon et al., 1991; de Kruijff, 1994; Tamm, 1991). The significance of such a reaction in vivo, however, is not clear. The prevailing concept includes the idea that the specific recognition of the targeting signals occurs via proteinaceous receptor components of the complex on the mitochondrial surface. These receptors have been identified and their role in translocation could be demonstrated (Fig. 2) (see below). The structural features of the presequences recognized by the receptors of the TOM complex are partly known. Recent studies have indicated

FIG. 2. Composition and specificity of the translocation systems of the mitochondrial membranes. The majority of mitochondrial preproteins carry positively charged matrix-targeting signals at their N-termini (presequences), which are recognized by receptor components Tom22/Tom20. From this so-called c/s site, the presequence is transferred through the GIP consisting of Tom40, Tom22, and the small Tom proteins. Upon reaching the intermembrane face of the outer membrane, the presequence binds to a trans site, which is constituted by Tom22, and possibly Tom40. A subset of preproteins, including the ADP/ATP carrier (AAC) and related proteins carrying internal targeting signals, are first bound to the specialized receptor Tom70. For further translocation, the TOM complex cooperates with the TIM23 complex and the TIM22 complex in the inner membrane. The transfer of preproteins via the TIM23 complex across the inner membrane strictly requires both, an electrochemical potential (A~) across the inner membrane and ATP in the matrix as energy sources. Insertion of the presequence into the TIM23 complex is thought to be driven electrophoretically by the membrane potential and complete transport of the precursor into the matrix is mediated by an ATPpowered import motor consisting of mtHsp70 and the nucleotide exchange factor Mgelp, which are attached to the inner outlet of the TIM23 complex. A number of preproteins with internal signals are guided by hetero-oligomeric complexes of small Tim proteins from the TOM complex across the aqueous intermembrane space to the TIM22 complex. The composition of these hetero-oligomeric complexes differs depending on whether they are soluble in the intermembrane space or are attached to the membrane integral portion of the TIM22 complex. The TIM22-mediated insertion of hydrophobic proteins into the inner membrane depends on the presence ofa AqJ but does not require ATE Abbreviations: Tom20 (20), Tom22 (22), Tom40 (40), Tom70 (70), Tom5 (5), Tom6 (6), Tom7 (7), mt-Hsp70 (70), Tim44 (44), Tirol7 (17), Tim23 (23), Mgelp (E), Tim22 (22), Tim54 (54), Timl8 (18), Tim9 (9), Tim10 (10), Tim12 (12), Tim8 (8), and Timl3 (13).

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that hydrophobic interactions rather than the positive charges appear to mediate binding of the presequence to Tom20, one of the major two TOM receptors (Fig. 2) (Abe et al., 2000). As the binding of the presequences is labile particularly under conditions of high ionic strength, it was assumed that weak electrostatic forces may act between the presequences and the receptor components (Mayer et al., 1995a). The cytoplasmic domain of Tom22, on the other hand, carries a cluster of 18 negatively charged residues. This region was proposed to interact with the positive charges of the presequences (Kiebler et al., 1993). The presequences are recognized not only on the mitochondrial surface but also on the inner face of the outer membrane, and in a further step, by components located at the surface of the inner membrane. In particular, binding by the TIM23 complex and translocation across the inner membrane appears to involve the positively charged amino acid residues of the presequence (Fig. 2) (Bauer et al., 1996). Whereas the presequences of the vast majority of matrix-targeted proteins are located at the amino-terminus, recently a mitochondrial matrix protein was identified that carries a cleavable targeting signal at its C-terminus. This C-terminal signal of the precursor of the yeast DNA helicase Hmilp is similar to classical N-terminal presequences and consists of a stretch of positively charged amino acids that has the potential to form an amphipathic a-helix (Lee et al., 1999). In contrast to the precursors carrying Nterminal presequences, this preprotein is imported in a reverse orientation with a C- to N-terminal direction, demonstrating that the import systems are able recognize the targeting signals irrespective of their position within the precursor protein. The presequences of most of the precursor proteins, including that of Hmilp, are cleaved offby the mitochondrial processing peptidase in the matrix (MPP) during or after their translocation across the inner membrane (Wang and Weiner, 1993, 1994; Arretz et al., 1991, 1994; Glaser and Dessi, 1999). In a number of cases the initial proteolytical processing, performed by MPP, is followed by an additional proteolytic maturation step in the matrix. A second portion is removed either by MPP, as with the precursor of F0-ATPase subunit 9 (Schmidt et al., 1984) or by the monomeric metalloprotease, MIP (mitochondrial intermediate peptidase), which removes an octapeptide from the N-termini generated by MPP (Kalousek et al., 1988; Isaya et al., 1992). 2. Variations on Targeting Signals for Sorting to Mitochondrial Subcompartments

Many preproteins destined for the inner and outer membrane and the intermembrane space carry N-terminal presequences or presequence-like

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signals in their mature parts. In addition to the positively charged targeting signals, more hydrophobic segments exist that mediate the sorting and insertion in the membrane compartments. Precursors destined for the outer membrane do not carry cleavable presequences, but they do contain targeting signals in their mature parts that are only partially characterized so far. The targeting signals of outer membrane proteins, like Tom70, which carries a single N-terminal anchor, has been analyzed in some detail. Yeast Tom70 exposes approximately 10 N-terminal amino acid residues into the intermembrane space, which are followed by a 20-residue membrane anchor and a large 60 kDa domain in the cytosol (Riezman et al., 1983; Millar and Shore, 1994). Both the information for targeting and for membrane integration are located in the first 30 residues (McBride et al., 1992). The structural features that target these preproteins to the outer membrane are not known, although they are bound via the receptor components, which also bind presequence-carrying preproteins. Insertion of these precursors into the outer membrane is mediated by hydrophobic stretches. As classical presequences, these stretches do not share distinct sequence motifs. Other signals for targeting to the outer membrane appear to present in C-terminal segments of proteins that are anchored to the membrane by hydrophobic segments located close to the C-terminus (Mitoma and Ito, 1992; Nguyen et al., 1993; Shore et al., 1995). The specificity of recognition and the mechanism of their insertion are not understood. Many intermembrane space proteins are initially synthesized without an N-terminal targeting signal. The internal signals are not known or only partially characterized, like for the intermembrane space protein cytochrome c heine lyase (CCHL) (Steiner et al., 1996). In the case of cytochrome b2 arrest at the level of the inner membrane by a stop-transfer sequence has been suggested. Other models imply partial or complete passing of such preproteins through the matrix space. Most inner membrane proteins and some intermembrane space proteins have positively charged matrix targeting signals at their N-termini that are complemented by more hydrophobic sorting signals either within the mature part of the protein or in tandem with the presequences. Three different kinds of such signals are known: first, sorting signals, which consist of hydrophobic segments with charged flanking regions that become arrested when they cross the inner membrane; second, precursors with a hydrophobic segment preceeding a hydrophobic transmembrane segment that becomes inserted in a kind of loop structure; and third, a hydrophobic segment in a preprotein that has a matrix targeting signal and becomes completely or partially imported into the matrix. Subsequently, the hydrophobic sorting signal then gets inserted into the inner membrane and adjacent segments

