Mitochondrial Heat Shock Protein (Hsp) 70 and Hsp10 Cooperate in the Formation of Hsp60 Complexes*

Background: The formation of Hsp60 complexes is poorly understood. Results: The biogenesis of Hsp60 complexes depends on mitochondrial (mt) Hsp70 and Hsp10. Conclusion: MtHsp70 interacts with Hsp10 to promote Hsp60 biogenesis. Significance: Coupling to partner proteins like Hsp10 modifies the functional specificity of mtHsp70. Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings.
Heat shock proteins of 70 kDa (Hsp70) fulfill several essential functions in prokaryotic and eukaryotic cells, like protein folding and transport. Hsp70 proteins prevent the aggregation of misfolded proteins and facilitate the removal of protein aggregates. To perform these various tasks, the chaperones bind transiently to hydrophobic patches exposed in non-native conformations of their client proteins (1)(2)(3).
Mitochondrial Hsp60 exists in single and double ring conformations, with one ring being composed of seven subunits (45)(46)(47)(48). Detailed structural and mechanistic insights have been obtained for the bacterial counterpart GroEL and its Hsp10 homolog GroES (1,3). The ring structure of the chaperonin provides a central cavity for folding of the enclosed client protein. The activity of the Hsp60 rings is driven by ATP-dependent conformational changes of the Hsp60 monomers. The heptameric Hsp10 ring forms the lid of the cavity and regulates the ATP-dependent reaction cycle of Hsp60 (47,49,50). Although Hsp60 is essential for cell survival (51), the assembly of the ring structures is poorly understood. MtHsp70 promotes the import of the Hsp60 precursor into the mitochondrial matrix (43). The subsequent formation of the Hsp60 ring structures depends on a pre-existing Hsp60 oligomer (44,52,53). Whether other factors support the formation of Hsp60 complexes is not known.
Despite its central role in mitochondrial biogenesis, comprehensive studies of the interaction partners of mtHsp70 are missing up to now. Here we performed affinity purification of His-tagged mtHsp70 and analyzed its binding partners by SILAC-based mass spectrometry. We found that mtHsp70 interacts with Hsp60 and Hsp10. Surprisingly, an mtHsp70-Hsp10 complex forms independently of Hsp60. We found that assembly of the Hsp60 precursor into the mature complexes is impaired in mutants of mtHsp70 and Hsp10. The unassembled Hsp60 precursor binds to both mtHsp70 and Hsp10. Therefore, we propose that coupling to Hsp10 enables mtHsp70 to promote the formation of the mature Hsp60 ring structures.

EXPERIMENTAL PROCEDURES
Yeast Strains and Growth Conditions-The yeast wild-type strains YPH499, YPH499 arg4⌬, and JK9 -3d (the wild type for hsp10ts and hsp60ts); the wild-type strain for ssc1-42; and the mutant strains mtHsp70 His , ssc1-42, hsp10ts, and hsp60ts have been described before (39,44,54). For SILAC-based mass spectrometric analysis of mtHsp70 His purification, a kanMX4 cassette was integrated into the ARG4 locus by homologous recombination in the yeast strain expressing mtHsp70 His . For biochemical studies, yeast cells were grown to logarithmic growth phase at 23°C or 30°C on YPG medium (1% (w/v) yeast extract, 2% (w/v) bacto peptone, and 3% (v/v) glycerol). For cycloheximide treatment, yeast cells were grown at 30°C in the presence of 50 g/ml cycloheximide for 2 h. For in vivo heat shock, hsp60ts, hsp10ts, and wild-type strain cells were shifted to 37°C for 2 h (44).
