The Preprotein Translocase of the Inner Mitochondrial Membrane: Evolutionary Conservation of Targeting and Assembly of Tim17

J. Mol. Biol. (1996) 262, 389–395

COMMUNICATION

The Preprotein Translocase of the Inner Mitochondrial Membrane: Evolutionary Conservation of Targeting and Assembly of Tim17 Ulf Bo¨mer1, Joachim Rassow1, Nicole Zufall1, Nikolaus Pfanner1* Michiel Meijer2 and Ammy C. Maarse2 1

Institut fu¨r Biochemie und Molekularbiologie,Universita¨t Freiburg, Hermann-HerderStrabe 7, D-79104, Freiburg Germany 2

Institut for Molecular Cell Biology, BioCentrum Amsterdam, 1098 SM Amsterdam, The Netherlands

The preprotein translocase of the inner mitochondrial membrane has only been described in Saccharomyces cerevisiae to date. We report that the essential subunit Tim17 is highly conserved in evolution. The targeting and assembly of yeast Tim17 as well as that of human and Drosophila melanogaster Tim17 were characterized with isolated yeast mitochondria. Targeting signals in the mature protein direct the Tim17 precursors to the receptor Tom70 on the mitochondrial surface. In a membrane potential-dependent step the precursors insert into the inner membrane, adopt a characteristic topology and assemble with Tim23. The mechanisms of targeting and assembly were indistinguishable between the Tim17s from distinct organisms, indicating a high evolutionary conservation. 7 1996 Academic Press Limited

*Corresponding author

Keywords: mitochondria; protein transport; Tim17; Tim23

The mitochondrial membranes contain specific machineries for import of nuclear-encoded precursor proteins. The preprotein translocase of the outer membrane (Tom) forms a dynamic complex with at least nine distinct subunits, several of which function as receptors for preproteins, whereas other subunits form a general import pore in the outer membrane (Ku¨brich et al., 1995; Lithgow et al., 1995; Lill & Neupert, 1996; Pfanner et al., 1996; Schatz & Dobberstein, 1996; Bo¨mer et al., 1996). Three subunits of the preprotein translocase of the inner mitochondrial membrane were shown to be essential for life, Tim17, Tim23 and Tim44 (Maarse et al., 1992; Pfanner et al., 1994; Ryan & Jensen, 1995). Possible additional subunits are not yet characterized at a molecular level (Berthold et al., 1995; Blom et al., 1995). Tim17 and Tim23 are thought to represent the core subunits of the translocation channel of the inner membrane. Subunits of the Tom-machinery have been identified in various organisms such as Saccharomyces cerevisiae, Neurospora crassa, human, rat and This paper is dedicated to Professor Helmut Holzer on the occasion of his 75th birthday. Abbreviations used: Dc, membrane potential; Tim, translocase of inner mitochondrial membrane; PCR, polymerase chain reaction. 0022–2836/96/390389–07 $18.00/0

potato (summarized by Pfanner et al., 1996). In contrast, subunits of the Tim-machinery have only been reported for S. cerevisiae so far. Therefore we performed a databank search with the TFASTAprogram (Pearson, 1990) and found a complete open reading frame from Drosophila melanogaster and partial open reading frames from human, Caenorhabditis elegans and Arabidopsis thaliana that may encode proteins with similarity to S. cerevisiae Tim17. We cloned the human cDNA. Figure 1A shows the nucleotide sequence and predicted amino acid sequence. Since we report below that the protein derived from the human sequence as well as that derived from the D. melanogaster sequence are targeted to the inner mitochondrial membrane and assemble with the preprotein translocase, they are termed Tim17 according to the new uniform nomenclature of mitochondrial protein import (Pfanner et al., 1996). A comparison of the amino acid sequences of the entire proteins from human, D. melanogaster and S. cerevisiae and the partial sequences from C. elegans and A. thaliana (Figure 1B) revealed a similarity of 70 to 82% between the various organisms, including 46 to 62% identical amino acid residues. This includes the presence of four hydrophobic segments in Tim17 that were predicted to function as membrane 7 1996 Academic Press Limited

390 anchor sequences (Figure 1A; Ku¨brich et al., 1994; Ryan et al., 1994). The conservation of primary structure between Tim17s from different organisms is remarkably high compared to other membrane components of the mitochondrial protein import machinery, such as Tom70, Tom40, Tom22 and

