INTRODUCTION

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The mitochondrion has an elaborate set of translocons on the outer and inner membranes to mediate the import of proteins from the cytosol (19, 21, 25, 30). Most mitochondrial proteins are synthesized as cytosolic precursors containing a cleavable N-terminal presequence that directs their import into mitochondria via the general protein import pathway. The precursor is escorted through the cytosol by chaperones, and then the hetero-oligomeric translocase of the outer membrane (TOM) mediates translocation across the outer membrane. Several components function as receptors, while others form the translocation pore. After passage through the outer membrane, the Tim17p-Tim23p (Tim, for translocase of inner membrane) complex of the inner membrane, together with the ATP-dependent import motor Hsp70, Tim44p, and mGrpE, mediates translocation across the inner membrane. Finally, a number of soluble proteins in the matrix involved in the proteolytic maturation and folding of the imported proteins may be required to complete assembly (19, 21, 25, 30).

The mitochondrion has a separate import pathway for inner membrane proteins, with components residing in the intermembrane space and inner membrane (2, 16, 21). Proteins destined for the inner membrane are escorted by cytosolic chaperones and then pass through the TOM complex to the intermembrane space. The intermembrane space complexes Tim9p-Tim10p and Tim8p-Tim13p function as putative chaperones to transfer the hydrophobic precursors across the intermembrane space to an inner membrane machinery specialized for the insertion of membrane proteins (1, 13, 15, 32). The inner membrane complex consists of Tim12p, Tim18p, Tim22p, Tim54p, and a small fraction of Tim9p and Tim10p, which together form a 300-kDa complex (1, 11-13, 15, 17, 32). Components Tim9p, Tim10p, Tim12p, Tim22p, and Tim54p are essential for viability (1, 11-13, 15, 17, 32).

A typical protein imported by this pathway is the ADP/ATP carrier (AAC), which contains six membrane-spanning regions (22). AAC lacks a cleavable N-terminal targeting sequence, instead carrying targeting information in discrete regions throughout the polypeptide chain (5, 22). Yeast has about three dozen members of the mitochondrial metabolite carrier family (20). The TIM22 pathway probably imports all of these as well as many other integral proteins of the mitochondrial inner membrane, including the import components Tim22p and Tim23p (3, 5, 18).

The small Tim proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) are approximately 25% identical and 40 to 50% similar, yet Tim9p partners exclusively with Tim10p and Tim8p partners with Tim13p in soluble intermembrane space complexes (1, 3, 14, 15). The Tim9p-Tim10p complex is 10-fold more abundant than the Tim8p-Tim13p complex (1, 14, 15, 32). Approximately 5% of Tim9p and Tim10p is associated with the 300-kDa Tim18p-Tim22p-Tim54p complex at the inner membrane, whereas 95% is soluble in the 70-kDa intermembrane space complex (14, 15). Tim9p and Tim10p bind to translocation intermediates of the mitochondrial carrier family, Tim17p and Tim22p, whereas Tim8p and Tim13p bind to Tim23p (3, 5, 18), suggesting that the battery of small Tim proteins may have different substrate specificities.

While previous studies have focused on investigating direct interactions with translocation intermediates, we investigated the role of the small Tim proteins by using a genetic approach. We constructed a yeast strain that is deficient in both 70-kDa Tim9p-Tim10p and Tim8p-Tim13p complexes and is kept viable by a functional version of Tim9p, designated Tim9^S67Cp, which was identified as a multicopy suppressor of a conditional tim10 mutant (15). Characterization of this strain revealed it was viable and that protein import into isolated mitochondria proceeded at a slower rate. Tim9p and Tim10p were bound to the inner membrane and seemingly were not present in a soluble 70-kDa complex. Both Tim9p and Tim10p could be cross-linked to an AAC translocation intermediate, indicating that they mediate protein import.