Isolation of Mitochondria and in Vitro Protein Import Assays-Mitochondria were isolated by differential centrifugation following a published procedure (39). Mitochondria were stored in SE buffer (10 mM MOPS/KOH (pH 7.2), 1 mM EDTA, and 250 mM sucrose) in a protein concentration of 10 mg/ml at Ϫ80°C until use. To generate 35 S-labeled precursor proteins, we used cell-free in vitro translation on the basis of rabbit reticulocyte lysate in the presence of 35 S-labeled methionine (Promega). Standard import reactions were performed following established assays (39,55). In brief, 35 S-labeled precursors (5-10% of the reaction volume) were incubated with isolated mitochondria at 25°C in import buffer (3% (w/v) BSA, 250 mM sucrose, 5 mM methionine, 80 mM KCl, 5 mM MgCl 2 , 10 mM MOPS/KOH (pH 7.2), and 2 mM KH 2 PO 4 ). Energy was added to the reaction mixture in the form of 2 mM ATP and 2 mM NADH (final concentration). The import reaction was stopped by addition of an AVO mixture (8 M antimycin A, 1 M valinomycin, and 20 M oligomycin, final concentrations) to dissipate the membrane potential. In the indicated control samples, the AVO mixture was added prior to import. For SDS-PAGE analysis, non-imported precursor proteins were removed by incubation with 50 g/ml proteinase K for 15 min on ice. Subsequently, mitochondria were reisolated, washed with SE buffer, and lysed with SDS-PAGE loading buffer. When the import reaction was studied by blue native electrophoresis, mitochondria were washed with SE buffer and solubilized under nondenaturing conditions using the non-ionic detergent digitonin to 1% (w/v) final concentration in lysis buffer (20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10% (v/v) glycerol, and 0.1 mM EDTA).
Affinity Purification of mtHsp70 His -Mitochondria from wildtype and mtHsp70 His cells were isolated and lysed with lysis buffer containing 1% (w/v) digitonin and 10 mM imidazole at a final protein concentration of 1 mg/ml. Lysis was performed for 15 min on ice. When indicated, solubilization was performed in the presence of 10 mM ADP or ATP in buffer A (20 mM Tris-HCl (pH 7.4), 50 mM KCl, 10 mM MgCl 2 , and 10% (v/v) glycerol) containing 1% (w/v) digitonin. In all cases, unsoluble material was removed by centrifugation. The mitochondrial lysate was incubated with Ni 2ϩ -NTA-agarose (Qiagen) under constant rotation for 1 h at 4°C. Unbound proteins were discarded, and the Ni 2ϩ -NTA-agarose beads were washed intensively with lysis buffer containing 0.1% (w/v) digitonin and 40 mM imidazole. To elute bound proteins, the Ni 2ϩ -NTA-agarose beads were incubated with lysis buffer containing 250 mM imidazole and 0.1% (w/v) digitonin.
For characterization of the binding of precursor proteins to mtHsp70 His , a 5-fold standard import reaction with the 35 Slabeled precursor was performed before affinity purification via Ni 2ϩ -NTA-agarose. The affinity purification followed the procedure described above. In the case of detection of protein complexes by blue native electrophoresis, bound proteins were eluted by addition of buffer A containing 20 mM ATP.
Mass Spectrometry and Data Analysis-Mass spectrometric analyses of affinity-purified mtHsp70 His complexes were performed as described previously (56) with minor modifications. Protein complexes (n ϭ 3) were precipitated with ice-cold acetone and resuspended in urea buffer (8 M urea in 50 mM NH 4 HCO 3 ). Cysteine residues were reduced with 5 mM Tris-2carboxyethylphosphine (30 min at 37°C) and subsequently alkylated with 50 mM iodoacetamide (30 min at room temperature in the dark). 50 mM NH 4 HCO 3 was added to reach a final urea concentration of 2 M. Proteins were then subjected to proteolysis with trypsin overnight at 37°C. Peptides were dried, reconstituted in 0.1% (v/v) trichloroacetic acid, and subjected to LC/MS analysis on an LTQ-Orbitrap XL (Thermo Scientific, Bremen, Germany) directly coupled to an UltiMate TM 3000 RSLCnano system (Thermo Scientific). Peptide separation was performed using a C18 reverse-phase nano LC column (50 cm ϫ 75 m; Acclaim PepMap; particle size, 2 m; pore size, 100 Å; Thermo Scientific). For peptide elution, a 130-min linear LC gradient ranging from 1.5-21% (v/v) acetonitrile and 2.5-35% (v/v) methanol in 4% (v/v) dimethyl sulfoxide/0.1% (v/v) formic acid was applied. The flow rate was 250 nl/min. Full scan MS spectra in the range of m/z 370 -1700 were acquired in the Orbitrap at a resolution of 60,000 at m/z 400.
