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Molecular and Cellular Biology, July 2008, p. 4251-4260, Vol. 28, No. 13
0270-7306/08/$08.00+0     doi:10.1128/MCB.02216-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Assembly Pathway of the Mitochondrial Carrier Translocase Involves Four Preprotein Translocases ^,

Karina Wagner,^1,2 Natalia Gebert,^1,2 Bernard Guiard,³ Katrin Brandner,¹ Kaye N. Truscott,^1,4 Nils Wiedemann,¹ Nikolaus Pfanner,^1* and Peter Rehling^1,5*

Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg, Germany,¹ Fakultät für Biologie, Universität Freiburg, D-79104 Freiburg, Germany,² Centre de Génétique Moléculaire, CNRS, 91190 Gif-sur-Yvette, France,³ Department of Biochemistry, La Trobe University, Melbourne 3086, Australia,⁴ Abteilung für Biochemie II, Universität Göttingen, D-37073 Göttingen, Germany⁵

Received 14 December 2007/ Returned for modification 28 February 2008/ Accepted 25 April 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.

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The majority of mitochondrial proteins are nucleus encoded and imported into mitochondria through protein translocase complexes (6, 7, 17, 25, 29, 33, 42). The translocase of the outer membrane (TOM complex) is the general entry gate for mitochondrial precursor proteins. Two translocases of the inner membrane (TIM), the presequence translocase (TIM23 complex) and the twin-pore carrier translocase (TIM22 complex), mediate signal-selective transport of precursor proteins. While the TIM23 complex translocates the majority of substrates into the matrix and inserts only a limited number of substrates into the inner membrane (7, 10, 13, 17, 25, 26, 40), the TIM22 complex is dedicated to the insertion of multispanning hydrophobic proteins into the inner membrane, including a large number of metabolite carriers (7, 17, 25, 27, 29, 46). The TIM22 complex is a voltage-dependent 300-kDa complex with three membrane-integral subunits, Tim18, Tim22, and Tim54. Tim22 forms the voltage-sensitive channels of the twin-pore translocase (21, 30). Tim54 was shown to play a role in the assembly of a protease complex (Yme1) of the inner membrane, yet the molecular mechanism of its action has not been elucidated (12). Thus, the molecular functions of Tim54 and Tim18 in the TIM22 complex are unknown (6, 7, 17, 25, 29).

The precursors of metabolite carriers are not directly transferred from the TOM complex to the TIM22 complex, but the TIM10 translocase complex of the intermembrane space binds to the precursors and functions in a chaperone-like manner to guide them through the aqueous space between outer and inner membranes. The hexameric TIM10 translocase is formed by the family of small Tim proteins. The soluble complex consists of three copies of Tim10 and three copies of Tim9 (41). A fraction of small Tim proteins, including Tim9, Tim10, and the homolog Tim12, associate with the TIM22 complex, forming a membrane-associated TIM10 chaperone. It is unknown which Tim subunits mediate the contact between the TIM10 chaperone and the membrane-integral portion of the TIM22 complex.

All subunits of the TIM22 complex are encoded in the nucleus and synthesized in the cytosol. Initial analysis of the biogenesis of TIM22 subunits has indicated that the precursors of Tim18 and Tim54 proteins utilize amino-terminal targeting signals and are imported via the presequence pathway (TIM23 complex) (15, 16, 19, 22). In contrast, Tim22 lacks an amino-terminal presequence and was proposed to be imported along the carrier pathway (22, 23, 35).

Proper assembly of inner mitochondrial membrane complexes is critical for mitochondrial function since this membrane is pivotal for cellular energy conversion through oxidative phosphorylation. It is crucial for the cell to assemble the protein complexes that reside in the inner membrane in a manner that excludes an uncontrolled flux of ions across the membrane in order to prevent a breakdown of the electrochemical proton gradient. This is especially true for protein complexes that contain channel-forming subunits such as the preprotein translocase complexes. However, it is currently unknown how the TIM complexes are assembled from newly imported subunits and if the assembly to oligomeric complexes is coupled to the import process.

We have dissected the in organello assembly pathways of all membrane-integral subunits of the TIM22 complex by establishing an efficient native system. We show here that at different steps of the TIM22 complex biogenesis pathway, four translocases are involved. Remarkably, each precursor follows a different assembly route. This involves a new posttranslocational function of the TIM10 complex. Moreover, we obtained evidence for a role of Tim18 in the assembly of the TIM22 complex and for cooperation of Tim54 with Tim10. We propose that the uncoupling of membrane insertion of subunits from their subsequent oligomeric assembly promotes efficient biogenesis of translocase complexes.

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Yeast strains, growth conditions, and isolation of mitochondria. All yeast strains used in this study were derivatives of the Saccharomyces cerevisiae strain YPH499. Temperature-sensitive alleles tim22-14 (YPH499 22-M4), tim54-11 (YPH-BG-54-1-1), tim54-16 (YPH-BG-54-1-6) and tim10-2 (YPH499 10-71-1) were generated by error-prone PCR (38). The tim18 strain was generated by homologous recombination of a kanMX6 cassette into the TIM18 locus. Liquid yeast cultures for the isolation of mitochondria were grown in YPG medium (1% yeast extract, 2% Bacto peptone, and 3% glycerol) at 30°C for the tim22-14, tim18, and PRY19 (tim18_ProtA) (30) mutants and the corresponding wild-type strains or at 24°C for all other temperature-sensitive strains. Isolation of mitochondria was performed essentially as described previously (24).