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al.

get exported into the intermembrane space. Apparently, it is the combination of the topologies of the charged sequences and the flanking hydrophobic membrane segments that determine targeting and membrane insertion. How this information is decoded by the components of the translocation systems of both membranes remains obscure. 3. Multiple Internal Targeting Signals

An exceptional case with respect to the structure of the targeting signals is the large family of mitochondrial cartier proteins of the inner membrane with the ATP/ADP carrier (AAC) as the most prominent member. The carrier proteins do not carry cleavable presequences, but are targeted by means of internal signals that are repeated three or in one case even six times within these carriers (Fig. 2). The ATP/ADP carrier is characterized by three domains each of about 100 amino acid residues (Saraste and Walker, 1982). Stretches of about 20 amino acids are present in the carboxy-terminal half of each domain predicted to form a-helices (Aquila et al., 1985) and resemble the classical mitochondrial presequences (Ito et al., 1985; Von Heijne, 1986; Smagula and Douglas, 1988). The internal signals appear to exert a cooperative effect in recruiting several receptors to one precursor molecule (Endres et al., 1999; Wiedemann et al., 2001). Only little is known, however, about the structural characteristics and the mode of action of these internal targeting signals. Studies have just begun to address the questions of how such precursors use the TOM complex and how they become inserted into the inner membrane by using the TIM22 machinery (Fig. 2).

B. THE TRANSLOCATIONSYSTEMOF THE OUTER MITOCHONDRIAL MEMBRANE---THE T O M COMPLEX

The TOM complex is composed of seven to eight protein subunits with different and distinct functions in the recogniton and the translocation of preproteins. All of them are integral membrane proteins (Fig. 2). They can be classified into receptor components, channel-forming components, and small membrane-spanning proteins with not yet clearly defined accessory functions. The preprotein receptors Tom70 and Tom20 expose hydrophilic domains of approximately 65 and 17 kDa, respectively, at the surface of the mitochondria, which recognize and bind the targeting signals of newly synthesized precursors present in the cytosol (S611ner et al., 1989, 1990; Hines et al., 1990; Kiebler et al., 1993; Ramage et al., 1993; Lithgow et al., 1994; Nakai and Endo, 1995; Honlinger et al., 1996). The Tom40 is the key structural component of the protein conduction channel, the GIP in the outer

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membrane that guides the precursors across the outer membrane in their unfolded conformation. It is essential for viability in yeast and Neurospora (Vestweber et al., 1989; Kiebler et al., 1990). Multiple copies of Tom40 are organized in the TOM core complex together with up to three small, membrane-embedded components, Tom5, Tom6, and Tom7 (Kassenbrock et al., 1993; Alconada et al., 1995; Cao and Douglas, 1995; Honlinger et al., 1996; Dietmeier et al., 1997) and Tom22, a subunit with hydrophilic domains exposed to both sides of the outer membrane (Kiebler et al., 1993; Lithgow et al., 1994; Nakai and Endo, 1995). The Tom22 fulfills two functions. It acts as receptor together with Tom20 and is a constituent of the GIP complex (Fig. 2) (Court et al., 1996; Kunkele et al., 1998b). Two further proteins Tom71, (Schlossmann et al., 1996) and Tom37 (Gratzer et al., 1995) were found in the yeast S. cerevisiae. Tom70 and Tom71 are structurally closely related (53% sequence identity, 70% similarity) (Bomer et al., 1996a; Schlossmann et al., 1996). So far, no protein with homology to Tom71 and Tom37 could be detected in any other higher eukaryotic organism. The recent isolation and purification of the TOM holo complex of Neurospora crassa has provided further insight into the composition, structure, and function of the TOM complex (Kunkele et al., 1998b; Ahting et al., 1999; Stan et al., 2000; Ahting et al., 2001). The isolated holo complex contained the established import receptors (Tom70 and Tom20) as well as the TOM core complex, consisting of Tom40, Tom22, Tom6, and Tom7 (Kunkele et al., 1998b; Ahting et al., 1999). The Tom6 and Tom7 were found to be in direct contact with the major component of the pore, Tom40. In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit (Dembowski et al., 2001). The TOM core complex has the characteristics of the general insertion pore GIP; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner (Stan et al., 2000). Electron microscopic (EM) analysis and tomographic studies revealed single particles with one, two, and three putative channels. The majority of these complexes seem to contain two protein-conducting channels (Ahting et al., 1999). As estimated from three-dimensional reconstruction by electron tomography and from electrophysiological measurements, the size of the two open pores traversing the complex is roughly 2.1 nm and has a height of approximately 7 nm, which is large enough to allow translocation of a polypeptide chain (Ahting et al., 1999). A TOM subcomplex consisting exclusively of Tom40 of N. crassa has been isolated (Ahting et aL, 2001). Structural analyses as determined by circular dichroism measurements and Fourier transform infrared spectroscopy revealed 31% t-sheet topology and 22% a-helix (Ahting et al., 2001).