Simultaneously with completion of the full scan, up to five of the most intense peptide ions (z Ն ϩ2) were fragmented further by low-energy collision-induced dissociation in the linear ion trap. A dynamic exclusion time of 45 s was applied to prevent repeated fragmentation of previously selected precursor ions.
For protein identification and SILAC-based relative protein quantification, MS data were analyzed with MaxQuant (version 1.4.1.2) and its search algorithm Andromeda (57,58). MS/MS data were searched against the Saccharomyces Genome Database (version 02/03/2011) (59) using the following parameters: mass tolerances of 4.5 ppm for precursor and 0.5 Da for fragment ions, tryptic specificity, maximum of two missed cleavages, acetylation of protein N termini and oxidation of methionine as variable modifications, carbamidomethylation of cysteine residues as fixed modification, and Arg-10 and Lys-8 as heavy labels. A false discovery rate of Ͻ0.01 on the peptide and protein level was applied. Proteins were identified on the basis of at least one unique peptide with at least seven amino acids. For the calculation of mtHsp70 His /WT protein ratios, only unique peptides were taken into account, and the minimum number of ratio counts (i.e. SILAC peptide pairs) was one. Data were visualized by plotting the mean log 10 mtHsp70 His /WT ratios across all three replicates against the p value (determined using one-sided Student's t test) for each protein. Potential mtHsp70 His -interacting proteins were required to have a sequence coverage of Ͼ4%, a ratio mtHsp70/WT of Ͼ5, and a p value of Ͻ0.05.
Coimmunoprecipitation of Imported Precursor Proteins-Protein A-Sepharose (GE Healthcare) was coated with anti-Hsp10 antibodies or its corresponding preimmune serum by covalent coupling with dimethylpimelidate. To detect association to imported precursor proteins, a 5-fold import reaction was performed following the procedure described above. After washing, the mitochondria were lysed with lysis buffer containing 1% (w/v) digitonin at a final protein concentration of 1 mg/ml. After removal of unsoluble material, the mitochondrial lysate was incubated with the indicated beads for 1 h under constant rotation at 4°C. Unbound material was removed, and beads were washed intensively with lysis buffer containing 0.1% (w/v) digitonin. Finally, bound proteins were eluted at low pH using 0.1 M glycine (pH 2.5). Eluted samples were immediately neutralized with Tris base and analyzed by SDS-PAGE.
In the case of two-step purification, the 35 S-labeled Hsp60 precursor was imported into mtHsp70 His mitochondria. Subsequently, mitochondria were lysed and proteins were purified via Ni 2ϩ -NTA-agarose as described above. The elution sample in lysis buffer containing 250 mM imidazole was incubated with protein A-Sepharose coated with anti-Hsp10 or the corresponding preimmune serum. Coimmunoprecipitation was performed as described above.
Binding Assay to the Mge1 Affinity Matrix-For the preparation of the Mge1 affinity matrix, His-tagged Mge1 lacking the presequence was recombinantly expressed and purified as described previously (39). Purified Mge1 His was coupled to Ni 2ϩ -NTA-agarose. Subsequently, affinity beads were washed several times with lysis buffer containing 0.1% (w/v) digitonin and 10 mM imidazole. Mitochondria were lysed in 1% (w/v) digitonin in lysis buffer at a final protein concentration of 1 mg/ml. After removal of unsoluble material by centrifugation, the mitochondrial lysate was incubated with the Mge1 affinity matrix for 1 h under constant rotation at 4°C. Subsequently, unbound material was removed, and the beads were washed intensively with lysis buffer containing 0.1% (w/v) digitonin and 40 mM imidazole. To elute bound proteins, the affinity matrix was incubated with 250 mM imidazole and 0.1% (w/v) digitonin in lysis buffer.
Protein Aggregation Assay-To study protein aggregation in wild-type and mutant mitochondria, isolated mitochondria were solubilized in lysis buffer containing 1% (w/v) digitonin. After incubation on ice for 15 min, protein aggregates were collected by centrifugation for 10 min at 4°C and 18,000 ϫ g. Proteins of the supernatant were precipitated with trichloroacetic acid. All pellet fractions were denatured by solubilization with SDS-PAGE loading buffer and subjected to SDS-PAGE and Western blotting.