Import of radiolabeled precursor proteins. For in vitro transcription and translation, the open reading frames encoding Tim22, Tim18, and Tim54 were cloned into the pGEM4Z vector (Promega), downstream of either the Sp6 promoter (Tim22 and Tim18) or the T7 promoter (Tim54) (22). Radiolabeling of precursor proteins with [³⁵S]methionine was performed with the TNT Sp6 quick coupled transcription/translation system or the TNT T7 coupled reticulocyte lysate system (Promega). Import of radiolabeled precursor proteins was performed essentially as described previously (44), and the proteins or complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or blue native electrophoresis, respectively. Digital autoradiography was utilized for detection.

Blue native electrophoresis. Mitochondria were solubilized under nondenaturing conditions in digitonin-containing buffer (1% digitonin, 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF]) for 30 min at 4°C and then centrifuged for 10 min at 16,000 x g. After addition of 10x loading dye (5% Coomassie brilliant blue G-250, 500 mM -amino n-caproic acid in 100 mM Bis-Tris [pH 7.0]) to the supernatant, the samples were separated on a 6 to 16.5% gradient blue native gel (4, 5). For Western blot analysis, the proteins were transferred to a polyvinylidene difluoride membrane and proteins of interest were labeled with the appropriate antibodies, incubated with a horseradish-conjugated secondary antibody, and detected with the ECL enhanced chemiluminescence detection system (GE Healthcare).

Antibody shift/antibody depletion analysis. After import, mitochondria were swollen in 30 mM sucrose, 1 mM EDTA, and 10 mM morpholinepropanesulfonic acid (MOPS)-KOH (pH 7.2), and antisera against Tim22 and Tim18 or bovine serum albumin (BSA) as a negative control were added for binding to the protein complexes. Samples were incubated for 45 min on ice and subsequently solubilized in digitonin-containing buffer. Binding of antibodies to outer membrane protein complexes (Tom40 or porin) and inner membrane protein complexes (Tim23 or Tim12) was performed during solubilization of mitochondria in digitonin buffer by adding the appropriate antiserum. Protein complexes were separated by blue native electrophoresis and analyzed by autoradiography (4, 32). For antibody-depletion analysis, solubilized mitochondria were incubated with purified lyophilized antibodies (anti-Tim10 and anti-Atp20) for 5 min followed by the addition of protein A-Sepharose and a 30-min incubation at 4°C. Protein A-Sepharose was removed by centrifugation, and the supernatant was analyzed by blue native electrophoresis and digital autoradiography.

Pulse-chase import experiment. Radiolabeled proteins were imported for 5 min at 25°C into mitochondria (75-µg protein amount) in import buffer (3% BSA, 250 mM sucrose, 80 mM KCl, 5 mM MgCl₂, 2 mM KH₂PO₄, 5 mM methionine, 10 mM MOPS-KOH [pH 7.2]) in the presence of 2 mM ATP and NADH. To remove nonimported radiolabeled precursor proteins, samples were centrifuged (10 min, 4°C, 16,000 x g) and mitochondria were resuspended in fresh import buffer. The chase reaction was carried out by incubation for 20 min at different temperatures (25°C, 30°C, and 37°C). After the chase reaction, samples were subjected to blue native electrophoresis.

Carbonate extraction. After import, mitochondria were resuspended in 0.1 M sodium carbonate (pH 10.8 to 11.5) and incubated for 30 min on ice. Pellet and supernatant fractions were separated by centrifugation (60 min, 4°C, 100,000 x g). Upon trichloracetic acid precipitation of supernatant and total, all samples were subjected to SDS-PAGE.

Chemical cross-linking. Mitochondria (0.5 mg protein per lane) from PRY19 cells (30) that contain Tim18_ProtA were resuspended in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH [pH 7.2]) and incubated with ethylene glycol bis[succinimidylsuccinate] for 1 h at 16°C. Following quenching for 25 min on ice, samples were washed with SEM buffer, resuspended in digitonin buffer (0.8% digitonin, 20 mM Tris-HCl [pH 7.4], 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1 mM PMSF), and solubilized for 30 min at 4°C. After a clarifying spin, immunoglobulin G (IgG)-Sepharose was added to the supernatant and the mixture was incubated for 120 min at 4°C. Bound proteins were washed with digitonin buffer, eluted by treatment with tobacco etch virus protease for 90 min at 16°C, and separated by SDS-PAGE. For combined import, chemical cross-linking, and immunoprecipitation, [³⁵S]methionine-labeled Tim54 was imported into Tim18_ProtA mitochondria for 1 h at 35°C. After addition of ethylene glycol bis[succinimidylsuccinate] and incubation for 90 min at 16°C, the reactions were quenched for 10 min on ice. Solubilization was done in digitonin buffer (0.8% digitonin, 20 mM Tris-HCl [pH 7.4], 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, 1 mM PMSF) for 15 min at 4°C. Mitochondrial extracts were incubated with IgG-Sepharose for 1 h at 4°C for complex purification. After washing in digitonin buffer, elution was performed using SDS sample buffer lacking bromophenol blue (10 min at room temperature). Upon heating to 95°C for 5 min and dilution with sample buffer that contained 0.5% (wt/vol) Triton X-100 but lacked bromophenol blue and SDS, samples were subjected to immunoprecipitation with anti-Tim9, anti-Tim10, or anti-Tim12 antibodies covalently coupled to protein A-Sepharose and incubated for 1 h at room temperature. Protein A-Sepharose was sedimented by centrifugation, and bound proteins were analyzed by SDS-PAGE and digital autoradiography.