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Isolated Tom40 was functional and forms pores with channel-forming activities very similar to those found with both TOM core complex and TOM holo complex, supporting the view that Tom40 is the central constituent of the protein-conducting channel of the TOM complex. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase and it forms the protein-conducting channel in an oligomeric assembly. Early studies have provided insights into the specificity of the mitochondrial preprotein receptors (S611ner et al., 1989; Ramage et al., 1993). The Tom22 was shown to act in concert with Tom20, thereby forming a receptor assembly that preferentially binds preproteins with positively charged presequences and precursors destined to be inserted into the outer membrane (Fig. 2) (Mayer et aL, 1995a; Brix et aL, 1997; Abe et al., 2000). The so-called cis site of this receptor is involved in the recognition of precursor proteins on the surface of the outer membrane and provides an extended binding area on which the various targeting signals can dock, and thereby are guided into the outer membrane translocation pore (Lill et al., 1996). The cytosolic domain of Tom20 contains a hydrophobic groove that accommodates a positively charged arnphipathic a-helical matrix-targeting sequence. Although the positive charges are necessary for translocation of the presequence across the inner membrane, binding of the presequence to Tom20 is mediated by hydrophobic interactions (Abe et al., 2000). The molecular basis of binding o f a presequence to Tom22 are not known. However, binding to the cis site is readily reversible and weakened in its stability at increasing salt concentrations. This may indicate the involvement of weak electrostatic forces acting between the presequences and the receptor components (Haucke et al., 1995; Mayer et al., 1995b). Hydrophobic precursor proteins that carry internal targeting information, such as the members of the family of the mitochondrial carriers, are preferentially bound by the receptor components Tom70 and Tom71. As these signals occur repeatedly within one carrier preprotein, it was assumed that several Tom70 molecules simultaneously bind, thereby stabilizing a hydrophobic preprotein on the mitochondrial surface (Planner et al., 1987; Wiedemann et al., 2001; Schlossmann et al., 1994). The high tendency of hydrophobic precursors to aggregate is not only a problem at the surface of mitochondria but occurs also in the cytosol. This problem is overcome by the action of several components in the cytosol that maintain precursor proteins in an import-competent state and perhaps protect them from rapid proteolyfic degradation. The Tom70 may also act as a docking site for

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these cytosolic targeting factors, which comprise members of the cytosolic Hsp70 family and probably additional binding factors. MSF (mitochondrial import stimulating factor) is the best characterized factor so far. It belongs to the 14-3-3 protein family (Hachiya et al., 1994; Mihara and Omura, 1996). MSF was proposed to recognize the mitochondrial precursor proteins in the cytosol, forms a complex with them and targets them to the receptors on the outer surface of mitochondria in an ATP-dependent manner (Hines et al., 1990; Hines and Schatz, 1993; Hachiya et al., 1995; Komiya et al., 1997). ATP hydrolysis is likely required to facilitate release of the preproteins from factors such as cytosolic Hsp70 and MSF. This release may promote binding of the preprotein by receptors of the TOM complex. Binding of preproteins to the c/s site is followed by the transfer through the translocation channel or GIP, which allows interaction of the N-terminal targeting sequence with a second specific binding site located at the inner face of the outer membrane called trans site (Fig. 2). Insertion of the N-terminal part of the mature protein into GIP is accompanied by the unfolding of the following segments of the preprotein (Mayer et al., 1995b). The molecular nature of the trans site is not entirely clear. The Tom40 is considered to be the main component generating the trans site. In addition, Tom22 may contribute to this binding site. The presence of specific presequences binding sites were proposed for the intermembrane space portions of both proteins (Mayer et al., 1995a; Hill et al., 1998; Athing et al., 1999). What drives the translocation of the presequence across the outer membrane? Apparently, neither a membrane potential nor ATP are necessary for directing the presequence to the trans site. The energy derived from presequence binding could constitute the driving force for transfer across the outer membrane. The much higher affinity of the presequences to the trans site as compared to the cis site could provide the driving force for movement and determine its directionality (Mayer et al., 1995b). A related concept is the "acid chain hypothesis," which proposes that the positively charged presequences are recognized by the negatively charged clusters of the TOM components via ionic interactions. This concept is supported by the observation that purified cytosolic and intermembrane space domains of several Tom proteins and Tim23 interacted with mitochondrial precursors in a sequential manner. Other noncovalent forces, like hydrophobic forces also appear to play an important role in the interaction between matrix-targeted preproteins and TOM components. A modified model has been proposed in which preproteins are transferred in a stepwise manner along a chain of binding sites that guides the precursor across the outer membrane into the intermembrane space (binding chain hypothesis) (Pfanner and Geissler, 2001).

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C. THE PRESEQUENCETRANSLOCASEOF THE INNERMEMBRANE THE TIM23 COMPLEX At the inner outlet of the TOM channel presequence-containing preproteins are b o u n d at the trans site before they are sorted to the TIM23 complex, which mediates further translocation into the matrix and into the inner m e m b r a n e (Figs. 1 and 2). During transport, the T O M complex and the TIM23 complex are transiently linked by the translocating polypeptide chain, thereby forming so-called translocation contact sites (Fig. 3) (Donzeau et al., 2000). Insertion of the presequence into the TIM23 complex is thought to be driven electrophoretically by the m e m b r a n e potential (Martin et al., 1991; Bauer et al., 1996), and complete transport of the precursor into the matrix is mediated by an ATP-powered import m o t o r attached to the inner outlet of the TIM23 complex (Scherer et al., 1992; Kronidou et al., 1994; Schneider et al., 1994). The TIM23 complex consists of a membrane-integrated section that is composed by the subunits Tim23 and TimlT; and by a section attached to it at the matrix side, which is composed by the components Tim44, mtHsp70, and Mgel (Fig. 3) (Maarse et al., 1992; Dekker et al., 1993; Emtage andJensen, 1993; Maarse et al., 1994; Ryan et al., 1994). The Tim23 forms a receptor for the presequence in the interm e m b r a n e space and together with T i m l 7 a preprotein conducting channel across the inner m e m b r a n e (Berthold et al., 1995; Bauer et al., 1996). T h e Tim23 can be divided into a hydrophilic N-terminal and a hydrophobic C-terminal half. It is anchored in the inner m e m b r a n e by its C-terminal portion (Emtage andJensen, 1993; Donzeau et al., 2000), resulting in an N-out and C-out topology. In the N-terminal half o f Tim23 an intermediate domain can be discriminated from an N-terminal domain. The intermediate domain is exposed in the i n t e r m e m b r a n e space, whereas the N-terminal domain is penetrating the outer m e m b r a n e so that a small segment is exposed on the mitochondrial surface where it is accessible to added protease (Donzeau et al., 2000). Thus, Tim23 is the first mitochondrial protein with a two-membrane-spanning topology. The T i m l 7 is structurally related to Tim23 in its membrane-integrated portion but lacks a hydrophilic N-terminal portion (Maarse et al., 1994; Ryan et al., 1994). The T i m l 7 and Tim23 are organized as a dimeric complex (Bauer et al., 1996; Moro et al., 1999). How these components generate the protein conducting channel is not clear. T h e section of the TIM23 translocase at the inner face of the inner m e m b r a n e forms the import motor. The Tim44 is a hydrophilic peripheral m e m b r a n e protein associated with the inner face o f the inner m e m b r a n e and forms a dimer (Maarse et al., 1992; Blom et al., 1993; Milisav et al., 2001). In contrast to T i m l 7 and Tim23, Tim44 is not accessible to added proteases from the outer side of the inner