	MATERIALS AND METHODS

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Plasmids and strains. Standard genetic techniques were used for growth, manipulation, and transformation of yeast strains (8, 31). The Saccharomyces cerevisiae strains used in this study are listed in Table 1. CK13 and CK14 are strains that contain the temperature-sensitive tim10-1 allele (designated as tim10-1 in Fig. 1) and have been previously described (15). The strain CK18 contains allele tim9^199AT coding for protein Tim9^S67Cp and previously was identified because it restored growth at the restrictive temperature of 37°C for temperature-sensitive tim10-1 and tim12-1 strains (15). The strain CK26 (designated as Tim9^S67C) was constructed by transforming a centromeric plasmid harboring tim9^199AT into diploid strain CK22, in which one TIM9 allele is disrupted, followed by sporulation and auxotrophic selection.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Strain 70k grows similar to the parental strain. The 70k and tim10-1 strains and the parental (wild-type [WT]) strain (Table 1) were grown to mid-log phase at 25°C in liquid YPD. Cultures were serially diluted by a factor of 3 and spotted onto YPD plates. Plates were incubated for 3 days at 25 or 37°C. WT, GA74-1A (10); tim10-1, temperature-sensitive tim10-1 allele integrated into LEU2 locus (15); 70k, strain in which 70-kDa Tim9p-Tim10p and Tim8p-Tim13p complexes are not detectable by immunoblot analysis.

199AT

Import of radiolabeled proteins into isolated mitochondria and cross-linking studies. Mitochondria were purified from lactate-grown yeast cells (7) and assayed for in vitro protein import as described previously (24). Proteins were synthesized in a rabbit reticulocyte lysate in the presence of [³⁵S]methionine after in vitro transcription of the corresponding gene by SP6 or T7 polymerase. The reticulocyte lysate containing the radiolabeled precursor was incubated with isolated mitochondria at the indicated temperatures in import buffer (1-mg/ml bovine serum albumin, 0.6 M sorbitol, 150 mM KCl, 10 mM MgCl₂, 2.5 mM EDTA, 2 mM ATP, 2 mM NADH, 20 mM K⁺-HEPES [pH 7.4]). Where indicated, the potential across the mitochondrial inner membrane was dissipated with 1 µM valinomycin. Nonimported radiolabeled proteins were removed by treatment with 100 µg of trypsin or 50 µg of proteinase K per ml for 15 to 30 min on ice; trypsin was inhibited with 200 µg of soybean trypsin inhibitor per ml, and proteinase K was inhibited with 1 mM phenylmethylsulfonyl fluoride (PMSF), respectively.

Blue native gel electrophoresis. Mitochondria (2.5 mg/ml) were solubilized in a mixture containing 20 mM K⁺-HEPES (pH 7.4), 50 mM NaCl, 10% glycerol, 2.5 mM MgCl₂. 1 mM EDTA, 0.16% n-dodecylmaltoside (Boehringer Mannheim) for 30 min on ice. Insoluble material was removed by centrifugation at 100 000 × g for 10 min, and the solubilized proteins were analyzed by blue native gel electrophoresis on a 6 to 16% linear polyacrylamide gradient (4, 27, 28).

Coimmunoprecipitation assays. Monospecific antibody (10 to 20 µl per mg of mitochondria) against Tim12p was bound to protein A-Sepharose (30 µl wet volume per mg of mitochondria; Amersham Pharmacia Biotech) for 1 h in 1.0 ml of wash buffer (20 mM HEPES-KOH [pH 7.4], 0.2 M sucrose, 50 mM NaCl, 1 mM PMSF). The beads were washed two times to remove unbound antiserum. Mitochondria were solubilized as described for blue native gel electrophoresis and incubated with the antibody-bound protein A-Sepharose beads by gentle rotation for 2 h at 4°C. After the beads had been washed twice with wash buffer, bound proteins were extracted at 65°C with sodium dodecyl sulfate (SDS)-containing sample buffer and analyzed by Tricine-SDS-polyacrylamide gel electrophoresis (PAGE).