Translocase of Inner Mitochondrial Membrane

Tom20. With those proteins, similarities of 46 to 70% and identities of 25 to 40% between different organisms (human, S. cerevisiae, Neurospora crassa) were observed (Kiebler et al., 1990; Steger et al., 1990; Schneider et al., 1991; Ramage et al., 1993; Lithgow et al., 1994; Moczko et al., 1994; Ho¨nlinger

Figure 1. Cloning of TIM17 and comparison of the primary structures of Tim17 from different organisms. A, cDNA sequence and derived amino acid sequence of human TIM17. A human (H.s.) cDNA sequence (accession number Z46191) with homology to part of S. cerevisiae TIM17 was amplified by polymerase chain reaction (PCR) using as a template DNA of a cDNA library obtained from total RNA of IL-lb stimulated HUVEC. The expected 304 bp PCR product, which contains 5'-flanking sequences and coding sequences for the amino terminus of human Tim17, was used as a probe to isolate a near full-length cDNA clone (lacking only eight codons for the amino terminus of Tim17) by colony hybridization of Escherichia coli harbouring the same cDNA library. This cDNA clone was then used to construct a full-length clone of human TIM17 by fusing it with the 304 bp PCR fragment at a common KpnI restriction site. The DNA sequence of the entire gene was determined (accession number X97544 at EMBL nucleotide sequence database). The derived amino acid sequence is given in the one letter code. The predicted four hydrophobic segments are underlined. B, Comparison of the predicted amino acid sequences of human Tim17, D. melanogaster (D.m.) Tim17 (accession number L35645), S. cerevisiae (S.c.) Tim17 (accession number X77796), part of C. elegans (C.e.) Tim17 (accession numbers D74639, D75891, D74501) and part of A. thaliana Tim17 (accession number T45278). Identical residues are boxed. We sequenced the chromosomal DNA of D. melanogaster TIM17 (from two independent PCR clones) and found a Leu to Phe substitution at position 133 of the predicted protein in comparison to the data base entry L35645. C, Comparison of the predicted amino acid sequences of S. cerevisiae Tim23 (accession number X74161) and A. thaliana Tim23 (accession number U18126). Identical residues are boxed.

Translocase of Inner Mitochondrial Membrane

391

Figure 2. Import of human (h), D. melanogaster (d) and S. cerevisiae (y) Tim17 into isolated mitochondria. A, Import into rat liver mitochondria. The coding sequence of human TIM17 was cloned into pGEM4 for use in in vitro transcription. hTim17 and yTim17 (Maarse et al., 1994) were synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine/[35S]cysteine (So¨llner et al., 1991). Freshly isolated rat liver mitochondria (90 mg protein per sample) in HMS (20 mM Hepes (pH 7.2), 220 mM mannitol, 70 mM sucrose) containing 2 mM NADH were incubated with the radiolabelled preproteins at 27°C for five minutes (samples 1), 15 minutes (samples 2), 30 minutes (samples 3) and 60 minutes (samples 4 and 5; Stoltz et al., 1995). In samples 5, the membrane potential was dissipated by addition of 1 mM valinomycin before the 27°C incubation. After the import reaction, mitochondria were treated with 200 mg/ml trypsin for 20 minutes at 0°C to remove preproteins which were not translocated across the mitochondrial membranes. After incubation with soybean trypsin inhibitor (30-fold weight excess) for ten minutes, mitochondria were pelleted for ten minutes at 16,000 g and washed with HMS. Analysis was performed by Tricine-SDS-PAGE and digital autoradiography using a phosphor imaging system. Twenty per cent of the amount of reticulocyte lysate used for an import sample was applied as standard (Std; sample 6). B, Import into S. cerevisiae mitochondria. Radiolabeled hTim17, dTim17 (coding region cloned into pGEM4 for in vitro transcription and translation in reticulocyte lysate), and yTim17 were incubated with isolated energized yeast mitochondria (10 mg protein per sample) in bovine serum albumin (BSA)-containing buffer (3%, w/v, BSA, 250 mM sucrose, 60 mM KCl, 5 mM MgCl2 , 5 mM malate, 2 mM ATP, 20 mM KPi , 10 mM Mops (pH 7.2) for five minutes (samples 1), ten minutes (samples 2), 15 minutes (samples 3) and 30 minutes (samples 4 and 5) at 25°C. In samples 5, the membrane potential was dissipated by addition of 1 mM valinomycin. Mitochondria were treated with 100 mg/ml proteinase K for 30 minutes at 0°C. After incubation with 2 mM phenylmethylsuphonyl fluoride (PMS), mitochondria were reisolated, washed with SEM (250 mM sucrose, 10 mM Mops (pH 7.2), 1 mM EDTA), and analyzed by Tricine-SDS-PAGE and digital autoradiography. Standard, 20% reticulocyte lysate (samples 6). C, Import of Tim17 into yeast mitochondria depends on Tom70. Radiolabeled hTim17, dTim17, and yTim17 were imported into mitochondria isolated from wild-type (WT) or tom70D S. cerevisiae strains (Moczko et al., 1994) as described in B. The incubation times were two minutes (samples 1 and 5), five minutes (samples 2 and 6) and ten minutes (samples 3, 4, 7 and 8).

