mtHsp70 and the individual domains of mtHsp70 fold rapidly upon import into mitochondria
To analyze the biogenesis of mtHsp70 in mitochondria, we established an assay monitoring the folding state of newly imported mtHsp70 in mitochondria. After import of the radiolabeled mtHsp70 precursor protein into isolated mitochondria and solubilization of mitochondria, the folding state of imported mtHsp70 was assessed by treatment with trypsin because the folded native mtHsp70 is trypsin resistant (Sichting et al., 2005
). Indeed, almost all imported mtHsp70 was protected against trypsin, suggesting that the imported mtHsp70 folds rapidly in organello ().
Figure 1. The chaperone mtHsp70 and its individual domains fold rapidly in organello. (A–E) Folding of mtHsp70 (A), PBD-DHFR (B), ATPase-DHFR (C), ATPaseLinker-DHFR (D), and ATPaseA4-DHFR (E) upon import into mitochondria. Radiolabeled precursor proteins (more ...)
We also tested the de novo folding of the individual mtHsp70 domains. To mimic the two-domain structure of full-length mtHsp70, the individual domains were analyzed in the context of dihydrofolate reductase (DHFR) fusion proteins, from now on referred to as PBD-DHFR and ATPase-DHFR, respectively, in which the PBD and the ATPase domain were each fused with an 80-aa-residue-long spacer to the mouse DHFR. Trypsin treatment of imported PBD-DHFR generated two protease-resistant fragments, corresponding to the PBD and the DHFR domain (). The kinetics of import of PBD-DHFR and of the formation of the protease-resistant fragments were very similar, indicating that the PBD-DHFR folds immediately after its import into the mitochondrial matrix. In the case of the ATPase-DHFR construct, the DHFR domain was protease resistant, but the ATPase domain was almost completely protease sensitive (). Thus, the ATPase domain does not fold properly in the context of the DHFR fusion protein. On the contrary, the PBD folds independently of the ATPase domain.
Because the interdomain linker between the ATPase domain and PBD is required for the communication between both domains and affects the native conformation of the ATPase domain, we asked whether the interdomain linker has an effect on the de novo folding of the ATPase domain. We followed the folding of imported ATPaseLinker-DHFR protein and tested in parallel, as a control, the folding of the ATPaseA4-DHFR variant, in which the linker amino acid residues 412–415 were replaced by four alanine residues. The ATPase domain in imported ATPaseLinker-DHFR but not in the ATPaseA4-DHFR variant folded into a trypsin-resistant conformation (). In both cases, the folded DHFR domain was protease resistant. We conclude that the folding of the ATPase domain depends on the presence of the interdomain linker. This is supported by experiments using a variant of full-length mtHsp70 protein, mtHsp70A4, in which the linker residues of mtHsp70 were likewise replaced by four alanine residues. This variant generated in the folding assay only a 35-kD stable fragment that corresponds in size to the PBD (Fig. S1
). By immunoprecipitation experiments with antibodies recognizing specifically the ATPase domain or the C terminus of mtHsp70, we confirmed the fragment to be the PBD (Fig. S1). Thus, in contrast to the PBD, the ATPase domain in the mtHsp70A4 mutant was not able to fold into a protease-resistant form.
In summary, mtHsp70 folds rapidly after its import into mitochondria. Both domains are independent folding units. The ATPase domain folds in the context of mtHsp70 only in presence of the interdomain linker, whereas the PBD folds without the interdomain linker.
Hep1 is required for the folding of the ATPase domain of mtHsp70 in organello
We asked whether other components are involved in the folding process of mtHsp70 in the cell. Because Hep1 interacts with mtHsp70 and is crucial for maintaining native mtHsp70 in its functional state (Sichting et al., 2005
), we analyzed whether Hep1 also acts in the folding of mtHsp70. To this end, we imported radiolabeled mtHsp70 into mitochondria lacking Hep1 and checked its folding state by treating mitochondrial extracts with trypsin. A 35-kD stable fragment of mtHsp70 was observed but not the full-length mtHsp70 (). Thus, in contrast to wild-type mitochondria, the folding of mtHsp70 was impaired in mitochondria lacking Hep1. The stable fragment could be immunoprecipitated with antibodies against the C terminus of mtHsp70, demonstrating that it corresponds to the PBD (Fig. S2
). This shows that the PBD folds independently of Hep1, whereas the ATPase domain has a folding defect in the absence of Hep1.