Miscellaneous. Submitochondrial localization of proteins was determined as described previously (6). Mitochondrial proteins were analyzed by SDS-PAGE with a 10 or 16% polyacrylamide gel and a Tricine-based running buffer (29). Proteins were detected by immunoblotting with nitrocellulose or polyvinylidene difluoride (PVDF) membranes and visualization of immune complexes with ¹²⁵I-protein A. Protein concentration was assayed by the bicinchoninic acid method (Pierce) with bovine serum albumin as the standard.

	RESULTS

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A yeast strain with a minimal complement of small Tim proteins is viable. Tim9p and Tim10p potentially have multiple roles in mediating the import of inner membrane proteins because they are partner proteins in a soluble 70-kDa complex in the intermembrane space and components of a 300-kDa inner membrane complex with Tim12p, Tim18p, Tim22p, and Tim54p. This raises the question of where the essential role of Tim9p and Tim10p isin the intermembrane space or at the inner membrane? Furthermore, the intermembrane space contains the distinct 70-kDa Tim8p-Tim13p complex, which is not essential for viability. We have used a genetic approach to investigate whether the 70-kDa complexes are essential to mediate protein import or whether they facilitate import in a manner similar to the outer membrane receptors on the TOM complex.

199AT

199AT

199AT

The yeast strain 70k lacks the soluble 70-kDa intermembrane space complexes. In strain 70k, the abundance of the Tim proteins was quantitated by immunoblotting to determine the relative abundance of the individual components. Mitochondria were purified from strain 70k and the parental strain grown at 37°C, and increasing amounts of mitochondrial protein were separated by SDS-PAGE as indicated in Fig. 2. Because 37°C is the nonpermissive temperature for the tim10-1 mutant, Tim10p should be nonfunctional. The amount of Tim10p was decreased by 94% (Fig. 2A); quantitation was performed by scanning laser densitometry (data not shown). In contrast, the levels of abundance of mitochondrial proteins porin, Tim23p, Tim44p, and AAC did not differ significantly (Fig. 2B). Previously, we estimated that mitochondria contain approximately 120 pmol of Tim10p and Tim9p per milligram of total mitochondrial protein (15). Given this decrease, the 70k mitochondria contain approximately 5 pmol of Tim10p/mg of mitochondria. Neither Tim8p nor Tim13p was detected by immunoblotting (Fig. 2C), because deletion of TIM8 leads to loss of the Tim8p-Tim13p complex (14). The abundance of Tim9p, Tim12p, and Tim54p was decreased by approximately 50%, and that of Tim22p was decreased by approximately 75%. The steady-state levels of Tim23p and AAC, substrates of the TIM22 import pathway, were not significantly affected in strain 70k mitochondria compared to wild-type mitochondria. This observation suggests that Tim23p and AAC seemingly are imported efficiently in vivo or, alternatively, may have an increased stability in strain 70k.

View larger version (57K): [in this window] [in a new window]   FIG. 2.   Abundance of the TIM22 import components is decreased in strain 70k. (A) The parental strain (wild type [WT]) and strain 70k were grown at 37°C in lactate medium. The mitochondria were isolated, and aliquots corresponding to total micrograms of mitochondrial protein (µg prot) were analyzed by SDS-PAGE and immunoblotting with rabbit antisera monospecific for Tim10. Blots were treated with 125I-protein A and subjected to autoradiography. (B) As in panel A, except that equal amounts (50, 100, and 150 µg) of mitochondrial proteins are loaded. Antibodies are listed on the left. AAC, ADP/ATP carrier. (C) As in panel A, except that equal amounts of mitochondrial proteins are loaded.

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Strain 70k lacks the soluble Tim9-Tim10 70-kDa complex. (A) Mitochondria from the parental (wild type [WT]) strain (10) and the 70k strain (70k) were solubilized in 0.16% n-dodecylmaltoside and subjected to blue native gel electrophoresis (6 to 16% acrylamide) (27). Tim9 and Tim10 were detected by immunoblotting and incubation with 125I-protein A. (B) Blue native gel electrophoresis was performed as in panel A; the blot was incubated with monospecific sera against Tim22 and Tim54. In the immunoblot with anti-Tim22p antibody (-Tim22p), bands located above and below the 300-kDa band resulted from proteins that cross-react with the antisera (17).

