Compensation for a defective interaction of the hsp70 ssq1 with the mitochondrial Fe-S cluster scaffold isu

Ssq1, a specialized yeast mitochondrial Hsp70, plays a critical role in the biogenesis of proteins containing Fe-S clusters through its interaction with Isu, the scaffold on which clusters are built. Two substitutions within the Ssq1 substrate binding cleft, both of which severely reduced affinity for Isu, had very different effects in vivo. Cells expressing Ssq1(F462S), which had no detectable affinity for Isu, are indistinguishable from Deltassq1 cells, underscoring the importance of the Ssq1-Isu1 interaction in vivo. In contrast, cells expressing Ssq1(V472F), whose affinity for Isu is at least 10-fold lower than that of wild-type Ssq1, had only moderately reduced Fe-S enzyme activities and increased iron levels and grew similarly to wild-type cells. Consistent with the reduced affinity for Isu, the ATPase activity of Ssq1(V472F) was stimulated less well than that of Ssq1 upon addition of Isu and Jac1, the J-protein partner of Ssq1. However, higher concentrations of Jac1 or Isu1, which form a stable complex, could compensate for this defect in stimulation of Ssq1(V472F). Expression of Isu1 was up-regulated 10-fold in ssq1(V472F) compared with wild-type cells, suggesting that formation of a Jac1-Isu1 complex can overcome a lowered affinity of Ssq1 for Isu in vivo as well as in vitro.     

Sequence-specific interaction between mitochondrial Fe-S scaffold protein Isu and Hsp70 Ssq1 is essential for their in vivo function

Isu, the scaffold for assembly of Fe-S clusters in the yeast mitochondrial matrix, is a substrate protein for the Hsp70 Ssq1 and the J-protein Jac1 in vitro. As expected for an Hsp70-substrate interaction, the formation of a stable complex between Isu and Ssq1 requires Jac1 in the presence of ATP. Here we report that a conserved tripeptide, PVK, of Isu is critical for interaction with Ssq1 because amino acid substitutions in this tripeptide inhibit both the formation of the Isu-Ssq1 complex and the ability of Isu to stimulate the ATPase activity of Ssq1. These biochemical defects correlate well with the growth defects of cells expressing mutant Isu proteins. We conclude that the Ssq1-Isu substrate interaction is critical for Fe-S cluster biogenesis in vivo. The ability of Jac1 and mutant Isu proteins to cooperatively stimulate the ATPase activity of Ssq1 was also measured. Increasing the concentration of Jac1 and mutant Isu together but not individually partially overcame the effect of the reduced affinity of the Isu mutant proteins for Ssq1. These results, along with the observation that overexpression of Jac1 was able to suppress the growth defect of an ISU mutant, support the hypothesis that Isu is "targeted" to Ssq1 by Jac1, with a preformed Jac1-Isu complex interacting with Ssq1.     

Characterization of the interaction between the J-protein Jac1p and the scaffold for Fe-S cluster biogenesis, Isu1p

Jac1p is a conserved, specialized J-protein that functions with Hsp70 in Fe-S cluster biogenesis in mitochondria of the yeast Saccharomyces cerevisiae. Although Jac1p as well as its specialized Hsp70 partner, Ssq1p, binds directly to the Fe-S cluster scaffold protein Isu, the Jac1p-Isu1p interaction is not well understood. Here we report that a C-terminal fragment of Jac1p lacking its J-domain is sufficient for interaction with Isu1p, and amino acid alterations in this domain affect interaction with Isu1p but not Ssq1p. In vivo, such JAC1 mutations had no obvious phenotypic effect. However, when present in combination with a mutation in SSQ1 that causes an alteration in the substrate binding cleft, growth was significantly compromised. Wild type Jac1p and Isu1p cooperatively stimulate the ATPase activity of Ssq1p. Jac1p mutant protein is only slightly compromised in this regard. Our in vivo and in vitro results indicate that independent interaction of Jac1p and the Isu client protein with Hsp70 is sufficient for robust growth under standard laboratory conditions. However, our results also support the idea that Isu protein can be "targeted" to Ssq1p after forming a complex with Jac1p. We propose that Isu protein targeting may be particularly important when environmental conditions place high demands on Fe-S cluster biogenesis or in organisms lacking specialized Hsp70s for Fe-S cluster biogenesis.     