Figure 2. Folding of mtHsp70 in organello depends on the Hep1 chaperone. (A–C) Folding of mtHsp70 (A), ATPaseLinker-DHFR (B), and PBD-DHFR (C) upon import into mitochondria lacking Hep1. Radiolabeled precursor proteins were imported into Δhep1 and (more ...)
To corroborate these findings, we assessed the folding of the two mtHsp70 constructs, ATPaseLinker-DHFR and PBD-DHFR, in Δhep1 mitochondria. The ATPaseLinker domain did not become folded, in contrast to the situation with wild-type mitochondria (). As expected, the PBD was able to fold also in the Hep1-deficient mitochondria ().
In conclusion, these results demonstrate that the chaperone Hep1 is required for de novo folding of mtHsp70 in organello. It mediates the correct folding of the ATPase domain plus linker of mtHsp70.
Folding of mtHsp70 is independent of Hsp60, Hsp78, and the mtHsp70 chaperone system
The mitochondrial matrix contains several chaperones promoting protein folding such as the Hsp60 chaperonin, the mtHsp70 chaperone system, and Hsp78, a member of the Clp/Hsp100 family (Voos, 2009
). Do these mitochondrial chaperones assist in the de novo folding of mtHsp70? We addressed this question by using strains harboring deletions or temperature-sensitive mutants of these chaperones. Folding of mtHsp70 was not affected in the mif4
temperature-sensitive mutant (). In this mutant, Hsp60 does not assemble and therefore aggregates at nonpermissive temperature, as previously reported (Cheng et al., 1990
). Moreover, imported mtHsp70 folded properly with comparable kinetics in the presence and absence of Hsp78 (). Thus, neither Hsp60 nor Hsp78 are crucial for folding of mtHsp70. We also tested the mtHsp70 system itself. Deletion of the J domain cochaperone Mdj1 did not affect the folding rates of imported mtHsp70 (), suggesting that the folding of mtHsp70 is independent of the mtHsp70 system.
Figure 3. Defects in mitochondrial chaperone systems do not affect folding of mtHsp70. (A–C) Radiolabeled mtHsp70 precursor was imported into mitochondria isolated from the mif4 mutant cells (A), Δhsp78 cells (B), and Δmdj1 cells (C) and (more ...)
Collectively, besides Hep1, none of the tested chaperones appear to be involved in the de novo folding of mtHsp70. This suggests Hep1 to be the major, if not the only, chaperone mediating the folding of mtHsp70 in mitochondria.
Hep1 is required for de novo folding of mtHsp70 in vivo
To confirm the role of Hep1 in the biogenesis of mtHsp70 in intact cells, we performed a labeling experiment with wild-type cells and with cells lacking Hep1. Cells were grown in the presence of radioactive methionine to synthesize radiolabeled proteins. This enabled us to follow their biogenesis. Mitochondria were isolated and mitochondrial extracts were prepared. Treatment of these extracts with trypsin followed by the immunoprecipitation with antibodies directed against mtHsp70 allowed the specific detection of the folding state of the newly synthetized and imported mtHsp70 protein. In mitochondrial extracts from wild-type cells, the majority of mtHsp70 was trypsin resistant, indicating its presence in the folded state. In contrast, in mitochondrial extracts from Δhep1 cells, only a minor fraction of mtHsp70 was trypsin resistant, and a stable fragment corresponding to the PBD was generated (). Apparently, de novo folding of the ATPase domain of mtHsp70 was impaired in cells lacking Hep1. Thus, consistent with the observations in organello, Hep1 plays an important role in the de novo folding of mtHsp70 in intact cells.
Figure 4. Folding of mtHsp70 in vivo requires Hep1.
Δhep1 and wild-type (wt) cells were radiolabeled with [35S]methionine for 10 min. Then protein synthesis was stopped and mitochondria were isolated and solubilized. The lysate was incubated without and (more ...)