et al., 1995; Goping et al., 1995; Seki et al., 1995; Hanson et al., 1996). While this manuscript was being completed, we found that a full length open reading frame from A. thaliana was homologous to Tim23. The degree of similarity to S. cerevisiae Tim23 was 40% with 26% identical residues (Figure 1C) and therefore in the range of similarity found with the Tom proteins. To study whether human Tim17 (hTim17) represented a mitochondrial protein, we synthesized the protein in rabbit reticulocyte lysate in the presence of [35S]methionine/[35S]cysteine and incubated it with isolated mammalian mitochondria (from rat liver). hTim17 was transported to a protease-protected location in a time-dependent manner (Figure 2A, upper panel, lanes 1 to 4). The import was inhibited by dissipating the membrane potential Dc

across the inner membrane by the potassium ionophore valinomycin (Figure 2A, upper panel, lane 5), demonstrating that the import of hTim17 required a membrane potential. The mature imported hTim17 was of the same size as the precursor (Figure 2A, upper panel, compare lanes 1 to 4 versus lane 6), indicating that hTim17 was not proteolytically cleaved during import. These import characteristics of hTim17 into mammalian mitochondria are comparable to that of import of yeast Tim17 (yTim17) into yeast mitochondria (Figure 2B, lower panel; Maarse et al., 1994). Moreover, these import characteristics were also found for heterologous import reactions, yTim17 into mammalian mitochondria (Figure 2A, lower panel), and hTim17 into yeast mitochondria (Figure 2B, upper panel). We also cloned D. melanogaster

392

Figure 3. Imported human, Drosophila and yeast Tim17 acquire the correct topology. A, Imported hTim17, dTim17, and yTim17 are not extracted at alkaline pH. hTim17, dTim17, and yTim17 were imported into energized yeast mitochondria (80 mg protein) for 60 minutes. After treatment with proteinase K, mitochondria were reisolated and resuspended with 1 ml 0.1 M Na2 CO3 . After incubation on ice for 30 minutes, centrifugation at 266,000 g for one hour yielded a pellet and supernatant fraction (trichloroacetate-precipitated). The samples were analyzed by Tricine-SDS-PAGE and Western transfer to polyvinylidene difluoride was performed. Imported proteins were analyzed by autoradiography and marker proteins (cytochrome b2 (cyt. b2 ), ATP/ADP carrier (AAC), and Tim44) were determined by immunodecoration. B, Formation of a characteristic fragment f of imported Tim17. Radiolabelled hTim17, dTim17, and yTim17 were incubated with yeast mitochondria for 15 minutes (samples 1 and 4) and 60 minutes (samples 2, 3, 5 and 6). Samples 1 to 3 were diluted with nine volumes of isotonic SEM-buffer, samples 4 to 6 with nine volumes of hypotonic swelling buffer (10 mM Mops (pH 7.2), 1 mM EDTA) to generate