View larger version (97K): [in this window] [in a new window]   FIG. 4.   Tim22 and Tim12 remain associated in strain 70k. Mitochondria from the parental (wild type [WT]) strain and the 70k strain (70k) were solubilized in 0.16% n-dodecylmaltoside. The lysate was subjected to immunoprecipitation with protein A-Sepharose beads containing immobilized rabbit IgGs monospecific for Tim12p. After centrifugation, 200 µg each of total (T), unbound (S), and bound (B) proteins was analyzed by SDS-PAGE and immunoblotting for Tim54p, Tim22p, Tim12p, Tim9p, and Tim10p.

View larger version (76K): [in this window] [in a new window]   FIG. 5.   Tim9p and Tim10p remain associated with the inner membrane in strain 70k. Isolated mitochondria from the parental (wild type [WT]) strain and the 70k strain were incubated in 20 mM HEPES-KOH (pH 7.4) and the indicated sorbitol concentrations (0.6 to 0.06 M) at 4°C for 30 min, followed by addition of 1 mM phenylmethylsulfonyl chloride (PMSF) to lyse the outer membrane. After centrifugation, the supernatant (Sup) and pellet (Pel) were analyzed by SDS-PAGE and immunoblotting for Tim9p, Tim10p, and Tim12p (pellet fraction only). To confirm that the intermembrane space contents were released, proteinase K was added at 50 µg/ml (PEL + PK). In a separate control (unpublished data), the effectiveness of the protease was verified by addition of Triton X-100.

Tim9p and Tim10p mediate import of AAC in strain 70k mitochondria. In mitochondria derived from the temperature-sensitive tim10-1mutant, the abundance and rate of in vitro protein import of the carrier proteins are decreased by 95% (13). Because Tim23p and AAC steady-state levels were not significantly reduced in strain 70k, the import of Tim23p and AAC may not be defective in vivo. For precursors AAC and Tim23p, we tested the rate of in vitro import into isolated mitochondria followed by protease treatment and carbonate extraction to confirm that AAC and Tim23p were inserted into the inner membrane (Fig. 6). Import of Tim23p and AAC was two- to threefold slower in 70k mitochondria than in wild-type mitochondria. Mitochondria from strain 70k thus are able to import substrates of the TIM22 import pathway.

View larger version (38K): [in this window] [in a new window]   FIG. 6.   Strain 70k mitochondria import inner membrane proteins AAC and Tim23p, but at a decreased rate. In separate experiments, radiolabeled AAC and Tim23p were synthesized in vitro and incubated for 1, 2, and 4 min at 25°C in the presence or absence of a membrane potential () with wild-type (WT) or 70k mitochondria. Samples were treated with proteinase K to remove nonimported precursor, followed by addition of 1 mM PMSF. Samples were analyzed by SDS-PAGE and fluorography. S, standard (10% of the radioactive precursor added to each assay). The import rate was quantitated by densitometry, with 100% being set as the amount of precursor imported into wild-type mitochondria after 4 min.

View larger version (58K): [in this window] [in a new window]   FIG. 7.   Tim9p and Tim10p, but not Tim12p and Tom40p, can be cross-linked to an AAC translocation intermediate in strain 70k. (A) Radiolabeled AAC was synthesized in vitro and imported into wild-type (WT) or 70k mitochondria. A fraction of the import reaction mixture was removed as an untreated control (BMH), and the remainder was subjected to cross-linking with 0.5 mM BMH for 5 min on ice. After quenching, an aliquot was removed for direct analysis (+BMH). The remainder was denatured with SDS, and equal aliquots were incubated with protein A-Sepharose beads containing immobilized rabbit IgGs monospecific for Tim9p (9), Tim12p (12), and Tom40p (40). After centrifugation, bound proteins were eluted with SDS-containing sample buffer and analyzed by SDS-PAGE and fluorography. The arrow marked "AAC" denotes the position of monomeric AAC. S, standard (as defined in the Fig. 6 legend). (B) As in panel A, except that antibodies against Tim10p were used for immunoprecipitation. (C) As in panel A, with isolated mitochondria from strain Tim9S67C, which expresses Tim9S67Cp, Tim8p, Tim10p, and Tim13p (Table 1), and wild-type mitochondria. Antibodies against Tim9p were used for immunoprecipitation (IP). (D) As in panel B, except that the cross-linker MBS (0.1 mM) was used.