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FIG. 3. Dynamic interaction between the TOM complex and the TIM23 complex during import of preproteins with matrix-targeting signals into the mitochondrial matrix. Schematic outline of the formation oftranslocation contact sites. (1) The dimeric TIM23 complex contains two molecules of Tim23, Tirol7, and Tim44, which recruit two molecules of mtHsp70 to the outlet of the translocation channel. Tim23 is integrated into both mitochondrial membranes. The N-terminal domain of Tim23 is embedded into the outer membrane (OM), the intermediate domain dimerizes and forms a negatively charged presequence receptor in the IMS, and the C-terminal half is integrated in the inner membrane (IM). A precursor in association with the TOM complex is shown. The positively charged matrix targeting signal (zigzag) is bound to the trans site (hatched) at the inner side of the outer membrane. The TIM23 complex, tethered to the outer membrane via its N-terminal domain, screens by lateral diffusion the inner side of the outer membrane. (2) The presequence receptor domain of Tim23 encounters the TOM complex and triggers the release of the presequence from the trans site. (3) Binding of the presequence destabilizes the interaction of the dimerized intermediate domains, leading to the A@-dependent opening of the protein conducting channel of the TIM23 complex. The presequence is translocated across the inner membrane. Upon entering the matrix, further translocation is driven by ATP-dependent reaction cycles of the import motor consisting of mtHsp70 (70), Tim44 (44), and Mgelp (E). m e m b r a n e . T h e m t H s p 7 0 is a m a t r i x - l o c a l i z e d m i t o c h o n d r i a - s p e c i f i c m e m b e r o f t h e l a r g e H s p 7 0 p r o t e i n family. It a s s o c i a t e s w i t h T i m 4 4 i n a n A T P d e p e n d e n t m a n n e r a n d this is r e g u l a t e d by t h e n u c l e o t i d e e x c h a n g e f a c t o r Mgel. H o w c a n o n e e n v i s i o n t h e f u n c t i o n o f this m o l e c u l a r m a c h i n e in t h e translocation of preproteins? The TIM23 complex comprises four distinct f u n c t i o n a l e l e m e n t s (Fig. 3) ( B a u e r et al., 1996; D o n z e a u et al., 2 0 0 0 ) : (a) By v i r t u e o f its s i m u l t a n e o u s i n t e g r a t i o n i n t o two m e m b r a n e s , T i m 2 3 f o r m s contacts between the outer and inner mitochondrial membranes. Tethering

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the TIM23 complex to the outer membrane facilitates the transfer of preproteins from the TOM complex to the inner membrane translocase, thereby increasing the efficiencyof protein import. (b) The TIM23 complex receives the preprotein from the TOM complex and binds the N-terminal matrix-targeting signals (presequences) through the presequence receptor domain of Tim23. The receptor is formed upon dimerization of the intermediate domain of Tim23 in a membrane potential-dependent manner. The receptor domains are negatively charged and may interact with the positively charged amphipathic matrix-targeting signals. Upon interaction with the presequence, the dimer of the intermediate domain dissociates, thereby triggering the opening of the protein-conducting import channel across the inner membrane. (c) The channel is formed by the membrane-integrated portion of Tim17 and Tim23. Opening of the channel allows translocation of the presequence across the inner membrane in a membrane-potentialdependent manner. The components forming the channel interact with the unfolded preprotein in transit, but they do not tightly bind the precursor and thus allow oscillation of the presequence in the channel (Ungermann et al., 1994; Berthold et al., 1995; Dekker et al., 1997). (d) for further inward movement a molecular motor is attached at the inner side of the inner membrane. Two models for the action of this molecular motor are currently under debate. In the first model, a Tim44 dimer recruits two molecules ofmtHsp70 to the outlet of the protein-conducting channel (Kronidou et al., 1994; Rassow etal., 1994; Schneider etal., 1994; Schneider etaL, 1996). The Tim44 and the mtHsp70, in cooperation with its cochaperone Mgelp, may constitute a molecular ratchet that drives complete translocation of the polypeptide chains into the matrix with ATP as an energy source. Within this ratchet, mtHsp70 appears to trap incoming segments of unfolded precursor proteins. Thus retrograde movements of the translocating polypeptide chain in the channel are prevented. Repeated cycles of mtHsp70 binding and release, in a kind of "hand-over-hand" mechanism, may facilitate vectorial translocation into the matrix in a stepwise manner (Moro et aL, 1999; Schneider et al., 1994). In the second model, Tim44-bound mtHsp70 undergoes significant conformational changes, thereby pulling the polypeptide chain of a precursor through the translocation channel. It is not known, however, what the strength of a pulling force exerted by small conformational changes could be and so it is not clear whether such a mechanism would be sufficient to drive the import of an entire polypeptide chain and in particular to provide the energy for unfolding of folded domains of precursor proteins in transit (Voos et al., 1996; Huang et al., 1999; Voisine et al., 1999). In addition to the energy derived from ATP hydrolysis, a second driving force is required for protein translocation into the matrix. This energy is

