The mitochondrial proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in mitochondria

Mitochondria of the yeast Saccharomyces cerevisiae contain three different Hsp70 chaperones, Ssc1, Ecm10 and Ssq1. Ssc1 is an essential protein that mediates the import of nuclear-encoded proteins into the organelle and their subsequent folding. The nucleotide state of Ssc1 is thereby regulated by the nucleotide exchange factor Mge1. Here, we show that Mge1 interacts with Ssq1 in an ATP-dependent manner, suggesting that Mge1 also regulates Ssq1 function. In contrast to Ssc1, Ssq1 does not associate with the Tim44 subunit of the protein translocating complex, indicating a different function of both chaperones. Mutants in Ssq1 were reported to have low levels of iron sulfur (FeS) cluster-containing enzymes. Employing an assay that allowed us to monitor the conversion of the apoform of mitochondrial ferredoxin into its FeS-containing holoform, Ssq1 was demonstrated to be required for the FeS cluster assembly in mitochondria. The mitochondrial DnaJ homolog Jac1 is crucial for this process, whereas Mdj1 function is dispensable. Furthermore, the presence of frataxin is necessary for FeS cluster assembly into ferredoxin suggesting a role for frataxin at the level of the formation of holo-ferredoxin.     

The Hsp70 peptide-binding domain determines the interaction of the ATPase domain with Tim44 in mitochondria

Ssc1, a molecular chaperone of the Hsp70 family, drives preprotein import into the mitochondrial matrix by a specific interaction with the translocase component Tim44. Two other mitochondrial Hsp70s, Ssc3 (Ecm10) and Ssq1, show high sequence homology to Ssc1 but fail to replace Ssc1 in vivo, possibly due to their inability to interact with Tim44. We analyzed the structural basis of the Tim44 interaction by the construction of chimeric Hsp70 proteins. The ATPase domains of all three mitochondrial Hsp70s were shown to bind to Tim44, supporting the active motor model for the Hsp70 mechanism during preprotein translocation. The peptide-binding domain of Ssc1 sustained binding of Tim44, while the peptide-binding domains of Ssc3 and Ssq1 exerted a negative effect on the interaction of the ATPase domains with Tim44. A mutation in the peptide-binding domain of Ssc1 resulted in a similar negative effect not only on the ATPase domain of Ssc1, but also of Ssq1 and Ssc3. Hence, the determination of a crucial Hsp70 function via the peptide-binding domain suggests a new regulatory principle for Hsp70 domain cooperation.     

Ecm10, a novel hsp70 homolog in the mitochondrial matrix of the yeast Saccharomyces cerevisiae

Members of the heat shock protein 70 (Hsp70) family are found in most of the compartments of eukaryotic cells where they play essential roles in protein metabolism. In yeast mitochondria, two Hsp70 proteins are known: Ssc1 and Ssq1. We identified Ecm10 as a third Hsp70 protein in the mitochondrial matrix. Ecm10 shares 82% amino acid identity with Ssc1 and 54% with Ssq1. Overexpression of Ecm10 mitigates protein import defects in ssc1 mutants suggesting that Ecm10 can play a role in protein translocation. Like Ssc1, Ecm10 interacts with the nucleotide exchange factor Mge1 in an ATP-dependent manner. Deletion of ecm10 leads to synthetic growth defects with ssc1 mutations at low temperature. Our data suggest an overlapping function of Ecm10 and Ssc1.     

Two-step purification of mitochondrial Hsp70, Ssc1p, using Mge1(His)(6) immobilized on Ni-agarose

The most abundant mitochondrial homolog of Hsp70, Ssc1p, is involved in the import and folding of mitochondrial proteins. We have developed an easy and efficient method for purifying Ssc1p. Following a first step of anion exchange at pH 6.6, a column of Mge1(His)(6) immobilized on Ni(2+)-agarose provides an efficient second dimension that results in highly purified protein. The strong and specific interaction between Ssc1p and its cofactor protein, Mge1, ensures that primarily functional protein is isolated. Ssc1p purified by this method hydrolyzed ATP with a turnover rate of 0.3/min. The ATP hydrolysis was enhanced slightly by Mge1, about 5 times by Mdj1, and 12 times by both cofactors together. The CD spectrum of Ssc1p had a pattern and temperature dependence similar to those shown for other hsp70 homologs, with a midpoint of the major transition at approximately 70 degrees C.     