View larger version (50K): [in this window] [in a new window]   FIG. 8.   The cross-linked AAC import intermediate is protected by the outer membrane in strain 70k mitochondria. In vitro import and cross-linking with radiolabeled AAC into strain 70k mitochondria were performed as in Fig. 7A, followed by treatment with 100 µg of trypsin per ml on ice for 30 min. After the addition of 200 µg of soybean trypsin inhibitor (Tryp) per ml, immunoprecipitation (IP) with protein A-Sepharose beads containing immobilized rabbit IgGs monospecific for Tim9p (9) was performed as in Fig. 7A. S, standard (as defined in the legend to Fig. 6).

	DISCUSSION

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Given the complexity of the TIM22 import pathway for inner membrane proteins, we have used a genetic approach to investigate whether the 70-kDa complexes are essential for mediating protein import or merely facilitate import, in a manner similar to the outer membrane receptors of the TOM complex. The TIM22 import pathway contains two soluble 70-kDa complexes, Tim9p-Tim10p and Tim8p-Tim13p, and the 300-kDa complex at the inner membrane, composed of Tim12p, Tim18p, Tim22p, and Tim54p with a fraction of Tim9p and Tim10p (1, 11-13, 15, 17, 32). We have constructed a strain, 70k, in which the soluble 70-kDa complexes are not detectable. Deletion of TIM8 results in loss of the Tim8p-Tim13p complex, and heat treatment at 37°C inactivates Tim10p. Instead, Tim9^S67Cp seemingly maintains viability because this strain grows at a rate similar to the parental strain. Therefore, the reduced complement of small Tim proteins does not result in a decreased rate of growth. Although the rate of protein import for Tim23p and AAC into isolated 70k mitochondria is decreased by two- to threefold, protein import in vivo seemingly is not affected. Similar discrepancies between in vivo and in vitro protein import rates coupled with no observed differences in growth rate have been observed previously (33); alternatively, Tim23p and AAC may have increased stability in strain 70k. Taken together, the 70-kDa complexes seemingly facilitate import, perhaps in a manner similar to that of the outer membrane receptors of the TOM complex.

Tim9p and Tim10p in mitochondria from strain 70k remain associated with the inner membrane. While immunoprecipitation experiments failed to show that they were in a complex with Tim12p and Tim22p, they may be transiently associated with the TIM22 complex. In contrast, the abundance of Tim54p in strain 70k is not affected. Rather, Tim54p is in a smaller complex of approximately 140 kDa, as shown previously in a tim22 temperature-sensitive mutant and tim18 strain (17, 18).

Neupert and colleagues have shown that AAC can be arrested in the TOM complex by protease digestion (5), while Pfanner and colleagues reported that a protein generated by fusion between AAC and dihydrofolate reductase can be stably arrested with components of the TOM and TIM22 complexes (26). In our study, attempts to identify a translocation intermediate associated with the TOM complex or with Tim12p and Tim22p were not successful. Rather, the AAC translocation intermediate from strain 70k cross-linked with high specificity to Tim9p and Tim10p. The AAC translocation intermediate in strain 70k most likely is associated with the inner membrane, bound to Tim9p and Tim10p, because it was protected from exogenous protease and Tim9p and Tim10p were associated tightly with the inner membrane. Taken together, characterization of strain 70k strongly suggests that the 70-kDa Tim8p-Tim13p and Tim9p-Tim10p complexes are not essential to mediate protein import. Further genetic analysis will provide insights into how the small Tim proteins cooperate with the subunits of the TIM22 complex and TOM complex.