IMPORT OF MITOCHONDRIALPROTEINS

present in the form of a total proton-motive force across the inner membrane, which must be sealed against pronounced leakage of ions. Energization of the inner membrane is not only required for protein translocation but an absolute requirement for the mitochondria to perform oxidative phosphorylation. In contrast to the translocase of the outer membrane, the TIM23 complex and as described below the TIM22 complex require a tight regulation of opening and closing. A permanently opened TIM23 channel would otherwise impair the electrochemical gradient across the inner membrane and cause a breakdown of the oxidative phosphorylation. It is the electrical component A~ of the total proton-motive force that promotes the dimerization of the intermediate domain of the Tim23, thereby presumably sealing the channel (Bauer et al., 1996). On the other hand, A ~ is required for the transfer of the targeting sequence of a preprotein across the inner membrane (Martin et al., 1991; Pfanner and Neupert, 1985); it is, however, not necessary for the movement of the mature part of the preprotein through the import channel of the inner membrane (Schleyer and Neupert, 1985). As discussed above, the membrane potential may exert an electrophoretic effect on the positively charged presequences in such a manner that translocation of the targeting signal is triggered and a gating effect is exerted (Martin et al., 1991).

D. THE TRANSLOCASEFOR IMPORT OF CARRIERPROTEINS INTO THE MITOCHONDRIAL INNER MEMBRANE---THE T I M 2 2 COMPLEX

Integral inner membrane proteins that carry a classical matrix targeting signal use the TIM23 complex for insertion. This can occur either in a "translocation arrest" pathway or by the transfer into the matrix and insertion from the inner face with the help of the OXA1 translocase (Hell et al., 1997, 1998). However, a number of inner membrane proteins carrying internal targeting signals do not use the TIM23 complex but rather are transferred from the TOM complex to the TIM22 complex for insertion into the inner membrane (Sirrenberg et al., 1996) (Fig. 1). Mitochondrial carrier proteins constitute the major class of precursors that are imported via this pathway (Sirrenberg et al., 1996, 1998; Koehler et al., 1998a,b; Endres et al., 1999). In addition, the TIM22 complex appears to mediate the import of precursors of other hydrophobic membrane proteins such as Tim23, Tirol7, and Tim22, which do not belong to the class of mitochondrial carriers (Adam et al., 1999; Leuenberger et al., 1999; Paschen et al., 2000). The Tim22 is the central component of the TIM22 complex; it is structurally related to Tim17 and Tim23, suggesting that both TIM complexes

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have evolved from a common ancestor by gene duplication events (Sirrenberg et al., 1996; Bauer et al., 1999b). Two further membrane-integrated components of the TIM22 complex, Tim54 and Timl8, are known (Fig. 2) (Kerscher et al., 1997, 2000). Their functions are not clear so far. The Tim54 seems to influence the stability of Tim22 in the inner membrane, but may not directly interact with the preproteins during import (Kerscher et al., 1997). The Timl8 shows structural similarity to the subunit IV of complex II (succinate dehydrogenase) of the respiratory chain (Kerscher et al., 2000). During import, the membrane-integral portion of the TIM22 complex interacts with a set of small, structurally related proteins of the mitochondrial intermembrane space (Koehler et al., 1998a,b; Sirrenberg et al., 1998; Adam et al., 1999). The interaction of these small Tim proteins with the translocating preproteins is metal dependent (Sirrenberg et al., 1998). In yeast, five small intermembrane space proteins, Tim8, Tim9, Tirol0, Tirol2, and Tim13 have been identified. All of them contain a Cys4motif that binds Zn 2+ ions proposed to be required for the formation of typical zinc finger structures (Sirrenberg et al., 1998; Adam et al., 1999). In yeast, Tim9, Tim10, and Tim12 are essential for the cell viability, whereas Tim8 and Tirol3 have no obvious deletion phenotype. In particular, Tim9, Tim10, and Tim12 were shown to mediate the insertion of members of the mitochondrial carrier family into the inner membrane; the insertion of a subclass of hydrophobic inner membrane proteins, such as Tim23, involves the assistance of Tim8 and Tim13 (Leuenberger et al., 1999; Paschen et al., 2000). 1. Import of Carrier Proteins

The Tim9, Tirol0, and Timl2 are organized in two distinct heterooligomeric 70 kDa complexes (Fig. 4) (Koehler et al., 1998a, b; Sirrenberg et al., 1998; Adam et al., 1999). The TIM9.10 complex appears to contain three molecules of Tim9 and three molecules of Timl0. The TIM9.10.12 complex is probably composed of three molecules of Tim9, two molecules of Tirol0, and one molecule of Tim12. The TIM9.10.12 complex is firmly associated with the membrane integrated components of the TIM22 complex, whereas the TIM9.10 complex is mobile in the intermembrane space (Sirrenberg et al., 1998). The TIM22 complex cooperates with both the TIM9.10 and the TIM9.10.12 complex, which sequentially interact with hydrophobic precursors and maintain them in an insertion-competent conformation. Most recently, Luciano and co-worker (Luciano et al., 2001) were able to reconstitute the TIM9-10 complex by co-importing recombinantly expressed Tim9 and Tirol 0. Moreover, import of recombinant Timl 0 into an AAC import-deficient strain lacking the endogenous TIM9.10 complex