Miscellaneous-In general, the signals detected by the antibodies used in this study were confirmed intensively using wildtype and the corresponding mutant mitochondria. The antibodies have been described previously (39,44,60). For immunodetection, proteins were transferred by semidry Western blotting to a PVDF membrane (Millipore). After immunodecoration with the indicated antibodies, the signals were detected using the chemiluminescence kit (ECL, GE Healthcare). 35 S-labeled proteins and protein complexes were detected by digital autoradiography (Storm imaging system, GE Healthcare). Analysis and quantification of protein signals were performed with ImageQuant 5.2 software (GE Healthcare).

RESULTS AND DISCUSSION
MtHsp70 Interacts with Hsp60 and Hsp10 -To identify novel binding partners of the major mitochondrial Hsp70, we used a yeast strain expressing a His-tagged mtHsp70 (mtHsp70 His ). We have demonstrated previously that the Histagged mtHsp70 is functional and interacts with known cochaperones (39). Isolated mitochondria were lysed with the non-ionic detergent digitonin and subjected to affinity purification via Ni 2ϩ -NTA chromatography. The elution fractions from wild-type and mtHsp70 His mitochondria were mixed and analyzed by stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry (61) (Fig. 1A, supplemental Table S1). We used the SILAC ratios to determine proteins that were specifically enriched in the mtHsp70 His -bound fraction (Fig. 1A, supplemental Table S1). A large number of different proteins were copurified along with mtHsp70 His , comprising potential interaction partners and substrate proteins of the chaperone. Known partner proteins, like subunits of the TIM23 complex and several cochaperones, were enriched in the mtHsp70 His purification (Fig. 1A, supplemental Table S1). Surprisingly, Hsp10 and Hsp60 were found among the strongly enriched proteins. We analyzed the affinity purification by Western blotting and immunodetection using antibodies against various mitochondrial proteins (Fig. 1B). We could demonstrate the copurification of Hsp60 and Hsp10 along with mtHsp70 His , indicating that a fraction of Hsp60 and Hsp10 interacts with mtHsp70 (Fig. 1B). Known mtHsp70 interactors, like the cochaperones Mge1, Mdj1, and Mdj2 and components of the TIM23 complex, bound to mtHsp70 His with high efficiency. We also confirmed the binding of mtHsp70 to Cox4 and Mss51 (Fig. 1B) (38,39). MtHsp70 binds to several interaction partners, like Tim44, Cox4, Zim17, and Mge1, in an ATP-sensitive manner (13-15, 27, 31, 35, 39, 62). Therefore, we performed the affinity purification via mtHsp70 His in the presence of ATP or ADP. The association of both Hsp60 and Hsp10 to mtHsp70 His is abolished in the presence of ATP (Fig. 1C, lanes 4 and 8). As reported, binding of Mss51 to Hsp70 His is not affected upon addition of ATP, whereas other control proteins, like Tim23, Cox4, and Mge1, lose their contact with mtHsp70 (Fig. 1C, lanes 4 and 8) (38,39). Next we analyzed whether binding of the cochaperone Mge1 to mtHsp70 interferes with the association of Hsp60 and Hsp10. To this end, we recombinantly expressed tagged Mge1 (Mge1 His ) and coupled the purified protein to Ni 2ϩ -NTA-agarose (39). Subsequently, the Mge1-affinity matrix was incubated with lysed mitochondria. MtHsp70 bound in stoichiometric amounts to the Mge1-coated column, as detected by Coomassie staining (Fig. 1D, lane 4). Hsp60 and Hsp10, as well as control proteins, were copurified along with the Mge1 affinity matrix, indicating that the chaperonin system   binds to Mge1-bound mtHsp70 (Fig. 1D, lanes 4 and 8). Therefore, we conclude that Hsp10 and Hsp60 specifically bind to mature mtHsp70 in an ATP-sensitive manner. MtHsp70 and the chaperonins capture substrate proteins during protein folding (31,43,63). Along this line, it has been reported that mtHsp70 binds to the Hsp60 precursor to promote its import (43). We asked whether the association between the chaperones persists upon prolonged incubation and would, therefore, reflect a stable binding to the mature Hsp60 and Hsp10 rather than transient binding to premature forms. To this end, yeast cells expressing mtHsp70 His were incubated with cycloheximide, which blocks de novo synthesis of nucleus-encoded proteins (64). After incubation with cycloheximide, mitochondria were isolated, lysed with digitonin, and subjected to affinity purification via Ni 2ϩ -NTA-agarose (Fig. 2). Incubation with cycloheximide did not affect the association of Hsp60 and Hsp10 with mtHsp70 (Fig. 2, lanes 6 and 7). Similarly, the stable interactions with Mge1 and the presequence translocase were not affected (Fig. 2, lanes 6 and 7). We conclude that the association of mtHsp70 to Hsp60 and Hsp10 does not depend on the import of newly synthesized proteins. Therefore, these mitochondrial chaperones interact stably with each other.