Ssq1, a mitochondrial Hsp70 involved in iron-sulfur (Fe/S) center biogenesis. Similarities to and differences from its bacterial counterpart

The results of in vivo and in organellar experiments indicate that the Hsp70 Ssq1 and the J-protein Jac1 function together to assist in the biogenesis of iron-sulfur (Fe/S) centers in the mitochondrial matrix. Here we present biochemical evidence supporting this idea. Isu, the proposed scaffold on which Fe/S centers are assembled, is a substrate for both Jac1 and Ssq1. Jac1 and Isu1 cooperatively stimulate the ATPase activity of Ssq1. In addition, Jac1 facilitates the interaction of Ssq1 with Isu1 in the presence of ATP. These findings are consistent with the role in Fe/S biogenesis previously proposed for the bacterial Hsp70 Hsc66 and J-protein Hsc20 that interact with the bacterial Isu homologue IscU. However, unlike the bacterial Hsp70, we found that Ssq1 has a high affinity for nucleotide, and shares a nucleotide exchange factor, Mge1, with a second mitochondrial Hsp70, Ssc1. Thus, whereas the bacterial and mitochondrial chaperone systems share critical features, they possess significant biochemical differences as well.     

Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins.

The mitochondrial heat shock protein Hsp70 is essential for import of nuclear-encoded proteins, involved in both unfolding and membrane translocation of preproteins. mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown. We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44. One mutant mtHsp70 (Ssc1-2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild-type kinetics, yet were impaired in unfolding of preproteins. The other mutant Hsp70 (Ssc1-3p') bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1-3p' was defective in its ATPase domain and did not undergo a nucleotide-dependent conformational change, resulting in permanent binding to Tim44. The following conclusions are suggested. (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70-Tim44 complex. Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1-2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides. (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide-dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70. This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation.     

A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.     

Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.     

Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1

Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo.     

Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.     

The interplay between components of the mitochondrial protein translocation motor studied using purified components

The final step of protein translocation across the mitochondrial inner membrane is mediated by a translocation motor composed of 1) the matrix-localized, ATP-hydrolyzing, 70-kDa heat shock protein mHsp70; 2) its anchor to the import channel, Tim44; 3) the nucleotide exchange factor Mge1; and 4) a J-domain-containing complex of co-chaperones, Tim14/Pam18-Tim16/Pam16. Despite its essential role in the biogenesis of mitochondria, the mechanism by which the translocation motor functions is still largely unknown. The goal of this work was to carry out a structure-function analysis of the mitochondrial translocation motor utilizing purified components, with an emphasis on the formation of the Tim44-mHsp70 complex. To this end, we purified Tim44 and monitored its interaction with other components of the motor using cross-linking with bifunctional reagents. The effects of nucleotides, the J-domain-containing components, and the P5 peptide (CALLSAPRR, representing part of the mitochondrial targeting signal of aspartate aminotransferase) on the formation of the translocation motor were examined. Our results show that only the peptide and nucleotides, but not J-domain-containing proteins, affect the Tim44-mHsp70 interaction. Additionally, binding of Tim44 to mHsp70 prevents the formation of a complex between the latter and Tim14/Pam18-Tim16/Pam16. Thus, mutually exclusive interactions between various components of the motor with mHsp70 regulate its functional cycle. The results are discussed in light of known models for the function of the mitochondrial translocation motor.     

The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.     