IMPORT OF MITOCHONDRIAL PROTEINS

FIG.4. The TIM22 complex mediates insertion of the members of the carrier familyinto the inner membrane. Precursors of substrate carrier proteins such as the ADP/ATP carrier (AAC) contain internal targeting information. They are released from cytosolic ribosomes (stage I) and preferentially bind to the Tom70 receptor on the surface of the mitochondria (stage II). The precursor is then transferred to the general insertion pore of the TOM complex. Segments of the precursor that are translocated across the TOM complex are trapped by the TIM9-10 complex in the intermembrane space, resulting in partial translocation of the AAC across the outer membrane The precursor remains at this stage firmly bound to the TOM complex (stage IIIa). The precursor is then transferred to the TIM9.10.12 complex (stage IIIb) at the outer face of the inner membrane. Insertion of carrier proteins into the inner membrane is mediated by Tim22 in a A V-dependent manner (stage IV). Finally, the inserted AAC assembles into a functional dimer (Stage V; homodimerization). was a b l e to r e s t o r e i m p o r t a n d i n s e r t i o n o f A A C to a l m o s t wild-type levels ( L u c i a n o et al., 2001). I t was s h o w n t h a t t h e p r e c u r s o r s o f t h e c a r r i e r p r o t e i n s i n t e r a c t with t h e h e t e r o - o l i g o m e r i c 70 k D a zinc f i n g e r p r o t e i n c o m p l e x e s in t h e i n t e r m e m b r a n e s p a c e in a Z n 2 + - d e p e n d e n t m a n n e r ( S i r r e n b e r g et al., 1998). T h u s , t h e i n t e r a c t i o n o f t h e zinc f i n g e r s o f t h e s m a l l T i m p r o t e i n s with t h e i n t e r n a l signals c o u l d b e t h e m o l e c u l a r basis f o r t h e r e c o g n i t i o n o f t h e m i t o c h o n d r i a l c a r r i e r p r o t e i n s by t h e i m p o r t m a c h i n e r y . T r a n s l o c a t i o n a n d m e m b r a n e i n s e r t i o n o f t h e c a r r i e r p r o t e i n s involves the coordinated action of both the TOM complex and the TIM22 complex (Fig. 4) ( P l a n n e r a n d N e u p e r t , 1987; R y a n et al., 1999). T h e f o l l o w i n g p a t h way o f i m p o r t is p r o p o s e d o n t h e basis o f t h e available e x p e r i m e n t a l data: t h e cytosolic p r e c u r s o r o f a c a r r i e r is initially r e c o g n i z e d by t h e o u t e r m e m b r a n e r e c e p t o r T o m 7 0 o f t h e T O M c o m p l e x . T h e p r e c u r s o r is t h e n t r a n s f e r r e d to

BAUERet al. the GIP and partially translocated across the outer membrane. It interacts with the TIM9.10 complex in the intermembrane space but remains firmly bound to the TOM complex (Adam et al., 1999). Still in contact with the TOM complex, the precursor is then handed over to the TIM9.10.12 complex at the inner membrane (Adam et al., 1999). Subsequently, the TIM22 complex triggers the release of the carrier from the TOM complex and mediates the insertion into the inner membrane (Adam et al., 1999). In contrast to the TIM23-mediated import into the matrix, the insertion of the hydrophobic preproteins into the inner membrane depends on the presence of a membrane potential but does not require ATE Finally, the inserted carrier assembles into a functional dimer (Nelson et al., 1998). It is not clear whether hydrophobic preproteins are imported at translocation contact sites of the TOM complex and the TIM22 complex in a manner similar to the import of hydrophilic matrix-targeted precursors via the TIM23 complex (Donzeau et al., 2000). It appears likely that cartier proteins cross the outer membrane in a partially folded form, exposing loops at the inner outlet of the TOM channel into the intermembrane space. The translocation of carriers in a loop formation may lead to a cooperative effect of the internal import signals which are subsequently recognized by the small Tim proteins of the intermembrane space (Wiedemann et al., 2001). The complexes of small Tim proteins may act like molecular chaperones that stabilize the precursors of hydrophobic inner membrane proteins in the aqueous environment of the intermembrane space in that particular conformation and guide them to the TIM22 complex. Thus, translocation of carrier proteins does not involve a soluble translocation intermediate in the intermembrane space (Adam et al., 1999). 2. Import of Tim23 into the Inner Membrane

The Tim8 and Tim13 also form a hetero-oligomeric 70 kDa complex in the intermembrane space. This complex is supposed to contain three molecules of each, Tim8 and Tirol3, but none of the other Tim proteins (Koehler et aL, 1999). The Tim8-13 complex is not required for the biogenesis of the mitochondrial carrier protein but rather affects import of noncarrier proteins of the inner membrane such as Tim23 (Kerscher et aL, 1997; Leuenberger et al., 1999; Paschen et al., 2000). As most other inner membrane proteins, the precursors of Tim23 and Timl7 contain internal signals, which mediate insertion of the precursors into the inner membrane in the presence of a membrane potential, AqJ (Davis et aL, 1998; Kaldi et al., 1998). The hydrophilic N-terminal domain of Tim23 contains, in addition, a targeting signal that mediates its import independent of A ~ (Kaldi et al., 1998). The TIM8.13 complex is proposed to stabilize the Tim23 precursor in a translocation-competent conformation in the intermembrane space,

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thereby facilitating its A~-dependent insertion into the inner membrane mediated by the TIM22 complex. Thus, the TIM8-13 complex and the related TIM9.10 complex have a different substrate specificity but appear to function in a similar manner. In yeast, the assistance of the TIM8.13 complex is not required, however, for import of Tim23 under all conditions (Paschen et al., 2000). Under normal growth conditions, the membrane potential is sufficient to drive import and membrane insertion of Tim23 even without the assistance of the TIM8.13 complex. Only when the membrane potential is low, was the TIM8.13 complex found to be necessary to accumulate Tim23 precursor at the inner face of the outer membrane where it can contact the TIM22 complex to facilitate membrane insertion. This situation differs from that in humans (see below).