MtHsp70 Interacts with Hsp10 Independently of Hsp60 -We have shown that mtHsp70 interacts with Hsp60 and Hsp10.   The association of both chaperone systems may facilitate substrate channeling, as suggested for the bacterial homologs (65). However, Hsp10 interacts stably with Hsp60 in the presence, but not in the absence, of ATP or ADP (47,49,66,67). The observed binding of Hsp10 to mtHsp70 occurred without the addition of nucleotides and was lost in the presence of ATP (Fig.  1, B and C). Therefore, the mode of interaction of Hsp10 with Hsp60 and with mtHsp70 differs. We wondered whether Hsp10 associates with mtHsp70 independently of Hsp60. To address this question, we employed mitochondria isolated from hsp60ts cells, which were shifted to non-permissive growth conditions. In these hsp60ts mitochondria, the steady-state levels of Hsp60 are unchanged, but the vast majority of Hsp60 aggregates (Figs.  3, A and B) (44). Consequently, Hsp60 ring complexes cannot be detected in hsp60ts mitochondria by blue native gel electrophoresis (Fig. 3C, lane 3). We analyzed whether the interaction  (Cyt1, bottom panel) were imported into WT and ssc1-42 mitochondria at the indicated time points. Where indicated, isolated mitochondria were subjected to in vitro heat shock prior to import. In control reactions, the membrane potential (⌬) was dissipated prior to import. Proteins were detected by SDS-PAGE and autoradiography. p, precursor protein; m, mature protein.
of endogenous Hsp10 with mtHsp70 was affected in the mutant mitochondria by utilizing an Mge1 His affinity matrix. The association of mtHsp70 with the Mge1 His affinity matrix was largely unchanged in hsp60ts mitochondria (Fig. 3D, lanes 8 and 9). Surprisingly, copurification of Hsp10 to an Mge1 His affinity matrix in hsp60ts mitochondria remained unaltered (Fig. 3D,  lanes 5 and 6). In contrast, binding of Hsp60 to mtHsp70 was strongly diminished (Fig. 3D, lanes 5 and 6). Therefore, Hsp10 interacts with Mge1-bound mtHsp70 independently of Hsp60.