Translocase of Inner Mitochondrial Membrane

T1M17, expressed the protein (dTim17) in vitro and imported it into yeast mitochondria. Transport to a protease-protected location depended on a membrane potential and did not involve proteolytic cleavage (Figure 2B, middle panel), in agreement with the import properties found with yTim17 and hTim17. The import efficiencies of the heterologous import reactions were close (60 to 80%) to those observed with the homologous import reactions. Which targeting pathway into mitochondria is used by Tim17? Maarse et al. (1994) reported that a pretreatment of yeast mitochondria with trypsin reduced the import of yTim17, indicating an involvement of proteinaceous component(s) on the mitochondrial surface. The analysis of various preproteins had suggested the presence of two targeting pathways that involve protease-accessible components: the Tom20-Tom22 receptor or the Tom70-Tom37 receptor (Ku¨brich et al., 1995; Lithgow et al., 1995; Lill & Neupert, 1996). Both receptors have a partially overlapping specificity. The Tom20-Tom22 pathway seems to be the major one; the majority of preproteins use Tom20-Tom22 for a large fraction of import and Tom70-Tom37 only for a smaller fraction of import. This is reflected by the differential inhibitory effect of antibodies against Tom20 or Tom70 on protein import (Alconada et al., 1995; Ku¨brich et al., 1995; Lithgow et al., 1995; Lill & Neupert, 1996). In a first screen, however, we found that anti-Tom70 antibodies, but not anti-Tom20 antibodies, revealed an inhibitory effect on import of Tim17 (yeast, human and Drosophila) into yeast mitochondria (not shown). To test directly whether import of Tim17 required Tom70, we compared the import into mitochondria from wild-type yeast and from a yeast strain lacking the receptor Tom70 (Steger et al., 1990; Moczko et al., 1994). Figure 2C shows a significant reduction of import of hTim17, dTim17 and yTim17 into mitochondria lacking Tom70 (lanes 5 to 7). We conclude that the import of Tim17 from yeast, human and Drosophila preferentially involves the Tom70 pathway. Tim17 thus joins the relatively small group of preproteins that prefer the Tom70 pathway. Included in this group are the precursors of the integral inner membrane proteins ADP/ATP carrier and phosphate carrier (Dietmeier et al., 1993; Lithgow et al., 1995), so it may be speculated that the predicted presence of hydrophobic segments in the Tim17s favours an interaction with Tom70. The imported Tim17s were resistant to extraction with sodium carbonate (pH 11.5; Figure 3A, columns 1 to 3). The treatment at pH 11.5 releases soluble proteins (e.g. cytochrome b2 of the intermembrane space; Figure 3A, column 4) and peripheral membrane proteins (e.g. Tim44; Figure 3A, column 6), whereas integral membrane proteins are retained in the membrane sheets (e.g. ADP/ ATP carrier; Figure 3A, column 5; Fujiki et al., 1982; mitoplasts. All samples were then treated with 100 mg/ml proteinase K before Tricine-SDS-PAGE.

Translocase of Inner Mitochondrial Membrane

Figure 4. Assembly of in vitro imported Tim17 with Tim23. A, Co-precipitation of imported Tim17 with Tim23. hTim17, dTim17, and yTim17 were imported into yeast mitochondria (160 mg protein) for 60 minutes at 25°C. The mitochondria were treated with proteinase K, reisolated and dissolved in lysis buffer (1% digitonin, 10% glycerol, 50 mM NaCl, 2 mM EDTA, 30 mM Hepes/KOH (pH 7.4). Unsolubilized material was removed by centrifugation (30 minutes at 100,000 g). The supernatants were divided into halves and added to antibodies directed against the N terminus of Tim23 (samples 4; Ku¨brich et al., 1994) or preimmune antibodies (samples 3), both prebound to Protein A-Sepharose. The samples were shaken at 8°C for 60 minutes. The Sepharose was washed with lysis buffer three times and the bound proteins were analyzed by Tricine-SDS-PAGE and digital autoradiography. As a control, 5% of the material added to the antibodies was directly analyzed (samples 1). In parallel, another sample was incubated with the radiolabelled preprotein after dissipation of the membrane potential (−Dc), and the treatment with proteinase K was omitted. Then co-immunoprecipitation with antibodies directed against the N terminus of Tim23 was performed (samples 5). A control sample 2, corresponding to the amount of sample 1, was applied (without protease treatment). B, Efficiency of co-precipi-