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Assembly of Tim18, Tim22, and Tim54 into the carrier translocase. To analyze the biogenesis of the TIM22 complex, we synthesized and radiolabeled the precursor of the core component Tim22 in vitro. The precursor was imported into isolated and energized yeast mitochondria, and nonimported precursor was removed by subsequent treatment of the mitochondria with proteinase K. The mitochondria were reisolated and lysed with the mild detergent digitonin under conditions that maintain the integrity of the TIM22 complex, as well as that of the other mitochondrial preprotein translocases (1, 22, 30, 43). Upon separation of the protein complexes by blue native electrophoresis, a small amount of radiolabeled Tim22 was found in the mature 300-kDa TIM22 complex (Fig. 1A, lanes 2 to 6). In addition, the precursor of Tim22 accumulated in two low-molecular-mass forms on the native gels. Formation of these forms required a membrane potential () across the inner membrane (Fig. 1A, lanes 2 to 6 versus lane 1). As outlined below, these forms likely represent monomeric and dimeric forms of Tim22 inserted into the inner membrane and are referred to as Tim22_m and Tim22_d, respectively (Fig. 1A).

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FIG. 1. Membrane-integral components of the TIM22 complex assemble via different intermediates. 35S-radiolabeled Tim22 (A), Tim54 (B), and Tim18 (C) were imported into isolated tim22-14 mitochondria at temperatures of 16°C to 25°C in the presence or absence of and subsequently treated with 50 µg/ml proteinase K (Prot. K). After solubilization in digitonin buffer, samples were subjected to blue native electrophoresis and analyzed by digital autoradiography. Import of Tim22 (D), Tim54 (E), and Tim18 (F) into isolated tim54-11 and tim54-16 mitochondria and subsequent sample analysis was carried out as described for panels A to C. Tim22 (G), Tim54 (H), and Tim18 (I) were imported into tim18 mitochondria as described above. Arrowheads, low-molecular-mass form of Tim54; asterisks, low-molecular-mass intermediate of Tim18; WT, wild type.

We generated temperature-conditional yeast mutants of TIM22 by error-prone PCR. The tim22-14 mutant strain was selected. The strain was inhibited for growth at 37°C on nonfermentable medium (see Fig. S1A in the supplemental material). Upon growth at permissive temperature, mitochondria were isolated from tim22-14 and wild-type cells and analyzed for steady-state protein levels. The amount of Tim22 was moderately reduced in tim22-14 mitochondria, while other subunits of the TIM22 complex and further mitochondrial proteins were present in levels close to that of wild-type mitochondria (see Fig. S1B in the supplemental material). Remarkably, the mutant mitochondria showed a strongly increased efficiency of integration of imported Tim22 into the mature TIM22 complex (Fig. 1A, lanes 8 to 12). Concomitantly, the amounts of Tim22_m and Tim22_d were reduced, raising the possibility that the low-molecular-mass forms may represent assembly intermediates of Tim22, which are consumed to form the mature 300-kDa complex (Fig. 1A, lanes 8 to 12).

We asked if the tim22-14 mutation led to a special phenotype of an increased assembly of the TIM22 complex and therefore imported the radiolabeled precursors of the two other membrane-integral subunits of the complex, Tim54 and Tim18. Both proteins assembled into the 300-kDa TIM22 complex of wild-type mitochondria in the presence of a (Fig. 1B and C, lanes 2 to 6). In tim22-14 mutant mitochondria, however, the integration of Tim54 as well as Tim18 into the TIM22 complex was strongly inhibited (Fig. 1B and C, lanes 7 to 11). To exclude indirect defects of the tim22-14 mutant mitochondria on the presequence pathway, we imported the precursor of subunit β of the F_oF₁-ATP synthase (F₁β). The preprotein was imported with similar efficiency in wild-type and mutant mitochondria (see Fig. S1C in the supplemental material). Moreover, assessment of the membrane potential by fluorescence quenching revealed that the tim22-14 mitochondria generated a membrane potential that was only slightly lower than that of wild-type mitochondria, excluding that the severe inhibition of the assembly of Tim54 and Tim18 was due to a dissipation of the (see Fig. S1D in the supplemental material). Thus, tim22-14 mutant mitochondria show a differential effect on the assembly of the TIM22 complex. While the integration of the precursor of Tim22 into the complex is considerably enhanced, the precursors of Tim54 and Tim18 are inhibited in assembly into the mutant complex.