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.     

The Hsp70 chaperone Ssq1p is dispensable for iron-sulfur cluster formation on the scaffold protein Isu1p

The specialized yeast mitochondrial chaperone system, composed of the Hsp70 Ssq1p, its co-chaperone J-protein Jac1p, and the nucleotide release factor Mge1p, perform a critical function in the biogenesis of iron-sulfur (Fe/S) proteins. Using a spectroscopic assay, we have analyzed the potential role of the chaperones in Fe/S cluster assembly on the scaffold protein Isu1p in vitro in the presence of the cysteine desulfurase Nfs1p. In the absence of chaperones, the kinetics of Fe/S cluster formation on Isu1p were compatible with a chemical reconstitution pathway with Nfs1p functioning as a sulfide donor. Addition of Ssq1p improved the rates of Fe/S cluster assembly 3-fold. However, this stimulatory effect of Ssq1p required neither ATP nor Jac1p and could be fully attributed to the activation of the Nfs1p desulfurase activity by Ssq1p. Furthermore, chaperone-stimulated Fe/S cluster assembly did not involve the specific interaction between Isu1p and Ssq1p, since the effect was observed with Isu1p mutant proteins defective in this interaction, suggesting that nonspecific binding of Ssq1p to Nfs1p helped to prevent its unfolding. Consistent with this idea, these Isu1p mutants were capable of binding an Fe/S cluster in vivo but failed to restore the growth and Fe/S cluster assembly defects of a Isu1p/Isu2p-deficient yeast strain. Taken together, these data suggest that Ssq1p/Jac1p/Mge1p are not important for Fe/S cluster synthesis on Isu1p. Hence, consistent with previous in vivo data, these chaperones likely function in steps subsequent to the de novo synthesis of the Fe/S cluster on Isu1p.     

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.     

Evolution of mitochondrial chaperones utilized in Fe-S cluster biogenesis

Biogenesis of Fe-S clusters is an essential process [1]. In both Escherichia coli and Saccharomyces cerevisiae, insertion of clusters into an apoprotein requires interaction between a scaffold protein on which clusters are assembled and a molecular chaperone system--an unusually specialized mitochondrial Hsp70 (mtHsp70) and its J protein cochaperone [2]. It is generally assumed that mitochondria inherited their Fe-S cluster assembly machinery from prokaryotes via the endosymbiosis of a bacterium that led to formation of mitochondria. Indeed, phylogenetic analyses demonstrated that the S. cerevisiae J protein, Jac1, and the scaffold, Isu, are orthologous to their bacterial counterparts [3, 4]. However, our analyses indicate that the specialized mtHsp70, Ssq1, is only present in a subset of fungi; most eukaryotes have a single mtHsp70, Ssc1. We propose that an Hsp70 having a role limited to Fe-S cluster biogenesis arose twice during evolution. In the fungal lineage, the gene encoding multifunctional mtHsp70, Ssc1, was duplicated, giving rise to specialized Ssq1. Therefore, Ssq1 is not orthologous to the specialized Hsp70 from E. coli (HscA), but shares a striking level of convergence at the biochemical level. Thus, in the vast majority of eukaryotes, Jac1 and Isu function with the single, multifunctional mtHsp70 in Fe-S cluster biogenesis.     

Overlapping Binding Sites of the Frataxin Homologue Assembly Factor and the Heat Shock Protein 70 Transfer Factor on the Isu Iron-Sulfur Cluster Scaffold Protein

In mitochondria FeS clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14-kDa scaffold protein, and then transferred to recipient apoproteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either Yfh1-Isu or Nfs1-Isu interactions destabilizes the complex. Cluster transfer to recipient apoprotein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that the Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of FeS cluster proteins.     

