Genetic analysis of complex interactions among components of the mitochondrial import motor and translocon in Saccharomyces cerevisiae

A highly conserved, Hsp70-based, import motor, which is associated with the translocase on the matrix side of the inner mitochondrial membrane, is critical for protein translocation into the matrix. Hsp70 is tethered to the translocon via interaction with Tim44. Pam18, the J-protein co-chaperone, and Pam16, a structurally related protein with which Pam18 forms a heterodimer, are also critical components of the motor. Their N termini are important for the heterodimer's translocon association, with Pam18's and Pam16's N termini interacting in the intermembrane space and the matrix, respectively. Here, using the model organism Saccharomyces cerevisiae, we report the identification of an N-terminal segment of Tim44, important for association of Pam16 with the translocon. We also report that higher amounts of Pam17, a nonessential motor component, are found associated with the translocon in both PAM16 and TIM44 mutants that affect their interaction with one another. These TIM44 and PAM16 mutations are also synthetically lethal with a deletion of PAM17. In contrast, a deletion of PAM17 has little, or no genetic interaction with a PAM18 mutation that affects translocon association of the Pam16:Pam18 heterodimer, suggesting a second role for the Pam16:Tim44 interaction. A similar pattern of genetic interactions and enhanced Pam17 translocon association was observed in the absence of the C terminus of Tim17, a core component of the translocon. We suggest the Pam16:Tim44 interaction may play two roles: (1) tethering the Pam16:Pam18 heterodimer to the translocon and (2) positioning the import motor for efficient engagement with the translocating polypeptide along with Tim17 and Pam17.     

Interaction of the J-protein heterodimer Pam18/Pam16 of the mitochondrial import motor with the translocon of the inner membrane

Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial 70-kDa heat-shock protein (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18(IMS)) with Tim17, and the direct interaction of the J-domain of Pam18 with the J-like domain of Pam16. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in the N terminus of Pam16 were isolated and found to be due to mutations in a short segment of TIM44, the gene encoding the peripheral membrane protein that tethers mtHsp70 to the translocon. These data suggest a model in which Tim44 serves as a scaffold for precise positioning of mtHsp70 and its cochaperone Pam18 at the translocon.     

The presequence translocase-associated protein import motor of mitochondria. Pam16 functions in an antagonistic manner to Pam18

Transport of preproteins into the mitochondrial matrix requires the presequence translocase of the inner membrane (TIM23 complex) and the presequence translocase-associated motor (PAM). The motor consists of five essential subunits, the mitochondrial heat shock protein 70 (mtHsp70) and four cochaperones, the nucleotide exchange-factor Mge1, the translocase-associated fulcrum Tim44, the J-protein Pam18, and Pam16. Pam16 forms a complex with Pam18 and displays similarity to J-proteins but lacks the canonical tripeptide motif His-Pro-Asp (HPD). We report that Pam16 does not function as a typical J-domain protein but, rather, antagonizes the function of Pam18. Pam16 specifically inhibits the Pam18-mediated stimulation of the ATPase activity of mtHsp70. The inclusion of the HPD motif in Pam16 does not confer the ability to stimulate mtHsp70 activity. Pam16-HPD fully substitutes for wild-type Pam16 in vitro and in vivo but is not able to replace Pam18. Pam16 represents a new type of cochaperone that controls the stimulatory effect of the J-protein Pam18 and regulates the interaction of mtHsp70 with precursor proteins during import into mitochondria.     

Comparative Analysis of Putative Orthologues of Mitochondrial Import Motor Subunit: Pam18 and Pam16 in Plants

Pam18/Tim14 and Pam16/Tim16, highly conserved proteins among eukaryotes, are two essential subunits of protein import motors localized in the inner mitochondrial membrane. The heterodimer formed by Pam18 and Pam16 via their J-type domains serves a regulatory function in protein translocation. Here, we report that thirty-one Pam18 and twenty-six Pam16 putative orthologues in twelve plant species were identified and analyzed through bioinformatics strategy. Results data revealed that Pam18 and Pam16 were also highly conserved among plants including their J-type domains within the hydrophilic region. Key amino acid residues and an HPD motif of Pam18 were identical among the orthologues except OsPam18L5. N-myristoylation sites of Pam18 and casein kinase II phosphorylation sites of Pam 16 were more abundant, which might be important functional sites. Some Pam18 and Pam16 proteins contained a transmembrane region at the N-terminal region. Sub-cellular prediction results indicated that many orthologues localized at mitochondria. Gene expression analyses revealed that Pam18 and Pam16 in Arabidopsis might play roles in senescence and abiotic stress responses. Our detailed study provides a better understanding of Pam18 and Pam16 in plant kingdom.     

Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor

Import of mitochondrial matrix proteins involves the general translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated motor (PAM) drives the completion of preprotein translocation into the matrix. Five subunits of PAM are known: the preprotein-binding matrix heat shock protein 70 (mtHsp70), the nucleotide exchange factor Mge1, Tim44 that directs mtHsp70 to the inner membrane, and the membrane-bound complex of Pam16-Pam18 that regulates the ATPase activity of mtHsp70. We have identified a sixth motor subunit. Pam17 (encoded by the open reading frame YKR065c) is anchored in the inner membrane and exposed to the matrix. Mitochondria lacking Pam17 are selectively impaired in the import of matrix proteins and the generation of an import-driving activity of PAM. Pam17 is required for formation of a stable complex between the cochaperones Pam16 and Pam18 and promotes the association of Pam16-Pam18 with the presequence translocase. Our findings suggest that Pam17 is required for the correct organization of the Pam16-Pam18 complex and thus contributes to regulation of mtHsp70 activity at the inner membrane translocation site.     

The Pam18/Tim14-Pam16/Tim16 complex of the mitochondrial translocation motor: the formation of a stable complex from marginally stable proteins

The vast majority of mitochondrial proteins are imported from the cytosol. For matrix-localized proteins, the final step of translocation across the inner membrane is mediated by the mitochondrial translocation motor, of which mhsp70 is a key component. The ATP-dependent function of mhsp70 is regulated by a complex, composed of a J-protein (called Pam18 or Tim14) and a J-like protein (called Pam16 or Tim16), and the nucleotide exchange factor Mge1. In this study, we investigated the structural properties of a recombinant purified Pam18/Tim14-Pam16/Tim16 complex using cross-linking with the bifunctional reagent DSS and CD-spectroscopy. The results of the study show that both Pam18/Tim14 and Pam16/Tim16 are thermally unstable proteins that unfold at very low temperatures (T(m) values of 16.5 degrees C and 29 degrees C, respectively). Upon mixing the proteins in vitro, or when both proteins are co-overexpressed in bacteria, Pam18/Tim14 and Pam16/Tim16 form a heterodimer that is thermally more stable than the individual proteins (T(m) = 41 degrees C). Analysis of the properties of the complex in GdnHCl shows that dissociation of the heterodimer is the limiting step in achieving full denaturation.     

Toc12, a novel subunit of the intermembrane space preprotein translocon of chloroplasts

Translocation of proteins across membranes is essential for the biogenesis of each cell and is achieved by proteinaceous complexes. We analyzed the translocation complex of the intermembrane space from chloroplasts and identified a 12-kDa protein associated with the Toc machinery. Toc12 is an outer envelope protein exposing a soluble domain into the intermembrane space. Toc12 contains a J-domain and stimulates the ATPase activity of DnaK. The conformational stability and the ability to stimulate Hsp70 are dependent on a disulfide bridge within the loop region of the J-domain, suggesting a redox-regulated activation of the chaperone. Toc12 is associated with Toc64 and Tic22. Its J-domain recruits the Hsp70 of outer envelope membrane to the intermembrane space translocon and facilitates its interaction to the preprotein.     

Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity

The molecular chaperone and cytoprotective activities of the Hsp70 and Hsp40 chaperones represent therapeutic targets for human diseases such as cancer and those that arise from defects in protein folding; however, very few Hsp70 and no Hsp40 modulators have been described. Using an assay for ATP hydrolysis, we identified and screened small molecules with structural similarity to 15-deoxyspergualin and NSC 630668-R/1 for their effects on endogenous and Hsp40-stimulated Hsp70 ATPase activity. Several of these compounds modulated Hsp70 ATPase activity, consistent with the action of NSC 630668-R/1 observed previously (Fewell, S. W., Day, B. W., and Brodsky, J. L. (2001) J. Biol. Chem. 276, 910-914). In contrast, three compounds inhibited the ability of Hsp40 to stimulate Hsp70 ATPase activity but did not affect the endogenous activity of Hsp70. Two of these agents also compromised the Hsp70/Hsp40-mediated post-translational translocation of a secreted pre-protein in vitro. Together, these data indicate the potential for continued screening of small molecule Hsp70 effectors and that specific modulators of Hsp70-Hsp40 interaction can be obtained, potentially for future therapeutic use.     