We thus screened for TIM54 temperature-conditional yeast mutants in order to address the function of Tim54 in the TIM22 assembly process. We selected the tim54-11 and tim54-16 mutants, which were inhibited for growth at 37°C on nonfermentable medium (see Fig. S2A in the supplemental material). Upon growth at permissive temperature, the protein levels of selected mitochondrial proteins were analyzed in isolated mitochondria. The levels of Tim54 were decreased in the mutant mitochondria, while other subunits of the TIM22 complex and further mitochondrial proteins were present in wild-type amounts (see Fig. S2B in the supplemental material). The tim54 mutant mitochondria were not impaired in the import of presequence-containing proteins, as shown with the matrix-targeted model preprotein b₂(167)dihydrofolate reductase (see Fig. S2C in the supplemental material). The membrane potential of the tim54 mutant mitochondria was comparable to that of wild-type mitochondria (see Fig. S2D in the supplemental material). Assembly of the precursor of Tim22 into the TIM22 complex of both mutant mitochondria was blocked (Fig. 1D, lanes 5 to 7 and 9 to 11). Strikingly, assembly of Tim54 into the tim54 mutant mitochondria was significantly increased in comparison to its assembly into wild-type mitochondria (Fig. 1E). The precursor of Tim18 did not assemble into the TIM22 complex in tim54 mutant mitochondria (Fig. 1F, lanes 5 to 7 and 9 to 11), similar to the observation made with the precursor of Tim22. Thus, the tim54 mutant mitochondria yielded an assembly pattern that was complementary to the pattern observed for tim22 mutant mitochondria. Assembly of the wild-type precursor (Tim54 and Tim22, respectively) was strongly enhanced in mitochondria containing a mutant version of this protein, while assembly of the other two precursor proteins was inhibited.

To study if these findings may point to a general principle, we generated a mutant of the third membrane subunit of the TIM22 complex, i.e., tim18 mitochondria. In tim18 mitochondria, the assembly of imported Tim18 into the 300-kDa TIM22 complex was indeed significantly increased compared to that in wild-type mitochondria (Fig. 1I). The precursor of Tim22 efficiently assembled into the 250-kDa TIM22' complex of tim18 mutant mitochondria (Fig. 1G, lanes 7 to 11). (Due to the lack of Tim18, the TIM22' complex of tim18 mitochondria migrates faster on blue native gels [16, 19].) Interestingly, when the precursor of Tim54 was imported into tim18 mitochondria, the assembly of Tim54 into the 250-kDa TIM22' complex was strongly decreased (Fig. 1H), suggesting that Tim18 is involved in the assembly pathway of Tim54.

The precursor of Tim18 was also found in a low-molecular-mass form that was formed in a -dependent manner (Fig. 1C, lanes 2 to 6 versus lane 1). In tim18 mitochondria, the amount of the low-molecular-mass form was reduced while the formation of the mature TIM22 complex proceeded faster (Fig. 1I), suggesting that this form may represent an intermediate on the assembly pathway of Tim18. (In the case of Tim54, we also observed a low-molecular-mass precursor form on the blue native gels. However, this form was only partially affected by a dissipation of [Fig. 1B, E and H] and a large fraction of this form was sensitive to externally added protease [see Fig. 3A below]. Thus, the low-molecular-mass form of Tim54 likely does not represent one defined species on the assembly pathway but is probably formed from precursors at different stages of their import pathway.)

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FIG. 3. Tim54 forms a TOM-TIM intermediate. (A) Assembly of radiolabeled Tim54 in wild-type (WT) mitochondria. After import of Tim54, samples were left untreated or treated with 50 µg/ml proteinase K, solubilized in digitonin buffer, and then analyzed by blue native electrophoresis and digital autoradiography. (B) After import of radiolabeled Tim54, complexes were shifted with antisera against Tom40 and porin or incubated with BSA. As a control, Tom22 was imported and shifted as described for Tim54. Analysis was carried out by blue native electrophoresis and digital autoradiography. (C) Radiolabeled Tim54 was imported into isolated wild-type and tim50-1 mitochondria in the presence or absence of . After treatment with proteinase K, proteins were separated by SDS-PAGE and visualized by digital autoradiography. (D) 35S-labeled Tim54 was imported into wild-type and tim50-1 mitochondria. After treatment with digitonin-containing buffer, complexes were separated by blue native electrophoresis and subjected to digital autoradiography. (E) Tim54 was imported into wild-type mitochondria and subjected to antibody shift/depletion analysis as described in Materials and Methods. Samples were analyzed by blue native electrophoresis and digital autoradiography. Arrowheads, low-molecular-mass form of Tim54.

Taken together, we have established a native assay to monitor the assembly of the three membrane-integral subunits of the TIM22 complex. The assay revealed possible assembly intermediates for the precursors of Tim22 and Tim18 and a role of Tim18 in the assembly of Tim54. Strikingly, each of the mutant mitochondria assembles the imported wild-type precursor, which corresponds to the mutant subunit, with high efficiency while the assembly of other wild-type subunits is impaired.

Assembly of Tim22 occurs via low-molecular-mass intermediate forms. We used antibody shift blue native electrophoresis (14, 38, 43) to characterize the two low-molecular-mass forms of the Tim22 assembly pathway, Tim22_m and Tim22_d. The radiolabeled precursor of Tim22 was imported into mitochondria, and surface-bound precursors were removed by protease treatment. The mitochondria were subjected to swelling to rupture the outer membrane and permit access of antibodies to the intermembrane space and inner membrane. Upon addition of antibodies directed against a C-terminal epitope of Tim22, both small species were efficiently shifted (Fig. 2A, lanes 2 to 4). However, the mature TIM22 complex was not shifted by the antibodies (Fig. 2A, lanes 2 to 4), indicating that in the fully assembled complex the epitope of Tim22 was not accessible from the intermembrane space while it remained accessible in the two low-molecular-mass species.