MtHsp70 Promotes the Formation of Hsp60 Complexes-We speculated that the mtHsp70-Hsp10 interaction is involved in the biogenesis of Hsp60. So far, it has been reported that mtHsp70 is required for the import of Hsp60, but a potential role of the chaperone in the formation of Hsp60 complexes has not been addressed (43). To distinguish between the role of mtHsp70 in import or folding and assembly of the Hsp60 precursors, we used a temperature-sensitive mutant of mtHsp70, ssc1-42 (54). The ssc1-42 mutant mtHsp70 harbors six point mutations that are distributed randomly in the ATPase, substrate-binding, and C-terminal domains of the chaperone (I101T, I114N, I485L, N560D, V588A, and K626R). The mutant ssc1-42 grows normally at the permissive temperature but reveals a strong growth defect at 37°C on non-fermentable and fermentable carbon sources (Fig. 4A). We grew the cells at the permissive temperature and isolated mitochondria for biochemical characterization. Steady-state levels of mtHsp70 were mildly reduced in the mutant, whereas the levels of other mitochondrial proteins, including Hsp10 and Hsp60, were similar to wild-type mitochondria (Fig. 4B). We analyzed the levels of Hsp60 and Hsp10 by blue native electrophoresis. A previous study revealed that the single (Hsp60 7 ) and double (Hsp60 14 ) ring complexes of Hsp60 can be detected on a blue native gel (60). Here we show that the heptameric Hsp10 oligomer can also be resolved on blue native gels (Fig. 4C). The levels of the Hsp60 and Hsp10 complexes as well as of respiratory chain complexes were unaffected in ssc1-42 mutant mitochondria (Fig. 4C). To functionally characterize the mutant mtHsp70, we tested whether the protein import capacity is affected in ssc1-42 mitochondria. Model precursor proteins were labeled with [ 35 S]methionine in a cell-free translation system and incubated with isolated mitochondria under import conditions. Subsequently, non-imported precursor proteins were proteolytically removed. We selected two precursors for our import studies that are transported, via mtHsp70, into the mitochondrial matrix: the precursor subunit 9 of the F 1 F O -ATP synthase fused to DHFR (pSu9-DHFR) and the precursor of a cytochrome b 2 variant lacking the transmembrane segment fused to DHFR (b 2 ⌬167-DHFR). The import of both precursor proteins was moderately affected in the mutant mitochondria under permissive conditions (Fig. 4D, lanes 4, 5, 16, and 17). However, upon in vitro heat shock, the transport of both model substrates into the mutant mitochondria was largely compromised (Fig.  4D, lanes 10, 11, 22, and 23). In contrast, import of the 35 Slabeled precursor of cytochrome c 1 (Cyt1) was not affected by in vitro heat shock (Fig. 4D, lanes 34 and 35). The precursor of cytochrome c 1 is sorted into the inner membrane in an mtHsp70-independent manner. We conclude that the import capability of the mutated mtHsp70 can be specifically inactivated in ssc1-42 mitochondria by in vitro heat shock.
Next, we established an assay to study the biogenesis of 35 Slabeled Hsp60 in mitochondria. The import of the 35 S-labeled Hsp60 precursor occurs rapidly and is strictly dependent on the membrane potential (Fig. 5A, bottom panel). We employed blue native electrophoresis to study the assembly of the Hsp60 precursor into the mature complexes. Indeed, we observed an efficient integration of the 35 S-labeled Hsp60 precursor into two high molecular weight complexes (Fig. 5A, top panel), which correspond in size to the single (Hsp60 7 ) and double (Hsp60 14 ) rings of Hsp60 (Fig. 4C). Import and assembly studies at different temperatures revealed that formation of the mature Hsp60 complexes occurs in a delayed manner compared with the import of Hsp60 into the mitochondrial matrix (Fig. 5A). Interestingly, a low molecular weight form of 150 kDa can be detected at a lower temperature on blue native gels (Fig. 5A). Because the 150-kDa band remained stable upon solubilization with SDS, it most likely represents monomeric Hsp60 (Fig. 5B). Depending on the isoelectric point and capability to bind the Coomassie dye, the running behavior of water-soluble proteins on blue native gels can differ from molecular weight markers (68). This could explain the difference between the size of the potential monomeric Hsp60 of 60 kDa and its running behavior on the blue native gel above the 140 kDa mass marker. The blue native system allows following the formation of Hsp60 complexes from the unassembled Hsp60 precursor.