393 Blom et al., 1993). This indicates that the imported Tim17s were firmly embedded into mitochondrial membranes. Since the import of outer membrane proteins is independent of a Dc (Ku¨brich et al., 1995; Lithgow et al., 1995; Lill & Neupert, 1996), the requirement of import for a Dc shows that the Tim17s were transported to the inner membrane. We then asked whether the Tim17s imported in vitro to the mitochondrial inner membrane acquired the correct topology. Yeast mitochondria with imported yTim17 were subjected to swelling in order to rupture the outer membrane and open the intermembrane space. Added proteinase K then cleaved yTim17 to a smaller form of 014 kDa by removing 03 kDa (Figure 3B, lower panel, lanes 4 and 5). Formation of the proteolytic fragment was only possible when the import of yTim17 occurred in the presence of a Dc. yTim17 that associated with mitochondria in the absence of a Dc did not yield the fragment, but was further degraded (Figure 3B, lower panel, lanes 3 and 6). Ku¨brich et al. (1994) showed that the C terminus of yeast Tim17 is located on the intermembrane space side of the inner membrane and suggested that Tim17 spans the inner membrane four times with both termini being on the intermembrane space side. While the N terminus before the first hydrophobic segment is rather short, the C terminus comprises about 25 residues (3 kDa) beyond the last hydrophobic segment and is thus most likely cleaved off by proteinase K. The formation of the 14 kDa fragment can be considered as a specific criterion for assaying the correct insertion of Tim17 into the mitochondrial inner membrane. A comparable fragment was also observed for hTim17 (037 residues/4 kDa shorter than the full length protein) and dTim17 (042 residues/5 kDa shorter than the full length protein) imported into yeast mitochondria (Figure 3B, upper and middle panels, lanes 4 and 5), suggesting that they adopted the correct topology in the inner membrane. Tim17 forms a complex together with Tim23 (Berthold et al., 1995; Blom et al., 1995). We asked whether in vitro imported 35S-labelled Tim17 was able to assemble with pre-existing Tim23. Mitochondria with imported Tim17 were lysed with digitonin and subjected to co-immunoprecipitation with antibodies directed against Tim23. Thereby the 35S-labelled yTim17 was efficiently co-precipitated (Figure 4A, lower panel, lane 4). Also hTim17 and dTim17 (Figure 4A, upper and middle panels,

tation of imported Tim17 and control proteins with Tim23. Different preproteins, Tim17, ADP/ATP carrier (AAC) and F1-ATPase subunit b (F1 b), were imported into energized yeast mitochondria for 30 minutes at 25°C. After proteinase K treatment, co-immunoprecipitation with antibodies directed against Tim23 was performed as described in A. The fraction of yeast Tim17 co-precipitated with Tim23 was set to 1 (control).

394 lanes 4) assembled with yeast Tim23 with an efficiency close to that observed with yTim17 (Figure 4B, columns 1 to 3). Only background precipitation was observed with preimmune antibodies (Figure 4A, lanes 3). Tim17, which had been accumulated at mitochondria in the absence of a membrane potential (Figure 4A, lanes 2; no protease treatment), was not co-precipitated with Tim23 (Figure 4A, lanes 5), demonstrating that import of Tim17 into the inner membrane was required for its assembly with Tim23. There was a possibility that the observed co-precipitation did not reflect an assembly process, but was due to accumulation of transport intermediates at Tim23. This is unlikely as the efficiency of co-precipitation of imported Tim17 with Tim23 is an order of magnitude higher than that reported for co-precipitation of transport intermediates with Tim23 (Berthold et al., 1995). To get direct evidence we imported two other preproteins for the same time as Tim17 into isolated yeast mitochondria, namely the precursors of the integral inner membrane protein ADP/ATP carrier and the b-subunit of the F1-ATPase, a peripheral inner membrane protein. In both cases, the amount of 35 S-labelled protein co-precipitated with Tim23 was in the background range (Figure 4B, columns 4 and 5). We conclude that in vitro imported yTim17, hTim17 and dTim17 assemble with the preprotein translocase of the inner mitochondrial membrane. In summary, we report the first identification of a component of the preprotein translocase of the mitochondrial inner membrane in organisms different from S. cerevisiae. The primary structure of Tim17 is well conserved in evolution. The degree of similarities between human, D. melanogaster, C. elegans, S. cerevisiae and A. thaliana is significantly higher than that observed for any other component of the protein transport machinery of the mitochondrial membranes studied so far (several Tom proteins and the A. thaliana Tim23 identified here). This is also reflected in the targeting and assembly of Tim17 precursors from different organisms into isolated mitochondria. The mechanisms, including targeting via Tom70, Dc-dependent and presequence-independent insertion into the inner membrane, adoption of a typical topology and assembly with Tim23, are indistinguishable between preproteins from different organisms. These efficient heterologous import and assembly reactions of Tim17 are remarkable, as previous work showed that import of preproteins into mitochondria from other organisms (e.g. mammals and fungi) was often inefficient (Zara et al., 1992) and could lead to incorrect assembly (Schlossmann & Neupert, 1995). The high conservation of primary structure, targeting and assembly of Tim17 reported here and the finding that Tim17 is one of the few mitochondrial proteins essential for the viability of yeast (Maarse et al., 1994; Ryan et al., 1994) support the view that Tim17 plays a central role in mitochondrial protein import.