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FIG. 2. Tim22 assembly occurs through low-molecular-mass intermediates. (A) Antibody shift analysis of imported Tim22 in wild-type (WT) mitochondria. After swelling of mitochondria, complexes were shifted by incubation with increasing amounts of antisera against Tim22 and Tim18. After solubilization with digitonin buffer, protein complexes were separated by blue native electrophoresis and analyzed by digital autoradiography. (B) Pulse-chase analysis of Tim22 assembly. 35S-labeled Tim22 was imported into mitochondria as indicated in the scheme, and samples were analyzed by blue native electrophoresis and digital autoradiography. (C) Radiolabeled Tim22 was imported into wild-type and tim10-2 mitochondria in the presence or absence of a and treated with 50 µg/ml proteinase K. Import was carried out at 25°C after a 15-min preincubation of mitochondria at 37°C. Samples were subjected to SDS-PAGE and analyzed by digital autoradiography. (D) Import of Tim22 precursor protein was done as described for panel C, and samples were analyzed by blue native electrophoresis and digital autoradiography (left panel). Isolated wild-type and tim10-2 mitochondria were incubated at 37°C for 15 min prior to solubilization in digitonin buffer and separation of complexes by blue native electrophoresis. Western blot analysis was performed with the indicated antiserum (right panel).

The blue native gel mobility of Tim22_m correlates with that of a monomer of Tim22. When mitochondria were lysed in SDS buffer prior to blue native separation, all of Tim22 comigrated with Tim22_m (data not shown). Tim22_d may represent a homodimer or a heterodimer of Tim22. According to the gel mobility, Tim18 would be a possible partner protein in a heterodimer. (Below a range of 100 kDa, the blue native mobility of membrane proteins is slower than that of the soluble 66-kDa marker protein [30, 31, 38].) Antibodies directed against Tim18 shifted the mature TIM22 complex in a dose-dependent manner but did not alter the mobility of the low-molecular-mass species of Tim22 (Fig. 2A, lanes 5 to 7). Since the epitope of Tim18 was exposed in the 300-kDa TIM22 complex, it is unlikely that the epitope would be not accessible in a small complex. Together with the observation that Tim22_d was still formed in tim18 mitochondria (Fig. 1G), we conclude that Tim18 is not a constituent of Tim22_d. To probe for the possible presence of Tim10 in Tim22_d, we used an antibody depletion assay. After import of Tim22, solubilized mitochondria were mixed with anti-Tim10 antibodies, followed by depletion using protein A-Sepharose. The mature 300-kDa TIM22 complex was depleted, whereas Tim22_d was not affected in comparison to control antibodies (see Fig. S3A in the supplemental material), suggesting that Tim10 or the TIM10 chaperone complex were not present in Tim22_d. Currently, it cannot be excluded that other proteins are associated with Tim22 in Tim22_d; however, the available results are also compatible with a homodimer of Tim22.

In order to determine if Tim22_m and Tim22_d represented intermediates on the assembly pathway of Tim22, a pulse-chase analysis was performed. The precursor of Tim22 was imported into mitochondria for a short time such that the two low-molecular-mass species were formed, but only a small amount of the mature TIM22 complex (Fig. 2B, lane 4). The mitochondria were reisolated and incubated at increasing temperatures. During this chase, Tim22 assembled into the TIM22 complex in a temperature-dependent manner and the amounts of small Tim22 species concomitantly decreased (Fig. 2B, lanes 1 to 3). We conclude that Tim22_m and Tim22_d represent intermediates of the Tim22 biogenesis pathway, explaining the reduced amounts of the low-molecular-mass species when the integration of Tim22 into the TIM22 complex was accelerated in tim22-14 mitochondria (Fig. 1A).

The precursor of Tim22 is transported to the inner membrane via the carrier pathway (18, 22, 23, 36). We used tim10-2 mutant mitochondria that are impaired in the TIM10 chaperone complex of the intermembrane space (2, 8, 38). The cells were grown at permissive temperature, and mitochondria were isolated and subjected to a short heat shock. As expected, translocation of the precursor of Tim22 to a protease-protected location was impaired upon loss of Tim10 function in mitochondria (Fig. 2C) (18, 36). Blue native electrophoresis analysis of Tim22 import into tim10-2 mitochondria revealed a strong inhibition of formation of the two low-molecular-mass species as well as of the mature TIM22 complex (Fig. 2D). To exclude that the lack of Tim22 assembly into the TIM22 complex was due to dissociation of the preexisting TIM22 complex under the conditions applied, we performed a Western blot analysis of the temperature-shifted mitochondria. The TIM22 complex remained stable under these conditions (Fig. 2D, lanes 10, 12, and 14). In contrast, only small amounts of the soluble TIM10 complex were observed (Fig. 2D, lane 14 versus lane 13). Thus, formation of the low-molecular-mass intermediates of Tim22 requires a functional TIM10 complex. Moreover, the formation of the intermediates requires the inner membrane potential (Fig. 1A and 2D). The is required for the insertion of precursors into the inner membrane after their -independent transfer across the intermembrane space (21, 30), suggesting that the low-molecular-mass intermediates represent inner membrane-inserted forms of the precursor. (The formation of Tim22_d strongly depends on the presence of a , while a fraction of the monomeric Tim22_m can also be observed in the absence of a .) Tim22_m likely includes two monomeric species: a -dependent one that is inserted into the inner membrane and a -independent one that is protected against externally added proteinase K but not yet inserted into the inner membrane. The latter intermediate has also been found for other precursors using the carrier pathway (30, 31, 38). In order to obtain further evidence that the low-molecular-mass intermediates of Tim22 were integrated into the inner membrane, we performed a treatment at alkaline pH to extract soluble and peripheral membrane proteins. We used tim54-16 mitochondria, where the imported radiolabeled Tim22 was only present in Tim22_m and Tim22_d but not in the TIM22 complex (Fig. 1D, lanes 9 to 11), to selectively analyze the low-molecular-mass intermediates. The majority of imported [³⁵S]Tim22 was not extracted at alkaline pH and thus behaved like the integral membrane proteins Tom70 and preexisting Tim22, while Tim10 was found in the supernatant (see Fig. S3B in the supplemental material). Taken together with the dependence, these results indicate that the low-molecular-mass forms of Tim22 are inserted into the membrane.