Jid1 is a J-protein functioning in the mitochondrial matrix, unable to directly participate in endoplasmic reticulum associated protein degradation

J-proteins are a class of molecular chaperones that serve to stimulate the activity of Hsp70s and are often located to recruit Hsp70 to a particular cellular function. Protein degradation associated with the endoplasmic reticulum (ERAD) is one such cellular process that requires Hsp70 on both faces of the endoplasmic reticulum. At least five J-proteins, including Jid1 (DnaJ protein Involved in ER-associated Degradation), have been implicated in controlling ERAD. Here we show that Jid1 is confined within the mitochondrial matrix - Jid1 has the same topology as the J-proteins Pam18 and Mdj2, which stimulate mitochondrial Hsp70 to drive protein import into the mitochondrial matrix. The location of Jid1 within mitochondria makes it unavailable to participate directly in the regulation of ERAD.     

Pam16 has an essential role in the mitochondrial protein import motor

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.     

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.     

Dynamics of the regulation of Hsp90 by the co-chaperone Sti1

In eukaryotic cells, Hsp90 chaperones assist late folding steps of many regulatory protein clients by a complex ATPase cycle. Binding of clients to Hsp90 requires prior interaction with Hsp70 and a transfer reaction that is mediated by the co-chaperone Sti1/Hop. Sti1 furthers client transfer by inhibiting Hsp90's ATPase activity. To better understand how Sti1 prepares Hsp90 for client acceptance, we characterized the interacting domains and analysed how Hsp90 and Sti1 mutually influence their conformational dynamics using hydrogen exchange mass spectrometry. Sti1 stabilizes several regions in all three domains of Hsp90 and slows down dissociation of the Hsp90 dimer. Our data suggest that Sti1 inhibits Hsp90's ATPase activity by preventing N-terminal dimerization and docking of the N-terminal domain with the middle domain. Using crosslinking and mass spectrometry we identified Sti1 segments, which are in close vicinity of the N-terminal domain of Hsp90. We found that the length of the linker between C-terminal dimerization domain and the C-terminal MEEVD motif is important for Sti1 association rates and propose a kinetic model for Sti1 binding to Hsp90.     

SWI], the prion formed by the chromatin remodeling factor Swi1, is highly sensitive to alterations in Hsp70 chaperone system activity

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.     

Ancient gene duplication provided a key molecular step for anaerobic growth of Baker's yeast

Mitochondria are essential organelles required for a number of key cellular processes. As most mitochondrial proteins are nuclear encoded, their efficient translocation into the organelle is critical. Transport of proteins across the inner membrane is driven by a multicomponent, matrix-localized "import motor," which is based on the activity of the molecular chaperone Hsp70 and a J-protein cochaperone. In Saccharomyces cerevisiae, two paralogous J-proteins, Pam18 and Mdj2, can form the import motor. Both contain transmembrane and matrix domains, with Pam18 having an additional intermembrane space (IMS) domain. Evolutionary analyses revealed that the origin of the IMS domain of S. cerevisiae Pam18 coincides with a gene duplication event that generated the PAM18/MDJ2 gene pair. The duplication event and origin of the Pam18 IMS domain occurred at the relatively ancient divergence of the fungal subphylum Saccharomycotina. The timing of the duplication event also corresponds with a number of additional functional changes related to mitochondrial function and respiration. Physiological and genetic studies revealed that the IMS domain of Pam18 is required for efficient growth under anaerobic conditions, even though it is dispensable when oxygen is present. Thus, the gene duplication was beneficial for growth capacity under particular environmental conditions as well as diversification of the import motor components.     