E. MITOCHONDRIAL 'IIL~NSLOCASES IN MAMMALS In contrast to the rather comprehensive knowledge on fungal systems, relatively little is known about the import components in mammalian mitochondria. On the other hand, it was to be expected that the proteinimport systems of mammalian mitochondria are basically similar to that of S. cerevisiae or N. crassa. Precursors from fungi were observed to be imported into isolated mammalian mitochondria, and precursor protein from mammalian cells could be imported into fungal mitochondria. Furthermore, similar requirements for import in vitro were seen with mitochondria from both types of organisms. Mitochondrial preprotein imports depends on similar energy requirements. Several mammalian homologs of components of the yeast import system have been identified. These are the mammalian homologues of the TOM receptor components Tom20, Tom22, and Tom70 (Goping et al., 1995; Hanson et al., 1996; Alvarez-Dolado et al., 1999; Saeki et al., 2000), and of the central core component Tom40 (Suzuki et al., 2000). Most notably, some of the newly identified mammalian proteins, such as human Tom20, can act as functional homologues to the yeast components and complement the respective null phenotype. Furthermore, human Tom34 (Nuttall et al., 1997; Young et al., 1998; Chewawiwat et al., 1999), and metaxin (Armstrong et al., 1997; Abdul et al., 2000) have been desribed as components of the mitochondrial import machinery in the outer membrane. Both mammalian components have no apparent counterpart in fungi but appear to be involved in mitochondrial import pathways in mammalians. Recently, also human components of the inner membrane translocases, TIM23 and TIM22, have been identified and characterized in more detail (B6mer et al., 1996b; Ishihara and Mihara, 1998; Wada and

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al.

Kanwar, 1998; Bauer et al., 1999a,b; Rothbauer et aL, 2001). The structural composition of the TIM23 complex appears to be conserved from lower to higher eukaryotes; on the other hand, significant differences have been observed. In contrast to yeast, two Timl 7 homologues are expressed in mammalians, giving rise to two distinct functional TIM23 complexes in the inner mitochondrial membranes: Tim17a-Tim23 and Tim17b-Tim23 (Bauer et al., 1999a). The preservation of the gene indicates that the human TIM17 genes originated by duplication and subsequent translocation to another chromosome. The functional relevance of these structural differences is not clear. The expression of two distinct functional TIM23 translocases in higher eukaryotes might reflect the development of a higher complexity in the mitochondrial composition during evolution, and therefore the development of different requirements of preprotein import. Differences in the structural composition of the second inner membrane translocase, TIM22, have also been described. Whereas the human homolog of yeast Tim22 was recently identified (Bauer et al., 1999b), a Tim54 homologue appears not to be expressed in mammalian mitochondria. Most recently, the structural and functional analysis of the human TIM22 import pathway, in particular of the small zinc finger proteins of the Timl0 family, has allowed to elucidate the pathomechansim underlying a complex neurodegenerative syndrome.

III. Mitochondrial Biogenesisand Human Neurodegenerafive Diseases

A. DYSFUNCTIONOF MITOCHONDRIALPREPROTEINIMPORTAS A CAUSE OF PROGRESSIVE NEURODEGENERATION--MOHR-TRANEBJAERGSYNDROME

The small Tim components of the intermembrane space belong to an evolutionary conserved protein family from which more than 50 ORFs have been identified throughout the eukaryotic kingdom (Bauer et al., 1999b). Six members of this protein family were shown to be expressed in humans (Bauer et al., 1999b; Jin et al., 1999). Based on the sequence alignments, humans contain two Tim8 homologues (hTim8a, hTim8b), one Timl3 homologue, one Tim9 homologue, and two Timl0 homologues (hTiml0a, hTiml0b), but no obvious Timl2 homologue. All human homologues appear to be expressed in a wide range of adult and fetal human tissues (Bauer et al., 1999b). Similar to yeast, the human small Tim proteins form distinct oligomeric complexes in the intermembrane space ofmitochondria (Rothbauer et al., 2001). Human Tim8a is identical to DDP1, the deafness-dystonia peptide encoded on chromosome Xq22 (Jin et al., 1996; Koehler et al., 1999). Mutations

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in the DDP1 gene are associated with a severe X-linked neurodegenerative disorder, the Mohr-Tranebjaerg syndrome (MTS) (McKusick, no. 304700) (Tranebjaerg et al., 1995). The phenotype of MTS includes progressive posflingual sensorineural hearing loss, often in combination with a variety of neurological symptoms including dystonia, muscle weakness, dementia, and blindness. Most of the DDP1 mutations are loss-of-function mutations predicted to lead to an absent or a truncated gene product. So far, only one missense mutation was found, causing a cysteine to tryptophan exchange (C66W) within the Cys4 motif (Tranebjaerg et al., 2000). By analogy to the function of Tim8 and Timl3 in yeast, it was suggested that the Mohr-Tranebjaerg syndrome is a new type of mitochondrial disease caused by a defect in the biogenesis of the human TIM23 complex (Paschen et al., 2000). However, the TIM8.13 complex in yeast is not strictly required for the import of Tim23 (see above). A requirement of the TIM8.13 complex was only observed when membrane insertion of Tim23 was compromised (Paschen et al., 2000). If this is true also for the human DDP1.Timl3 complex, how can loss of DDP1 function in MTS patients lead to such a severe neurodegenerative phenotype? Recent data suggest, that the human DDPI.hTiml3 complex is functional in yeast. It rescues the growth defect observed at low temperature in the A8/A13 yeast deletion mutant (Paschen et al., 2000; Rothbauer et al., 2001). In contrast, expression of a mutant DDP1 carrying a C66W amino acid exchange (the only missense mutation observed in MTS patients) does not complement the yeast deletion phenotype (Hofmann et al., 2002). The C66W mutations presumably leads to a nonfunctional zinc finger (Hofmann et al., 2002). Studies on the mutant DDP1c66w revealed that it does not accumulate in the intermembrane space of mitochondria from patient cell lines (C. K6hler, personal communication). This suggests that the mutant DDP1 protein is not able to fold properly and is rapidly degraded; this also explains the full-blown clinical phenotype observed in a patient harboring the mutant C66W allele on the X chromosome. The human DDPI.hTiml3 complex facilitates the import of Tim23 precursor across the outer membrane at low A~P in a manner similar to yeast (Fig. 5). However, import of human Tim23 into isolated yeast mitochondria required the assistance of the DDPI-hTiml3 complex even when A ~ was high (Rothbauer et al., 2001). Under these conditions the import of yeast Tim23 is not dependent on the TIM8.13 complex. Apparently, import of human Tim23 into yeast mitochondria and into mammalian mitochondria requires a higher membrane potential than import of yeast Tim23. This is probably due to a weaker import signal in the C-terminal portion of human Tim23 (Paschen et al., 2000). The biogenesis of human Tim23 may