Translocase of Inner Mitochondrial Membrane

Acknowledgements We are grateful to Dr H. Clevers for D. melanogaster chromosomal DNA, Dr A. J. van Zonneveld for the human cDNA bank, Dr Carmen Brizio and Michaela Stoltz for rat liver mitochondria, and Dr Peter Dekker for critical comments on the manuscript. This work was supported by the Sonderforschungsbereich 388 and the Fonds der Chemischen Industrie.

References Alconada, A., Ga¨rtner, F., Ho¨nlinger, A., Ku¨brich, M. & Pfanner, N. (1995). Mitochondrial receptor complex from Neurospora crassa and Saccharomyces cerevisiae. Methods Enzymol. 260, 263–286. Berthold, J., Bauer, M. F., Schneider, H. C., Klaus, C., Dietmeier, K., Neupert, W. & Brunner, M. (1995). The MIM complex mediates preprotein translocation across the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system. Cell, 81, 1085–1093. Blom, J., Ku¨brich, M., Rassow, J., Voos, W., Dekker, P. J. T., Maarse, A. C., Meijer, M. & Pfanner, N. (1993). The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane. Mol. Cell. Biol. 13, 7364–7371. Blom, J., Dekker, P. & Meijer, M. (1995). Functional and physical interactions of components of the yeast mitochondrial inner-membrane import machinery (MIM). Eur. J. Biochem. 232, 309–314. Bo¨mer, U., Pfanner, N. & Dietmeier, K. (1996). Identification of a third yeast mitochondrial Tom protein with tetratrico peptide repeats. FEBS Letters, 382, 153–158. Dietmeier, K., Zara, V., Palmisano, A., Palmieri, F., Voos, W., Schlossmann, J., Moczko, M., Kispal, G. & Pfanner, N. (1993). Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria. J. Biol. Chem. 268, 25958–25964. Fujiki, Y., Hubbard, A. L., Fowler, S. & Lazarow, P. B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmatic reticulum. J. Cell Biol. 93, 97–102. Goping, I. S., Millar, D. G. & Shore, G. C. (1995). Identification of the human mitochondrial protein import receptor, huMas20p. Complementation of Dmas20 in yeast. FEBS Letters, 373, 45–50. Hanson, B., Nuttall, S. & Hoogenraad, N. (1996). A receptor for the import of proteins into human mitochondria. Eur. J. Biochem. 235, 750–753. Ho¨nlinger, A., Ku¨brich, M., Moczko, M., Ga¨rtner, F., Mallet, L., Bussereau, F., Eckerskorn, C., Lottspeich, F., Dietmeier, K., Jacquet, M. & Pfanner, N. (1995). The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins. Mol. Cell. Biol. 15, 3382–3389. Kiebler, M., Pfaller, R., So¨llner, T., Griffiths, G., Horstmann, H., Pfanner, N. & Neupert, W. (1990). Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature, 348, 610–616. Ku¨brich, M., Keil, P, Rassow, J., Decker, P. J. T., Blom, J., Meijer, M. & Pfanner, K. (1994). The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site. FEBS Letters, 49, 222–228.