The mutants of Tim54 revealed a differentiation between Tim22_m and Tim22_d. The formation of Tim22_d but not of Tim22_m was markedly decreased in the tim54-11 mutant mitochondria (Fig. 1D, lanes 5 to 7). Thus, Tim22_m is formed despite the mutation in Tim54, while the generation of Tim22_d depends on the function of Tim54. These results suggest that the precursor of Tim22 is inserted into the inner membrane as a monomeric form and then converted to Tim22_d in a Tim54-dependent manner.

Tim54 forms a TOM-TIM supercomplex during import. To analyze early steps in the import of Tim54, mitochondria were incubated with the radiolabeled precursor and separated by blue native electrophoresis without treating the mitochondria with protease. We found a large protein complex containing the precursor of Tim54 in addition to the TIM22 complex (Fig. 3A, lanes 2 to 6). This complex was fully sensitive to a treatment with protease (Fig. 3A, lanes 8 to 12), indicating that the complex was exposed on the mitochondrial surface, in contrast to the mature TIM22 complex. Surprisingly, the formation of this complex strictly depended on a across the inner membrane (Fig. 3A, lanes 2 to 6 versus lane 1), raising the possibility that the precursor of Tim54 was accumulated in a two-membrane-spanning fashion in a TOM-TIM supercomplex.

A TOM-TIM supercomplex of a preprotein in transit has so far been reported for arrested model preproteins, which contain a stably folded passenger protein (3, 4, 11, 28, 34, 45). The large Tim54 complex would represent the first TOM-TIM intermediate of an authentic preprotein that is visualized by blue native electrophoresis. Thus, rigorous controls were required to demonstrate the two-membrane accumulation of the precursor of Tim54. First, we analyzed if the Tim54-containing complex contained the TOM complex of the outer membrane by using antibody shift blue native electrophoresis. Antibodies directed against Tom40, the central channel-forming subunit of the TOM complex (see Fig. S3C in the supplemental material), quantitatively shifted the complex (Fig. 3B, lane 2), while antibodies against porin did not (Fig. 3B, lane 3). In a control reaction, we imported the receptor Tom22 into mitochondria. Tom22 assembled into the TOM complex and was efficiently shifted with anti-Tom40 antibodies (Fig. 3B, lane 5). The size of the Tim54-containing complex, however, was larger than that of the TOM complex (Fig. 3B, lane 1 versus 4). We conclude that the precursor of Tim54 was accumulated in a TOM-containing complex that apparently contained additional components.

Import of Tim54 into mitochondria occurs through the presequence translocase (22, 23). In agreement with this, in conditional tim50-1 mutant mitochondria (1) the import of Tim54 was decreased compared to that in wild-type mitochondria (Fig. 3C). The level of the large Tim54-containing complex was strongly reduced in the mutant mitochondria (Fig. 3D, lanes 4 and 5). Thus, functional Tim50 is critical for the formation of the Tim54-containing complex. Together with the strict dependence, we conclude that an active inner membrane is required to form this large complex and thus the precursor of Tim54 is likely accumulated in a two-membrane-spanning fashion in a TOM-TIM supercomplex. This supercomplex may contain either the TIM23 complex or the TIM22 complex. To directly probe for the presence of TIM complexes, we used specific antibodies. Antibodies against Tim10 and Tim12 removed the TIM22 complex, as expected, but did not influence the supercomplex, while antibodies against Tim23 affected the supercomplex (Fig. 3E). We conclude that the precursor of Tim54 was accumulated in a TOM-TIM23 supercomplex.

Assembly of Tim54 depends on the TIM10 complex. The TIM10 translocase complex is critical for the transport of proteins with internal targeting signals from the TOM complex to the TIM22 translocase (8, 18, 23, 36, 38). For a few preproteins with amino-terminal targeting signals, dependence on the soluble TIM translocases of the intermembrane space was also reported (23, 37). When we imported Tim54 into tim10-2 mutant mitochondria, assembly of Tim54 into the TIM22 complex was severely affected (Fig. 4A, lanes 4, 5, 10, and 11). However, the TOM-TIM supercomplex of Tim54 was efficiently formed (Fig. 4A, lanes 4 and 5). This indicated that the block of Tim54 biogenesis in tim10-2 mitochondria occurred after the -dependent translocation of Tim54 into mitochondria. To test this, we compared the Tim54 import efficiency between wild-type and tim10-2 mitochondria by treatment with proteinase K and analysis by SDS-PAGE. Tim54 was transported to a protease-protected location in tim10-2 mitochondria with an efficiency that was close to that of wild-type mitochondria (Fig. 4B). Tim54 imported into tim10-2 mitochondria was resistant to extraction at alkaline pH like an integral membrane protein (Fig. 4C), indicating that the precursor was inserted into the membrane. Since the TIM22 complex remained stable in tim10-2 mitochondria (Fig. 2D), we concluded that the assembly of Tim54 into the TIM22 complex was selectively affected by inactivation of the TIM10 complex at a postmembrane insertion stage. As a control, we analyzed the assembly of Tim18 in wild-type and tim10-2 mitochondria. Tim18 was imported and assembled independently of Tim10 (Fig. 4D). In summary, while the transport of Tim54 into the inner membrane is not affected by inactivation of the TIM10 complex, assembly of Tim54 into the carrier translocase depends on the TIM10 complex. This mutant defect is selective for Tim54, since Tim18 biogenesis is not affected.