Having established an assay to separately analyze the import and assembly of the 35 S-labeled Hsp60 precursor, we asked which step of Hsp60 biogenesis is affected in ssc1-42 mutant mitochondria. The 35 S-labeled Hsp60 precursor is imported into untreated and in vitro heat shock-treated ssc1-42 mitochondria, although with reduced efficiency (Fig. 6, A and B). Time (min) 10 30 Time (min) 10 30 Time (min) 10 30 -heat shock    35 S-labeled Hsp60 was imported into WT and ssc1-42 mitochondria at the indicated time points. Where indicated, isolated mitochondria (Mito) were subjected to in vitro heat shock, or the membrane potential (⌬) was dissipated prior to import. Proteins were analyzed by SDS-PAGE (SDS) or blue native (BN) electrophoresis and autoradiography. B, quantification of the import and assembly (Hsp60 14 ) experiments in A. The longest import time point in WT mitochondria was set to 100% (control). Mean Ϯ S.E. of four independent experiments is depicted. C, 35 S-labeled Hsp60 was imported into WT and ssc1-42 mitochondria for a short time (Pulse), followed by further incubation after block of the import (Chase). Proteins were analyzed by SDS-PAGE or blue native electrophoresis and autoradiography. D, 35 S-labeled Hsp60 was imported into WT and mtHsp70 His mitochondria for a short time (Pulse), followed by further incubation after block of the import (Chase). Mitochondria were lysed with digitonin, and proteins were purified via Ni 2ϩ -NTA-agarose. Bound proteins were eluted with ATP and analyzed by SDS-PAGE or blue native electrophoresis and autoradiography.
This observation supports the role of mtHsp70 in the import of the Hsp60 precursor (42). Strikingly, the assembly of 35 S-labeled Hsp60 is more severely impaired in both untreated and heat-treated ssc1-42 mitochondria (Fig. 6, A and B), indicating an additional role of mtHsp70 in the formation of Hsp60 complexes. To test this notion, 35 S-labeled Hsp60 was imported into wild-type and mutant mitochondria for a short time period (Fig. 6C, Pulse). Subsequently, the membrane potential was depleted to prevent further import of 35 S-labeled Hsp60 precursors. Mitochondria were reisolated and incubated under import conditions to allow the assembly of the imported 35 Slabeled Hsp60 (Fig. 6C, Chase). Although 35 S-labeled Hsp60 was efficiently integrated into the mature complexes in wildtype mitochondria, its assembly was blocked in ssc1-42 mutant mitochondria (Fig. 6C). To substantiate a role of mHsp70 in the formation of Hsp60 complexes, we analyzed which oligomeric state of Hsp60 binds to mtHsp70. 35 S-labeled Hsp60 was imported into mtHsp70 His mitochondria for a short time period (pulse), followed by dissipation of the membrane potential and further incubation (chase). After the import reaction, mitochondria were lysed with digitonin and subjected to affinity purification via Ni 2ϩ -NTA agarose. Bound proteins were eluted from mtHsp70 His by incubation with ATP. After a short import period, mtHsp70 His binds to unassembled 35 S-labeled Hsp60 (Fig. 6D, lane 5), whereas, upon chase incubation, a heptameric ring of 35 S-labeled Hsp60 associates with mtHsp70 His (Fig. 6D, lane 6). We conclude that mtHsp70 binds to two different assembly stages of Hsp60, revealing an important role of mtHsp70 in the formation of the Hsp60 complexes.
MtHsp70 Cooperates with Hsp10 to Promote the Formation of the Hsp60 Ring Structures-We wondered whether Hsp10 functions in the assembly of Hsp60 complexes as well. To this end, we imported 35 S-labeled Hsp60 into hsp10ts mitochondria (44). Indeed, similar to ssc1-42 mitochondria, the assembly of Hsp60 into the high molecular weight complexes is compromised in hsp10ts mitochondria, whereas the overall import efficiency is not altered (Fig. 7A). Steady-state levels of Hsp60 complexes in hsp10ts mitochondria are unaffected (Fig. 3C), pointing to a direct role of Hsp10 in the biogenesis of Hsp60. To substantiate a role of Hsp10 in the biogenesis of Hsp60 complexes, we wondered whether Hsp10 is capable to bind to the unassembled Hsp60 precursor. Therefore, we imported 35 Slabeled Hsp60 at 2°C to prevent formation of the mature Hsp60 complexes (Fig. 5A). Under these conditions, the 35 S-labeled Hsp60 precursor is coimmunoprecipitated by antisera against Hsp10, indicating that Hsp10 interacts with an unassembled   35 S-labeled Hsp60 was imported into WT and hsp10ts mitochondria at the indicated time points. Proteins were analyzed by SDS-PAGE (SDS) or blue native (BN) electrophoresis and autoradiography. Where indicated, the membrane potential (⌬) was dissipated prior to import. B, 35 S-labeled Hsp60 was imported into WT mitochondria at 2°C, followed by coimmunoprecipitation with antibodies against Hsp10 (␣Hsp10) or preimmune serum (PI). Load, 1%; elution, 100%. C, 35 S-labeled Hsp60 was imported into WT and hsp60ts mitochondria (Mito) at 2°C, followed by coimmunoprecipitation with antibodies against Hsp10 or preimmune serum. Load, 1%; elution, 100%. D, 35 S-labeled Hsp60 was imported into WT and hsp60ts mitochondria at the indicated time points. After import, the samples were analyzed by SDS-PAGE or blue native electrophoresis and autoradiography. E, 35 S-labeled Hsp60 was imported into WT and mtHsp70 His mitochondria at 2°C, followed by affinity purification via Ni 2ϩ -NTA-agarose. Bound proteins were subjected to coimmunoprecipitation with antibodies against Hsp10 or preimmune serum. Load, 0.5%; elution Ni 2ϩ -NTA-agarose, 0.5%; elution coimmunoprecipitation, 100%.