Thermal adaptation of the yeast mitochondrial Hsp70 system is regulated by the reversible unfolding of its nucleotide exchange factor

The Hsp70 protein switches during its functional cycle from an ADP-bound state with a high affinity for substrates to a low-affinity, ATP-bound state, with concomitant release of the client protein. The rate of the chaperone cycle is regulated by co-chaperones such as nucleotide exchange factors that significantly accelerate the ADP/ATP exchange. Mge1p, a mitochondrial matrix protein with homology to bacterial GrpE, serves as the nucleotide exchange factor of mitochondrial Hsp70. Here, we analyze the influence of temperature on the structure and functional properties of Mge1p from the yeast Saccharomyces cerevisiae. Mge1p is a dimer in solution that undergoes a reversible thermal transition at heat-shock temperatures, i.e. above 37 degrees C, that involves protein unfolding and dimer dissociation. The thermally denatured protein is unable to interact stably with mitochondrial Hsp70, and therefore is unable to regulate its ATPase and chaperone cycle. Crosslinking of wild-type mitochondria reveals that Mge1p undergoes the same dimer to monomer temperature-dependent shift, and that the nucleotide exchange factor does not associate with its Hsp70 partner at stress temperatures (i.e. > or =45 degrees C). Once the stress conditions disappear, Mge1p refolds and recovers both structure and functional properties. Therefore, Mge1p can act as a thermosensor for the mitochondrial Hsp70 system, regulating the nucleotide exchange rates under heat shock, as has been described for two bacterial GrpE proteins. The thermosensor activity is conserved in the GrpE-like nucleotide exchange factors although, as discussed here, it is achieved through a different structural mechanism.     

Residues of Tim44 involved in both association with the translocon of the inner mitochondrial membrane and regulation of mitochondrial Hsp70 tethering

Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.     

Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1

Hsp70 chaperones mediate folding of proteins and prevent their misfolding and aggregation. We report here on a new kind of Hsp70 interacting protein in mitochondria, Hep1. Hep1 is a highly conserved protein present in virtually all eukaryotes. Deletion of HEP1 results in a severe growth defect. Cells lacking Hep1 are deficient in processes that need the function of mitochondrial Hsp70s, such as preprotein import and biogenesis of proteins containing FeS clusters. In the mitochondria of these cells, Hsp70s, Ssc1 and Ssq1 accumulate as insoluble aggregates. We show that it is the nucleotide-free form of mtHsp70 that has a high tendency to self-aggregate. This process is efficiently counteracted by Hep1. We conclude that Hep1 acts as a chaperone that is necessary and sufficient to prevent self-aggregation and to thereby maintain the function of the mitochondrial Hsp70 chaperones.     

Primary sequence that determines the functional overlap between mitochondrial heat shock protein 70 Ssc1 and Ssc3 of Saccharomyces cerevisiae

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can "make or break" mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.     

Real‐time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP‐hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate‐binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate‐ and nucleotide‐binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide‐free state. The nucleotide‐exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.     

Mitochondrial import driving forces: enhanced trapping by matrix Hsp70 stimulates translocation and reduces the membrane potential dependence of loosely folded preproteins

The mitochondrial heat shock protein Hsp70 (mtHsp70) is essential for driving translocation of preproteins into the matrix. Two models, trapping and pulling by mtHsp70, are discussed, but positive evidence for either model has not been found so far. We have analyzed a mutant mtHsp70, Ssc1-2, that shows a reduced interaction with the membrane anchor Tim44, but an enhanced trapping of preproteins. Unexpectedly, at a low inner membrane potential, ssc1-2 mitochondria imported loosely folded preproteins more efficiently than wild-type mitochondria. The import of a tightly folded preprotein, however, was not increased in ssc1-2 mitochondria. Thus, enhanced trapping by mtHsp70 stimulates the import of loosely folded preproteins and reduces the dependence on the import-driving activity of the membrane potential, directly demonstrating that trapping is one of the molecular mechanisms of mtHsp70 action.     

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.     

A bichaperone (Hsp70-Hsp78) system restores mitochondrial DNA synthesis following thermal inactivation of Mip1p polymerase

Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae. We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system. Furthermore, the activity of purified Mip1p was also thermosensitive; however, the purified components of the mitochondrial bichaperone system (Ssc1p, Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under moderate heat shock conditions as well as to reactivate thermally inactivated Mip1p. Interestingly, the reactivation of endogenous Mip1p contributed more significantly to the restoration of mtDNA synthesis than did import of newly synthesized Mip1p from the cytosol. These observations suggest an important link between function of mitochondrial chaperones and the propagation of mitochondrial genomes under ever-changing environmental conditions.