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FIG. 5. Role of the DDP1-hTim13 complex in the import of human Tim23 and proposed pathomechanism of the Mohr-Tranebjaerg syndrome. Import into intact mitochondria (left panel) : the Tim23 precursor (dashed line) is translocated across the TOM complex and trapped by the DDPI.hTiml 3 complex in the intermembrane space. The TIM22 complex interacts with the accumulated precursor and mediates efficient insertion into the inner membrane at normal levels of A ~ and with reduced efficiency at low levels of AqJ. Loss of DDP1 function (right panel): in the absence of the DDPI-hTiml3 complex (Mohr-Tranebjaerg syndrome), the hTim23 precursor cannot be trapped in the intermembrane space and accumulates bound to the receptors on the surface of the mitochondria (dashed line). Due to the reduced concentration of translocation intermediates in the intermembrane space, insertion of hTim23 into the inner membrane by the TIM22 complex is compromised.

therefore be more dependent on a functional DDPI-hTiml3 complex than biogenesis of yeast Tim23. It can be speculated that mutations in DDP1 could significantly affect the biogenesis of the TIM23 complex in humans. In the absence of a functional DDPl.hTim13 complex, the Tim23 precursor cannot be trapped in the intermembrane space of human mitochondria (Fig. 5). A direct interaction of the Tim23 precursor with the TIM22 complex might be rather inefficient and the equilibrium shifted toward retrograde translocation. Thus, membrane insertion might require multiple rounds of interaction of the TIM22 complex with the TOM-bound precursor of Tim23. Accordingly, the

IMPORT OF MITOCHONDRIAL PROTEINS

Mohr-Tranebjaerg syndrome may be primarily a result of an impaired import of Tim23 into the inner membrane (Fig. 5). Thus, Mohr-Tranebjaerg syndrome is considered the first example demonstrating that defects in the mitochondrial import machinery can lead to a mitochondrial disease, thereby suggesting a fundamentally new pathogenetic mechanism for progressive neurodegeneration. As the clinical features of MTS resemble typical defects in mitochondrial oxidative phosphorylation (OXPHOS), the underlying mechanism causing the disease phenotype may be similar, at least in part. The Tim23 is an essential component of the TIM23 complex, and it is required for the import of a variety of components necessary for the translocation, assembly, and integrity of the OXPHOS system of mitochondria. Therefore, it can easily be envisioned that a defect in targeting preproteins to the mitochondrial matrix may indirectly affect the mitochondrial OXPHOS activity and energy production by malfunctional shuttling of ATP or other metabolites required for functional integrity of mitochondria. This is supported by the fact that nerve cells, in particular those of the cochlea and the basal ganglia, are sensitive to insufficient ATP supply and many mitochondrial diseases cause neurological movement disorders and inner ear deafness.

B. DEFECTSOF QUALITYCONTROLOF MITOCHONDRIALINNERMEMBRANE PROTEINS-----HEREDITARYSPASTICPARAPLEGIA The biogenesis of mitochondria is not only dependent on the import and sorting of nuclear-encoded preproteins to their correct destination, but also on the removal of mistargeted, misfolded, or malfunctional preproteins, AAA-proteases are a conserved class of ATP-dependent proteases that mediate the degradation of integral membrane proteins in bacteria, mitochondria, and chloroplasts (Beyer, 1997; Langer et al., 2001). They combine proteolytic and chaperone-like activities, thereby forming a membrane-integrated quality-control system. Two proteolytic complexes are present in the mitochondrial inner membrane. These complexes are composed of homologous subunits but expose their catalytic sites to opposite membrane surfaces. The m-AAA-protease is active at the matrix side and is composed of Afg3 (also known as Ytal0) and Rcal (Ytal2) (Arlt et al., 1996). The i-AAA-protease, which contains Ymel, probably in a homooligomeric complex, faces the intermembrane space (Leonhard et al., 1996). Inactivation of AAA-proteases causes severe defects in various organisms. Recently, the disease gene of an autosomal recessive form of hereditary spastic paraplegia (HSP) was shown to encode a mitochondrial protein named paraplegin, which is highly homologous to the yeast AAA-proteases Afg3,

BAUER et al. R c a l , a n d Ymel. Patients with m u t a t i o n s in p a r a p l e g i n exhibit progressive spasticity o f the lower limbs d u e to d e g e n e r a t i o n o f corticospinal axons (Casari et al., 1998). I n yeast, inactivation o f Afg3 o r R c a l impairs b o t h d e g r a d a t i o n o f nonass e m b l e d i n n e r m e m b r a n e proteins as well as the assembly o f respiratory chain c o m p l e x e s a n d o f the ATP synthase (Paul a n d Tzagoloff, 1995; Arlt et al., 1996, 1998). Pleiotropic defects, i n c l u d i n g i m p a i r e d respiration a n d a b b e r a n t m i t o c h o n d r i a l m o r p h o l o g y , were also d e t e c t e d in yeast cells lacking the i-AAA-protease s u b u n i t Ymel (Thorsness et al., 1993). I n agreem e n t with the observed r e q u i r e m e n t o f AAA-proteases for respiratory c h a i n assembly in yeast, muscle biopsies f r o m patients h a r b o r i n g m u t a t i o n s in p a r a p l e g i n revealed m i t o c h o n d r i a l O X P H O S defects. Thus, an i m p a i r e d quality c o n t r o l o f m i t o c h o n d r i a l i n n e r m e m b r a n e proteins d u e to c o m p r o m i s e d c h a p e r o n e or protease f u n c t i o n leads to imp a i r e d O X P H O S f u n c t i o n a n d n e u r o d e g e n e r a t i o n in h u m a n s . Defects in paraplegin m a y cause an a c c u m u l a t i o n o f n o n a s s e m b l e d subunits o f respiratory c h a i n c o m p l e x e s o r ATP-synthase, a n d m a y p r o m o t e n u c l e a t i o n in neurodegeneration.

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