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 two mitochondrial heat shock proteins 70, Ssc1 and Ssq1, compete for the cochaperone Mge1

Two members of the heat shock protein 70 kDa (Hsp70) family, Ssc1 and Ssq1, perform important functions in the mitochondrial matrix. The essential Ssc1 is an abundant ATP-binding protein required for both import and folding of mitochondrial proteins. The function of Ssc1 is supported by an interaction with the preprotein translocase subunit Tim44, the cochaperone Mdj1, and the nucleotide exchange factor Mge1. In contrast, only limited information is available on Ssq1. So far, a basic characterization of Ssq1 has demonstrated its involvement in the maintenance of mitochondrial DNA, the maturation of the yeast frataxin (Yfh1) after import, and assembly of the mitochondrial Fe/S cluster. Here, we analyzed the biochemical properties and the interaction partners of Ssq1 in detail. Ssq1 showed typical chaperone properties by binding to unfolded substrate proteins in an ATP-regulated manner. Ssq1 was able to form a specific complex with the nucleotide exchange factor Mge1. In particular, complex formation in organello was enhanced significantly when Ssc1 was inactivated selectively. However, even under these conditions, no interaction of Ssq1 with the two other mitochondrial Hsp70-cochaperones, Tim44 and Mdj1, was observed. The Ssq1-Mge1 interaction showed a lower overall stability but the same characteristic nucleotide-dependence as the Ssc1-Mge1 interaction. A quantitative analysis of the interaction properties indicated a competition of Ssq1 with Ssc1 for binding to Mge1. Perturbation of Mge1 function or amounts resulted in direct effects on Ssq1 activity in intact mitochondria. We conclude that mitochondria represent the unique case where two Hsp70s compete for the interaction with one nucleotide exchange factor.     

Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p

The mitochondrial proteins Isu1p and Isu2p play an essential role in the maturation of cellular iron-sulfur (Fe/S) proteins in eukaryotes. By radiolabelling of yeast cells with 55Fe we demonstrate that Isu1p binds an oxygen-resistant non-chelatable Fe/S cluster providing in vivo evidence for a scaffolding function of Isu1p during Fe/S cluster assembly. Depletion of the cysteine desulfurase Nfs1p, the ferredoxin Yah1p or the yeast frataxin homologue Yfh1p by regulated gene expression causes a strong decrease in the de novo synthesis of Fe/S clusters on Isu1p. In contrast, depletion of the Hsp70 chaperone Ssq1p, its co-chaperone Jac1p or the glutaredoxin Grx5p markedly increased the amount of Fe/S clusters bound to Isu1p, even though these mitochondrial proteins are crucial for maturation of Fe/S proteins. Hence Ssq1p/Jac1p and Grx5p are required in a step after Fe/S cluster synthesis on Isu1p, for instance in dissociation of preassembled Fe/S clusters from Isu1p and/or their insertion into apoproteins. We propose a model that dissects Fe/S cluster biogenesis into two major steps and assigns its central components to one of these two steps.     

Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation

Genetic and biochemical studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide containing the IscU recognition region bound to the HscA substrate binding domain has been determined. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Additional genetic and biochemical studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.     

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.     

Yeast mitochondrial protein, Nfs1p, coordinately regulates iron-sulfur cluster proteins, cellular iron uptake, and iron distribution.

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missense NFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.     

Molecular Chaperones HscA/Ssq1 and HscB/Jac1 and Their Roles in Iron-Sulfur Protein Maturation

ABSTRACT

Genetic and biochemical studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide containing the IscU recognition region bound to the HscA substrate binding domain has been determined. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Additional genetic and biochemical studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.     