Translocase of Inner Mitochondrial Membrane

Ku¨brich, M., Dietmeier, K. & Pfanner, N. (1995). Genetic and biochemical dissection of the mitochondrial protein-import machinery. Curr. Genet. 27, 393–403. Lill R. & Neupert, W. (1996). Mechanisms of protein import across the mitochondrial outer membrane. Trends Cell Biol. 6, 56–61. Lithgow, T., Junne, T., Suda, K., Gratzer, S. & Schatz, G. (1994). The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast. Proc. Natl Acad. Sci. USA, 91, 11973–11977. Lithgow, T., Glick, B. S. & Schatz, G. (1995). The protein import receptor of mitochondria. Trends Biochem. Sci. 20, 98–101. Maarse, A. C., Blom, J., Grivell, L. A. & Meijer, M. (1992). MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria. EMBO J. 11, 3619–3628. Maarse, A. C., Blom, J., Keil, P., Pfanner, K. & Meijer, M. (1994). Identification of the essential yeast protein MIM17, an integral mitochondrial inner membrane protein involved in protein import. FEBS Letters, 349, 215–221. Moczko, M., Ehmann, B., Ga¨rtner, F., Ho¨nlinger, A., Scha¨fer, E. & Pfanner, N. (1994). Deletion of the receptor Mom19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria. J. Biol. Chem. 269, 9045–9051. Pearson, W. R. (1990). Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183, 63–98. Pfanner, N., Craig, E. A. & Meijer, M. (1994). The protein import machinery of the mitochondrial inner membrane. Trends Biochem. Sci. 19, 368–372. Pfanner, N., Douglas, M. G., Endo, T., Hoogenraad, N. J., Jensen, R. E., Meijer, M., Neupert, W., Schatz, G., Schmitz, U. K. & Shore, G. C. (1996). Uniform nomenclature for the protein transport machinery of the mitochondrial membranes. Trends Biochem. Sci. 21, 51–52. Ramage, L., Junne, T., Hahne, K., Lithgow, T. & Schatz, G. (1993). Functional cooperation of mitochondrial protein import receptors in yeast. EMBO J. 12, 4115–4123.

395 Ryan, K. R. & Jensen, R. E. (1995). Protein translocation across mitochondrial membranes: what a long, strange trip it is. Cell, 83, 517–519. Ryan, K. R., Menold, M. M., Garett, S. & Jensen, R. E. (1994). SMS1, a high-copy suppressor of the yeast mas6 mutant, encodes an essential inner membrane protein required for mitochondrial protein import. Mol. Biol. Cell, 5, 529–538. Schatz, G. & Dobberstein, B. (1996). Common principles of protein translocation across membranes. Science, 271, 1519–1526. Schlossmann, J. & Neupert, W. (1995). Assembly of the preprotein receptor Mom72/Mas70 into the protein import complex of the outer membrane of mitochondria. J. Biol. Chem. 270, 27116–27121. Schneider, H., So¨llner, T., Dietmeier, K., Eckerskorn, C., Lottspeich, F., Tru¨lzsch, B., Neupert, W. & Pfanner, N. (1991). Targeting of the master receptor MOM19 to mitochondria. Science, 254, 1659–1662. Seki, N., Moczko, M., Nagase, T., Zufall, N., Ehmann, B., Dietmeier, K., Scha¨fer, E., Nomura, N. & Pfanner, N. (1995). A human homolog of the mitochondrial protein import receptor Mom19 can assemble with the yeast mitochondrial receptor complex. FEBS Letters, 375, 307–310. So¨llner, T., Rassow, J. & Pfanner, N. (1991). Analysis of mitochondrial protein import using translocation intermediates and specific antibodies. Methods Cell Biol. 34, 345–358. Steger, H. F., So¨llner, T., Kiebler, M., Dietmeier, K. A., Pfaller, R., Tru¨lzsch, K. S., Tropschug, M., Neupert, W. & Pfanner, N. (1990). Import of ADP/ATP carrier into mitochondria: two receptors act in parallel. J. Cell Biol. 111, 2353–2363. Stoltz, M., Rysavy, P., Kalousek, F. & Brandsch, R. (1995). Folding, flavinylation, and mitochondrial import of 6-hydroxy-d-nicotine oxidase fused to the presequence of rat dimethylglycine dehydrogenase. J. Biol. Chem. 270, 8016–8022. Zara, V., Palmieri, F., Mahlke, K. & Pfanner, N. (1992). The cleavable presequence is not essential for import and assembly of the phosphate carrier of mammalian mitochondria but enhances the specificity and efficiency of import. J. Biol. Chem. 267, 12077–12081.

Edited by M. Yaniv (Received 13 May 1996; received in revised form 11 July 1996; accepted 16 July 1996)