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FIG. 4. Tim54 assembly is dependent on the TIM10 complex. (A) Assembly of Tim54 in tim10-2 mutant mitochondria. Radiolabeled Tim54 was imported into isolated wild-type (WT) and tim10-2 mitochondria in the presence or absence of a after a 15-min preincubation at 37°C. Where indicated, mitochondria were treated with proteinase K. Complexes were separated by blue native electrophoresis and visualized by digital autoradiography. (B) 35S-labeled Tim54 was imported as described in panel A, including treatment with proteinase K. Samples were analyzed by SDS-PAGE and digital autoradiography. (C) Radiolabeled Tim54 was imported. The mitochondria were treated with proteinase K and subjected to treatment with carbonate. Total (T), pellet (P), and supernatant (S) were analyzed by SDS-PAGE and digital autoradiography ([35S]Tim54) or immunolabeling (Tom70, Mge1). (D) Radiolabeled Tim18 was imported into wild-type and tim10-2 mitochondria as described in panel A. Arrowhead, low-molecular-mass form of Tim54; asterisk, low-molecular-mass intermediate of Tim18.

We asked if the TIM10 complex indirectly affected the assembly of Tim54 or if TIM10 and Tim54 interacted with each other in organello. Mitochondria containing a protein A-tagged version of Tim18 (30) were subjected to chemical cross-linking. The TIM22 complex was isolated by IgG affinity chromatography and analyzed by SDS-PAGE. Antibodies directed against Tim54, as well as antibodies directed against Tim10, labeled a band of about 65 kDa (Fig. 5A, lanes 2 and 4), suggesting that Tim54 may be in proximity to Tim10. To obtain further evidence, we imported radiolabeled Tim54 into Tim18_ProtA mitochondria. Upon purification of the TIM22 complex, bound proteins were eluted under denaturing conditions and subjected to immunoprecipitation with antibodies against Tim9, Tim10, or Tim12. The 65-kDa cross-linking product was specifically precipitated with antibodies against Tim10, but not with antibodies against Tim9 or Tim12 (Fig. 5B). Thus, Tim10 is in close proximity to Tim54 in the TIM22 complex, supporting the view that Tim10 plays a direct role in the assembly of Tim54.

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FIG. 5. Tim54 and Tim10 interact at the carrier translocase. (A) Tim18ProtA mitochondria were subjected to chemical cross-linking as described in Materials and Methods. The TIM22 complex was purified by IgG-Sepharose chromatography. After washing and elution, proteins were separated by SDS-PAGE and analyzed by immunolabeling with antibodies against Tim54 and Tim10. Circle, unspecific cross-reaction of anti-Tim10. (B) After import of radiolabeled Tim54 into Tim18ProtA mitochondria, proteins were cross-linked as described in Materials and Methods and the TIM22 complex was purified with IgG-Sepharose. After washing and elution of the proteins, an immunoprecipitation was performed with antibodies against Tim9, Tim10, and Tim12. Samples were analyzed by SDS-PAGE and digital autoradiography.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We developed an in organello assay to monitor the biogenesis and assembly pathway of each membrane-integral subunit of the mitochondrial carrier translocase. We show that each precursor follows a different assembly pathway. Remarkably, up to four preprotein translocase complexes are required for import and assembly. Thus, for the precursor of Tim54, all known protein import translocases of mitochondria, TOM, TIM23, TIM10, and TIM22, are required, representing one of the most complex protein assembly pathways of mitochondria known to date.

Despite the diversity of the import routes, an important principle emerged, which is uncoupling of translocation into the inner membrane from the subsequent assembly to the oligomeric complex. The mitochondrial inner membrane has to translocate entire polypeptide chains and assemble membrane-spanning proteins into oligomeric complexes. Each of these processes bears the danger of disturbing the barrier of the inner membrane that is essential to maintain the electrochemical proton gradient. The proton gradient is the main driving force for the bulk of cellular ATP synthesis, as well as for the import of precursor proteins, and a leakage of the inner membrane would be deleterious. Since membrane insertion of an individual unfolded polypeptide chain and the assembly of different proteins into a large complex are mechanistically quite different processes, a combination of both processes would substantially increase the risk of a nonspecific ion leakage. We thus analyzed how translocation and assembly are separated for the individual precursor proteins.