Frataxin and mitochondrial carrier proteins, Mrs3p and Mrs4p, cooperate in providing iron for heme synthesis

Frataxin is a conserved mitochondrial protein implicated in cellular iron metabolism. Deletion of the yeast frataxin homolog (YFH1) was combined with deletions of MRS3 and MRS4, mitochondrial carrier proteins implicated in iron homeostasis. As previously reported, the Deltayfh1 mutant accumulated iron in mitochondria, whereas the triple mutant (DeltaDeltaDelta) did not. When wild-type, Deltamrs3/4, Deltayfh1, and DeltaDeltaDelta strains were incubated anaerobically, all strains were devoid of heme and protected from iron and oxygen toxicity. The cultures were then shifted to air for a short time (4-5 h) or a longer time (15 h), and the evolving mutant phenotypes were analyzed (heme-dependent growth, total heme, cytochromes, heme proteins, and iron levels). A picture emerges from these data of defective heme formation in the mutants, with a markedly more severe defect in the DeltaDeltaDelta than in the individual Deltamrs3/4 or Deltayfh1 mutants (a "synthetic" defect in the genetic sense). The defect(s) in heme formation could be traced to lack of iron. Using a real time assay of heme biosynthesis, porphyrin precursor and iron were presented to permeabilized cells, and the appearance and disappearance of fluorescent porphyrins were followed. The Mrs3/4p carriers were required for rapid iron transport into mitochondria for heme synthesis, whereas there was also evidence for an alternative slower system. A different role for Yfh1p was observed under conditions of low mitochondrial iron and aerobic growth (revealed in the DeltaDeltaDelta), acting to protect bioavailable iron within mitochondria and to facilitate its use for heme synthesis.     

Characterization of iron-sulfur protein assembly in isolated mitochondria. A requirement for ATP,NADH, and reduced iron

To study the biochemical requirements for maturation of iron-sulfur (Fe/S) proteins, we have reconstituted the process in vitro using detergent extracts from Saccharomyces cerevisiae mitochondria. Efficient assembly of biotin synthase as a model Fe/S protein required anaerobic conditions, dithiothreitol, cysteine, ATP, and NADH. Cysteine is utilized by the cysteine desulfurase Nfs1p to release sulfan sulfur; ATP presumably reflects the function of the Hsp70 family chaperone Ssq1p; and NADH is used for reduction of the ferredoxin Yah1p involved in Fe/S protein biogenesis. Hence, our assay system faithfully reproduces the in vivo pathway. We have further investigated the involvement of various mitochondrial proteins suspected to participate in Fe/S protein biogenesis. In mitochondrial extracts depleted in Isa1p, Fe/S protein formation was severely decreased. A similar strong decline was observed with extracts from Delta yfh1 mitochondria, indicating that both Isa1p and the yeast frataxin homologue, Yfh1p, are crucial for biogenesis of mitochondrial Fe/S proteins. Conversely, the activities of mitochondrial extracts from Delta nfu1 cells were only moderately reduced, suggesting a dispensable role for Nfu1p. Finally, iron utilized for Fe/S protein formation was imported into the matrix of intact mitochondria in ferrous form in a membrane potential-dependent transport step. Our results represent the first in vitro reconstitution of the entire pathway of Fe/S protein maturation.     

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.     

Co‐evolution‐driven switch of J‐protein specificity towards an Hsp70 partner

Molecular mechanisms by which protein–protein interactions are preserved or lost after gene duplication are not understood. Taking advantage of the well–studied yeast mtHsp70:J–protein molecular chaperone system, we considered whether changes in partner proteins accompanied specialization of gene duplicates. Here, we report that existence of the Hsp70 Ssq1, which arose by duplication of the gene encoding multifunction mtHsp70 and specializes in iron–sulphur cluster biogenesis, correlates with functional and structural changes in the J domain of its J–protein partner Jac1. All species encoding this shorter alternative version of the J domain share a common ancestry, suggesting that all short JAC1 proteins arose from a single deletion event. Construction of a variant that extended the length of the J domain of a ‘short’ Jac1 enhanced its ability to partner with multifunctional Hsp70. Our data provide a causal link between changes in the J protein partner and specialization of duplicate Hsp70.     

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe-S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe-S cluster biogenesis.