Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.     

Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins

Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes phenotypes such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thioredoxin-like domain is fused to the glutaredoxin domain. Here we have shown that Grx3 locates at the nucleus and that the thioredoxin-like domain is required for such location. We have addressed the functional divergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mutant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thioredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can interchange their biological activities when compartment barriers are surpassed.     

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.     

Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin

Grx5 is a yeast mitochondrial protein involved in iron-sulfur biogenesis that belongs to a recently described family of monothiolic glutaredoxin-like proteins. No member of this family has been biochemically characterized previously. Grx5 contains a conserved cysteine residue (Cys-60) and a non-conserved one (Cys-117). In this work, we have purified wild type and mutant C60S and C117S proteins and characterized their biochemical properties. A redox potential of -175 mV was calculated for wild type Grx5. The pKa values obtained by titration of mutant proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for Cys-117. When Grx5 was incubated with glutathione disulfide, a transient mixed disulfide was formed between glutathione and the cystein 60 of the protein because of its low pKa. Binding of glutathione to Cys-60 promoted a decrease in the Cys-117 pKa value that triggered the formation of a disulfide bond between both cysteine residues of the protein, indicating that Cys-117 plays an essential role in the catalytic mechanism of Grx5. The disulfide bond in Grx5 could be reduced by GSH but at a rate at least 20 times slower than that observed for the reduction of glutaredoxin 1 from E. coli, a dithiolic glutaredoxin. This slow reduction rate could suggest that GSH may not be the physiologic reducing agent of Grx5. The fact that wild type Grx5 efficiently reduced a glutathiolated protein used as a substrate indicated that Grx5 may act as a thiol reductase inside the mitochondria.     

Catalysis of thiol/disulfide exchange. Glutaredoxin 1 and protein-disulfide isomerase use different mechanisms to enhance oxidase and reductase activities

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.     

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1⁺ gene encoding a putative A-type Fe-S scaffold, in addition to mas5⁺ and hsc1⁺ genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2⁺, but not isu1⁺ and ssc1⁺, complemented the growth phenotype of Δgrx5 mutant as isa1⁺ did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.     

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression of SSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.     

AtGRXcp, an Arabidopsis chloroplastic glutaredoxin, is critical for protection against protein oxidative damage

Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H2O2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage.     

Prokaryotic and eukaryotic monothiol glutaredoxins are able to perform the functions of Grx5 in the biogenesis of Fe/S clusters in yeast mitochondria

The Saccharomyces cerevisiae monothiol glutaredoxin Grx5 participates in the mitochondrial biogenesis of iron-sulfur clusters. Grx5 homologues exist in organisms from bacteria to humans. Chicken (cGRX5) and human (hGRX5) homologues contain a mitochondrial targeting sequence, suggesting a mitochondrial localization for these two proteins. We have compartmentalized the Escherichia coli and Synechocystis sp. homologues, and also cGRX5 and hGRX5, in the mitochondrial matrix of a yeast grx5 mutant. All four heterologous proteins rescue the defects of the mutant. The chicken cGRX5 gene was significantly expressed throughout the embryo stages in different tissues. These results underline the functional conservation of Grx5 homologues throughout evolution.     

An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1

Depletion of the mitochondrial matrix protein frataxin is the molecular cause of the neurodegenerative disease Friedreich ataxia. The function of frataxin is unclear, although recent studies have suggested a function of frataxin (yeast Yfh1) in iron/sulphur (Fe/S) protein biogenesis. Here, we show that Yfh1 specifically binds to the central Fe/S-cluster (ISC)-assembly complex, which is composed of the scaffold protein Isu1 and the cysteine desulphurase Nfs1. Association between Yfh1 and Isu1/Nfs1 was markedly increased by ferrous iron, but did not depend on ISCs on Isu1. Functional analyses in vivo showed an involvement of Yfh1 in de novo ISC synthesis on Isu1. Our data demonstrate a crucial function of Yfh1 in Fe/S protein biogenesis by defining its function in an early step of this essential process. The iron-dependent binding of Yfh1 to Isu1/Nfs1 suggests a role of frataxin/Yfh1 in iron loading of the Isu scaffold proteins.     

During FeS cluster biogenesis,ferredoxin and frataxin use overlapping binding sites on yeast cysteine desulfurase Nfs1

In mitochondria, cysteine desulfurase (Nfs1) plays a central role in the biosynthesis of iron–sulfur (FeS) clusters, cofactors critical for activity of many cellular proteins. Nfs1 functions both as a sulfur donor for cluster assembly and as a binding platform for other proteins functioning in the process. These include not only the dedicated scaffold protein (Isu1) on which FeS clusters are synthesized but also accessory FeS cluster biogenesis proteins frataxin (Yfh1) and ferredoxin (Yah1). Yfh1 has been shown to activate cysteine desulfurase enzymatic activity, whereas Yah1 supplies electrons for the persulfide reduction. While Yfh1 interaction with Nfs1 is well understood, the Yah1–Nfs1 interaction is not. Here, based on the results of biochemical experiments involving purified WT and variant proteins, we report that in Saccharomyces cerevisiae, Yah1 and Yfh1 share an evolutionary conserved interaction site on Nfs1. Consistent with this notion, Yah1 and Yfh1 can each displace the other from Nfs1 but are inefficient competitors when a variant with an altered interaction site is used. Thus, the binding mode of Yah1 and Yfh1 interacting with Nfs1 in mitochondria of S. cerevisiae resembles the mutually exclusive binding of ferredoxin and frataxin with cysteine desulfurase reported for the bacterial FeS cluster assembly system. Our findings are consistent with the generally accepted scenario that the mitochondrial FeS cluster assembly system was inherited from bacterial ancestors of mitochondria.     

Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive

Biogenesis of mitochondrial iron-sulfur (Fe/S) cluster proteins requires the interaction of multiple proteins with the highly conserved 14-kDa scaffold protein Isu, on which clusters are built prior to their transfer to recipient proteins. For example, the assembly process requires the cysteine desulfurase Nfs1, which serves as the sulfur donor for cluster assembly. The transfer process requires Jac1, a J-protein Hsp70 cochaperone. We recently identified three residues on the surface of Jac1 that form a hydrophobic patch critical for interaction with Isu. The results of molecular modeling of the Isu1-Jac1 interaction, which was guided by these experimental data and structural/biophysical information available for bacterial homologs, predicted the importance of three hydrophobic residues forming a patch on the surface of Isu1 for interaction with Jac1. Using Isu variants having alterations in residues that form the hydrophobic patch on the surface of Isu, this prediction was experimentally validated by in vitro binding assays. In addition, Nfs1 was found to require the same hydrophobic residues of Isu for binding, as does Jac1, suggesting that Jac1 and Nfs1 binding is mutually exclusive. In support of this conclusion, Jac1 and Nfs1 compete for binding to Isu. Evolutionary analysis revealed that residues involved in these interactions are conserved and that they are critical residues for the biogenesis of Fe/S cluster protein in vivo. We propose that competition between Jac1 and Nfs1 for Isu binding plays an important role in transitioning the Fe/S cluster biogenesis machinery from the cluster assembly step to the Hsp70-mediated transfer of the Fe/S cluster to recipient proteins.     

Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins

Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron-sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.     

The Yeast Saccharomyces cerevisiae Contains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40–52% identity and 61–76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using β-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.     

Localization and function of three monothiol glutaredoxins in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe contains two dithiol glutaredoxins (Grx1 and Grx2) and genes for three putative monothiol glutaredoxins (grx3, 4, and 5). We investigated the expression, sub-cellular localization, and functions of the three monothiol glutaredoxins. Fluorescence microscopy revealed that Grx3 is targeted to nuclear rim and endoplasmic reticulum, Grx4 primarily to the nucleus, and Grx5 to mitochondria. Null mutation of grx3 did not significantly affect growth and resistance against various oxidants, whereas grx5 mutation caused slow growth and sensitivity toward oxidants such as hydrogen peroxide, paraquat, and diamide. The grx2grx5 double mutation, deficient in all mitochondrial glutaredoxins, caused further retardation in growth and severe sensitivity toward all the oxidants tested. The grx4 mutation was not viable, suggesting a critical role of Grx4 for the physiology of S. pombe. Overproduction of Grx3 and Grx5, but not the truncated form of Grx5 without mitochondrial target sequence, severely retarded growth as Grx2 did, supporting the idea that Grx2, 3, and 5 are targeted to organellar compartments. Our results propose a distinct role for each glutaredoxin to maintain thiol redox balance, and hence the growth and stress resistance, of the fission yeast.     

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.     

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H₂O₂-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.     

A mammalian monothiol glutaredoxin, Grx3, is critical for cell cycle progression during embryogenesis

Glutaredoxins (Grxs) have been shown to be critical in maintaining redox homeostasis in living cells. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified. However, the biological and physiological functions of this group of proteins have not been well characterized. Here, we characterize a mammalian monothiol Grx (Grx3, also termed TXNL2/PICOT) with high similarity to yeast ScGrx3/ScGrx4. In yeast expression assays, mammalian Grx3s were localized to the nuclei and able to rescue growth defects of grx3grx4 cells. Furthermore, Grx3 inhibited iron accumulation in yeast grx3gxr4 cells and suppressed the sensitivity of mutant cells to exogenous oxidants. In mice, Grx3 mRNA was ubiquitously expressed in developing embryos, adult tissues and organs, and was induced during oxidative stress. Mouse embryos absent of Grx3 grew smaller with morphological defects and eventually died at 12.5 days of gestation. Analysis in mouse embryonic fibroblasts revealed that Grx3(-/-) cells had impaired growth and cell cycle progression at the G(2) /M phase, whereas the DNA replication during the S phase was not affected by Grx3 deletion. Furthermore, Grx3-knockdown HeLa cells displayed a significant delay in mitotic exit and had a higher percentage of binucleated cells. Therefore, our findings suggest that the mammalian Grx3 has conserved functions in protecting cells against oxidative stress and deletion of Grx3 in mice causes early embryonic lethality which could be due to defective cell cycle progression during late mitosis.     

The Nfs1 interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in mitochondria

Formation of iron/sulfur (Fe/S) clusters, protein translocation and protein folding are essential processes in the mitochondria of Saccharomyces cerevisiae. In a systematic approach to characterize essential proteins involved in these processes, we identified a novel essential protein of the mitochondrial matrix, which is highly conserved from yeast to human and which we termed Isd11. Depletion of Isd11 caused a strong reduction in the levels of the Fe/S proteins aconitase and the Rieske protein, and a massive decrease in the enzymatic activities of aconitase and succinate dehydrogenase. Incorporation of iron into the Fe/S protein Leu1 and formation of the Fe/S cluster containing holoform of the mitochondrial ferredoxin Yah1 were inhibited in the absence of Isd11. This strongly suggests that Isd11 is required for the assembly of Fe/S proteins. We show that Isd11 forms a stable complex with Nfs1, the cysteine desulfurase of the mitochondrial machinery for Fe/S cluster assembly. In the absence of Isd11, Nfs1 is prone to aggregation. We propose that Isd11 acts together with Nfs1 in an early step in the biogenesis of Fe/S proteins.