Hep1 is crucial for the folding of mtHsp70 in a reconstituted system
We reconstituted the folding process of mtHsp70 using purified components to elucidate the molecular mechanisms of how mtHsp70 folds in mitochondria. To this end, recombinant mtHsp70 was denatured by precipitation and resuspension in buffer containing urea. The sample was diluted in ATP-containing buffer lacking urea with and without addition of recombinant Hep1. Refolding of mtHsp70 was monitored by its trypsin resistance. In the presence of equimolar amounts of Hep1, mtHsp70 became resistant against trypsin in the timescale of minutes (, left). In contrast, virtually no trypsin-resistant full-length mtHsp70 was detected in absence of Hep1 (, right). Thus, Hep1 promotes refolding of denatured mtHsp70 in the presence of ATP in vitro. Interestingly, two fragments of mtHsp70 with apparent molecular masses of ~45 and 35 kD were observed when samples were taken from the refolding mix containing Hep1 at early time points and treated with trypsin. These fragments represent folding intermediates of mtHsp70 that correspond, according to their molecular masses, to the ATPase domain and the PBD, respectively. Apparently, each of the domains of mtHsp70 adopts independently a trypsin-resistant conformation in the de novo folding of mtHsp70 before the full-length protein folds into its native, fully protease-protected state. Upon incubation in the absence of Hep1, only the 35-kD fragment was detected, indicating the presence of a folded PBD. This suggests that the PBD is able to fold without any chaperone, whereas the ATPase domain requires Hep1 for its folding in vitro.
Figure 5. Isolated mtHsp70 folds in the presence of isolated Hep1 and adenine nucleotides into its active form. (A) Unfolded mtHsp70 was diluted into ATP-containing buffer with (left) or without (right) the addition of recombinant Hep1. At the time points indicated, (more ...)
Is the rate of refolding of mtHsp70 dependent on the ratio of unfolded mtHsp70 to the added Hep1? The kinetics of refolding was measured at molar ratios of mtHsp70 to Hep1 of 1:1 to 1:0.25 over a period of 120 min. The initial rates of refolding increased with increasing amount of Hep1; the efficiency of refolding, however, was at the same level of ~100% in the samples with a molar ratio of mtHsp70 to Hep1 of 1:0.5 and of 1:1 (). In samples with a lower Hep1 concentration and a molar ratio of 1:0.25, the efficiency of refolding was ~85%. During incubation with Hep1, mtHsp70 may adopt a conformation that is refractory to refolding and/or Hep1 may lose functional activity. In summary, the data suggest that a Hep1 molecule can support folding of more than one mtHsp70 molecule.
To test whether folding of mtHsp70 in vitro leads to acquisition of catalytic activity, we performed an ATPase activity assay with purified native mtHsp70 and with fractions of mtHsp70 that were subjected to the refolding procedure in the presence and absence of Hep1. mtHsp70 refolded in the presence of Hep1 regained ~70% of the activity of the native mtHsp70 protein (). In contrast, mtHsp70 remained inactive when subjected to the refolding procedure in the absence of Hep1 (). Thus, the trypsin-resistant conformation adopted by mtHsp70 upon refolding in the presence of Hep1 obviously represents the active form of the protein.
Are nucleotides involved in the folding process? To address this, we tested the effects of different nucleotides on the folding of mtHsp70 in the presence of Hep1. ATP and ADP promoted the folding of mtHsp70 to the protease-resistant, native form with similar efficiencies. In the presence of a nonhydrolyzable ATP analogue, ATPγS, mtHsp70 became trypsin resistant as well, albeit with slightly reduced efficiency. Trypsin-protected mtHsp70 was formed very inefficiently in the presence of AMP-PNP, another nonhydrolyzable ATP analogue (). Folded mtHsp70 was also not observed in the absence of any nucleotides. In conclusion, the de novo folding of mtHsp70 requires nucleotides in addition to Hep1. Because ADP and ATPγS act similarly to ATP, it is rather the binding of nucleotides than the hydrolysis of ATP that appears to be needed for folding. The different effects of the nucleotides suggest that a certain conformer of the ATPase domain is formed as an intermediate during the folding process, which is specifically recognized by nucleotides, thereby triggering further folding to the native state of mtHsp70.