(i) The precursor of Tim22 is transferred from outer to inner membranes by the TIM10 translocase complex of the intermembrane space and forms low-molecular-mass assembly intermediates in the inner membrane before the subsequent integration into the 300-kDa TIM22 complex. The -dependent insertion into the inner membrane takes place at the level of the low-molecular-mass intermediates and is thus uncoupled from the assembly into the TIM22 complex. Electrophysiological analysis of the TIM22 complex in comparison to reconstituted Tim22 alone indeed showed that the Tim22 channel is highly active in the TIM22 complex and performs rapid gating transitions in response to membrane potential and targeting peptides, while Tim22 alone shows a significantly lower activity (30), suggesting that the uncoupling of membrane insertion and oligomerization reduces the risk of an unspecific leakage of ions. (ii) The precursor of Tim54 is transferred across the intermembrane space by a different mechanism. Cooperation between TOM and TIM23 complexes leads to a two-membrane-spanning preprotein-TOM-TIM23 supercomplex. The -dependent insertion of Tim54 into the inner membrane is required for formation of this supercomplex and occurs before the assembly of Tim54 into the TIM22 complex. The separation of translocation and assembly was directly shown by an unexpected new function of the TIM10 translocase complex of the intermembrane space. The TIM10 complex is not required for the translocation of Tim54 from outer to inner membranes but for its subsequent incorporation into the 300-kDa complex. (iii) The precursor of Tim18, which is also imported by the presequence route (9, 16, 19), is inserted into the inner membrane in a -dependent manner, forming a low-molecular-mass form. This form is consumed when the assembly of Tim18 into the TIM22 complex is enhanced, indicating that the low-molecular-mass form represents the inner membrane-inserted intermediate form. Interestingly, the TIM10 translocase is needed for neither translocation nor assembly of Tim18.

A recent study on the biogenesis of a subunit of the F_oF₁ ATP synthase of Escherichia coli revealed that oligomerization was not a prerequisite for membrane insertion of the protein (20) and the sorting and assembly machinery of the mitochondrial outer membrane complex performs the tasks of membrane insertion and assembly in consecutive steps (43). We propose that uncoupling of membrane insertion and oligomeric assembly may represent a general mechanism for the biogenesis of membrane protein complexes.

The assembly of each wild-type precursor of a Tim protein into the TIM22 complex was strongly enhanced when mutant mitochondria were used that were defective in exactly this subunit. The assembly of the other two subunits was not enhanced in the mutant mitochondria but decreased in most cases. This behavior was observed for each of the three membrane-integral subunits. A related result was observed for one subunit of the TIM23 complex (39); however, it could not be decided if this represented a unique case for this subunit or a more general principle. The systematic analysis of all three membrane subunits of the TIM22 complex now provides the basis to formulate a general principle for the assembly of a hetero-oligomeric membrane protein complex. We propose that a destabilization of the TIM22 complex in the mutant mitochondria facilitates the integration of individual newly imported subunits. Under wild-type conditions, an incoming precursor protein has to replace a preexisting subunit or to associate with nonassembled pools of both other subunits that are likely small under wild-type conditions. Thus, the efficiency of integration into the wild-type complex is limited. When the TIM22 complex is labilized by a mutation of an individual subunit, the incoming wild-type subunit can much more easily replace the preexisting (mutant) subunit. Moreover, destabilization of the complex will increase the pools of nonassembled subunits and thus the incoming wild-type subunit can rapidly associate with the other two subunits to form the holo-translocase.

Our studies reveal evidence for the possible molecular functions of Tim18 and Tim54 in the TIM22 complex. Tim18 has been found as a stoichiometric subunit of the TIM22 complex (16, 19); however, its role remained unclear. While the precursor of Tim22 still assembled into a smaller TIM22 complex in mitochondria lacking Tim18, the assembly of the precursor of Tim54 was inhibited. The assembly of Tim54 was not blocked completely, consistent with the viability of yeast cells lacking Tim18 (16, 19), but was strongly retarded in the in organello assay, suggesting that Tim18 may be involved in the efficient integration of Tim54 into the TIM22 complex. Tim54 exposes a large domain to the intermembrane space (15) and was thus an interesting candidate for the docking site of small Tim proteins at the TIM22 complex; however, experimental evidence has been lacking. We observed efficient cross-linking of Tim54 to Tim10, suggesting that the link between intermembrane space translocase and the membrane portion of the TIM22 complex is mediated by Tim10 and Tim54. The contact of Tim54 with Tim10 is not only needed for docking of the TIM10 complex to the TIM22 complex but also for the incorporation of the Tim54 precursor into the TIM22 complex. Thus, the small Tim protein of the intermembrane space is critical for the assembly of the membrane-integral portion of the carrier translocase, suggesting that the cooperation of Tim54 and Tim10 represents an important element for both biogenesis and function of the TIM22 complex.

Taken together, each subunit of the twin-pore carrier translocase follows a different assembly pathway. Along the assembly pathways, distinct intermediate complexes are formed and inserted into the inner membrane before the association with the large complex. We speculate that a dimeric form of the central pore-forming subunit Tim22 is the initial building block with which Tim54 associates in a TIM10- and Tim18-dependent manner. Thus, the multistep maturation pathway of the carrier translocase separates the steps of membrane insertion and oligomeric assembly.

 
We thank M. van der Laan, A. Chacinska, and C. Meisinger for discussion and comments on the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 746, Gottfried Wilhelm Leibniz Program, Max Planck Research Award, and the Fonds der Chemischen Industrie.

 
* Corresponding author. Mailing address for Nikolaus Pfanner: Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg, Germany. Phone: 49-761-203-5224. Fax:49-761-203-5261. E-mail: nikolaus.pfanner{at}biochemie.uni-freiburg.de. Mailing address for Peter Rehling: Abteilung für Biochemie II, Universität Göttingen, Heinrich-Düker-Weg 12, D-37073 Göttingen, Germany. Phone: 49-551-39-5947. Fax: 49-551-39-5979. E-mail: peter.rehling{at}medizin.uni-goettingen.de

Published ahead of print on 5 May 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, July 2008, p. 4251-4260, Vol. 28, No. 13
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