The Hsp70 Ssz1 modulates the function of the ribosome-associated J-protein Zuo1

J-proteins are obligate partners of Hsp70s, forming a ubiquitous class of molecular chaperone machinery. The ribosome-associated Hsp70 of yeast Ssb binds nascent polypeptides as they exit the ribosome. Here we report that the ribosome-associated J-protein Zuo1 is the partner of Ssb. However, Zuo1 efficiently stimulates the ATPase activity of Ssb only when in complex with another Hsp70, Ssz1. Ssz1 binds ATP, but none of the 11 different amino acid substitutions in the ATP-binding cleft affected Ssz1 function in vivo, suggesting that neither nucleotide binding nor hydrolysis is required. We propose that Ssz1's predominant function in the cell is to facilitate Zuo1's ability to function as a J-protein partner of Ssb on the ribosome, serving as an example of an Hsp70 family member that has evolved to carry out functions distinct from that of a chaperone.     

Mitochondrial protein import motor: differential role of Tim44 in the recruitment of Pam17 and J-complex to the presequence translocase

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane translocase.     

Activation of pleiotropic drug resistance by the J-protein and Hsp70-related proteins, Zuo1 and Ssz1

Ssz1 (Pdr13) and Zuo1, members of the Hsp70 and J-protein molecular chaperone families, respectively, form a heterodimer and function on the ribosome with the Hsp70, Ssb, presumably assisting folding of newly synthesized polypeptides. As it has also been reported that Ssz1 induces pleiotropic drug resistance (PDR) when overexpressed, a possible role for Zuo1 in PDR was investigated. The C-terminal domain of Zuo1, which is dispensable for Zuo1's chaperone function on the ribosome, is both necessary and sufficient for PDR induction by Zuo1. A single domain of Ssz1, the N-terminal ATPase domain, is sufficient for PDR induction as well, indicating that Ssz1 does not function as a chaperone in PDR. No role for Ssb was found in PDR; overexpression did not affect PDR, nor was its presence required for Ssz1's or Zuo1's effect on PDR. As our results also indicate that Ssz1 and Zuo1 must be free of ribosomes to induce PDR, we propose that Ssz1's and Zuo1's function in PDR is distinct from their role as ribosome-associated co-chaperones and may be regulatory in nature.     

Stimulation of the weak ATPase activity of human hsp90 by a client protein.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the folding and assembly of a limited set of "client" proteins, many of which are involved in signal transduction pathways. In vivo, it is found in complex with additional proteins, including the chaperones Hsp70, Hsp40, Hip and Hop (Hsp-interacting and Hsp-organising proteins, respectively), as well as high molecular mass immunophilins, such as FKBP59, and the small acidic protein p23. The role of these proteins in Hsp90-mediated assembly processes is poorly understood. It is known that ATP binding and hydrolysis are essential for Hsp90 function in vivo and in vitro.Here we show, for the first time, that human Hsp90 has ATPase activity in vitro. The ATPase activity is characterised using a sensitive assay based on a chemically modified form of the phosphate-binding protein from Escherichia coli. Human Hsp90 is a very weak ATPase, its activity is significantly lower than that of the yeast homologue, and it has a half-life of ATP hydrolysis of eight minutes at 37 degrees C. Using a physiological substrate of Hsp90, the ligand-binding domain of the glucocorticoid receptor, we show that this "client" protein can stimulate the ATPase activity up to 200-fold. This effect is highly specific and unfolded or partially folded proteins, which are known to bind to Hsp90, do not affect the ATPase activity. In addition, the peroxisome proliferator-activated receptor, which is related in both sequence and structure to the glucocorticoid receptor but which does not bind Hsp90, has no observable effect on the ATPase activity.We establish the effect of the co-chaperones Hop, FKBP59 and p23 on the basal ATPase activity as well as the client protein-stimulated ATPase activity of human Hsp90. In contrast with the yeast system, human Hop has little effect on the basal rate of ATP hydrolysis but significantly inhibits the client-protein stimulated rate. Similarly, FKBP59 has little effect on the basal rate but stimulates the client-protein stimulated rate further. In contrast, p23 inhibits both the basal and stimulated rates of ATP hydrolysis.Our results show that the ATPase activity of human Hsp90 is highly regulated by both client protein and co-chaperone binding. We suggest that the rate of ATP hydrolysis is critical to the mode of action of Hsp90, consistent with results that have shown that both over and under-active ATPase mutants of yeast Hsp90 have impaired function in vivo. We suggest that the tight regulation of the ATPase activity of Hsp90 is important and allows the client protein to remain bound to Hsp90 for sufficient time for activation to occur.     

Mitochondrial protein biogenesis: The essential functions of chaperones and their cofactors during preprotein translocation

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.     

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.