Next, we analyzed the de novo folding of the individual domains of mtHsp70 in the reconstitution system. Denatured ATPaseLinker and PBD were diluted in buffer with and without Hep1 and ATP. The ATPaseLinker protein became trypsin resistant only in the presence of Hep1 and ATP (). In contrast, the PBD efficiently refolded even without the addition of Hep1 and ATP (). These findings are consistent with the results obtained from the analysis of the folding of the full-length protein in organello and in vitro. Apparently, it is the ATPase domain in full-length mtHsp70 that requires Hep1 and nucleotide for its de novo folding.
Can the function of Hep1 be taken over by Mge1, the nucleotide exchange factor of mtHsp70? It was previously reported that Mge1 is able to keep mtHsp70 soluble upon coexpression in Escherichia coli
(Momose et al., 2007
). Therefore, we tested the refolding of mtHsp70 in the presence of Mge1 in vitro. In contrast to Hep1, a fourfold molar excess of Mge1 compared with mtHsp70 did not result in folded mtHsp70 and ATPaseLinker (). Thus, interaction partners of mtHsp70 such as Mge1 appear not to promote de novo folding of mtHsp70. We conclude that the role of Hep1 in the folding of mtHsp70 reflects a specific chaperone function of Hep1.
Hep1 interacts with a folding intermediate of mtHsp70
A chaperone function of Hep1 in the de novo folding process of mtHsp70 implies a transient interaction of Hep1 with mtHsp70. We tested for such an interaction by chemical cross-linking with glutaraldehyde. To this end, we incubated the purified proteins in the absence or presence of ATP in the refolding buffer with or without cross-linking agent. Samples were analyzed by SDS-PAGE and Coomassie staining or immunodecoration with antibodies against Hep1 and mtHsp70. Upon incubation of mtHsp70 with Hep1, a cross-linked adduct containing Hep1 and mtHsp70 was generated (, A [lanes 8, 13, and 18] and B [lanes 5 and 11]). Importantly, much more adduct was formed in the absence than in the presence of ATP (, lanes 8, 9, 13, and 14). Because nucleotides such as ATP are needed to generate folded mtHsp70, a folding intermediate containing Hep1 was most likely trapped in the refolding process in the absence of ATP. In the presence of ATP, a large fraction of the intermediate was converted to fully folded mtHsp70, which was detected as a sharp band on the Coomassie blue–stained gel and in the immunodecoration with mtHsp70 (, lane 19). The weak and diffuse signal of mtHsp70 in the lanes lacking either Hep1 or ATP probably reflects the unfolded or partly folded nature of the protein, which allowed extensive modification and intramolecular cross-linking of the protein. In contrast to Hep1, the nucleotide exchange factor Mge1 did not form a cross-linked adduct with refolding mtHsp70 (, lanes 5 and 9). Apparently, not every interaction partner of the ATPase domain of native mtHsp70 has the ability to interact with refolding mtHsp70. This lack of interaction between Mge1 and refolding mtHsp70 is consistent with the observation that Mge1 cannot replace Hep1 in the folding of mtHsp70 (). In conclusion, binding of Hep1 to a folding intermediate of mtHsp70 is a crucial step of the de novo folding pathway of mtHsp70 that precedes the nucleotide-dependent step.
Figure 6. A folding intermediate of mtHsp70 interacts with Hep1. (A and B) Unfolded mtHsp70 was diluted into the buffer containing Hep1 in twofold molar excess over mtHsp70 in the presence or absence of ATP, as indicated (A and B), or in a twofold molar excess (more ...)
We further analyzed the effect of nucleotides on the formation of the cross-linked adduct. Addition of ATP or ADP after formation of the mtHsp70–Hep1 complex led to release and addition of ATPγS to partial release of mtHsp70 from Hep1, whereas almost no mtHsp70 dissociated from Hep1 upon addition of AMP-PNP (). These observations are consistent with the results obtained for the folding of mtHsp70 in the presence of various nucleotides ().
In summary, our results confirm a transient interaction of Hep1 with mtHsp70 during de novo folding of mtHsp70. Binding of nucleotide triggers release of Hep1 and further folding of the intermediate to native mtHsp70. Thus, Hep1 fulfils a crucial chaperone function in the de novo folding pathway of mtHsp70.