form of Hsp60 (Fig. 7B, lanes 3 and 4). Supporting this view, coimmunoprecipitation of the 35 S-labeled Hsp60 precursor by Hsp10-specific antiserum is not affected in hsp60ts mitochondria (Fig. 7C, lanes 5 and 6). In contrast, the imported 35 Slabeled Hsp60 precursor does not assemble into the mature Hsp60 complexes in hsp60ts mitochondria, likely because of aggregation and malfunction of the mutated endogenous Hsp60 (Figs. 7D and 3, B and C) (44,52). We conclude that Hsp10 promotes early steps in the formation of Hsp60 complexes. This observation is supported by the increased protease accessibility of imported Hsp60 in the Hsp10 temperature-sensitive strain (hsp10ts) (44). We asked whether mtHsp70 and Hsp10 cooperate in the biogenesis of Hsp60. To address this question, we imported 35 S-labeled Hsp60 at 2°C into mtHsp70 His mitochondria to prevent the formation of mature Hsp60 complexes. Subsequently, the imported Hsp60 was copurified along with mtHsp70 His via Ni 2ϩ -NTA-agarose (Fig. 7E, lane  2). Bound proteins were eluted and subjected to coimmunoprecipitation with Hsp10-specific antibodies or their corresponding preimmune serum. Following this approach, we found that a portion of the mtHsp70-bound Hsp60 was associated with Hsp10 (Fig. 7E, lane 3). We conclude that a fraction of Hsp10 interacts with mtHsp70 to promote the biogenesis of Hsp60.

CONCLUSIONS
MtHsp70 mediates multiple functions in mitochondrial protein homeostasis (2,4). To carry out its different tasks mtHsp70 interacts with various interaction partners. Recent studies have indicated more specialized functions of mtHsp70 in the biogenesis of cytochrome c oxidase. Here mtHsp70 interacts with Mss51 and Cox4, which do not belong to the classical cochaperones of Hsp70 (38,39). These observations indicate that further interaction partners of the chaperone may exist. Indeed, we found a robust interaction of mtHsp70 with Hsp60 and Hsp10. In bacteria, an association of the Hsp70 protein DnaK with the Hsp60 homolog GroEL has been reported (65). Similar to bacteria, the interaction between the two different types of chaperones may facilitate the transfer of substrate proteins. Strikingly, the interaction of Hsp10 with mtHsp70 occurs independently of Hsp60. MtHsp70 and Hsp10 cooperate to facilitate the integration of the Hsp60 precursor into the mature Hsp60 complexes. Two populations of mtHsp70 mediate Hsp60 biogenesis. First, mtHsp70 drives the import of Hsp60 at the presequence translocase. Second, mtHsp70 and Hsp10 promote the maturation of Hsp60 complexes. We propose that coupling to dedicated partner proteins like Hsp10, Tim44, Cox4, or Mss51 enables mtHsp70 to carry out a plethora of specific functions in mitochondrial biogenesis.