Thio-modification of yeast cytosolic tRNA requires a ubiquitin-related system that resembles bacterial sulfur transfer systems

The wobble uridine in yeast cytosolic tRNA(Lys2)(UUU) and tRNA(Glu3)(UUC) undergoes a thio-modification at the second position (s(2) modification) and a methoxycarbonylmethyl modification at the fifth position (mcm(5) modification). We previously demonstrated that the cytosolic and mitochondrial iron-sulfur (Fe/S) cluster assembly machineries termed CIA and ISC, including a cysteine desulfurase called Nfs1, were essential for the s(2) modification. However, the cytosolic component that directly participates in this process remains unclear. We found that ubiquitin-like protein Urm1 and ubiquitin-activating enzyme-like protein Uba4, as well as Tuc1 and Tuc2, were strictly required for the s(2) modification. The carboxyl-terminal glycine residue of Urm1 was critical for the s(2) modification, indicating direct involvement of the unique ubiquitin-related system in this process. We also demonstrated that the s(2) and mcm(5) modifications in cytosolic tRNAs influence each other's efficiency. Taken together, our data indicate that the s(2) modification of cytosolic tRNAs is a more complex process that requires additional unidentified components.     

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.     

Functional characterization of the eukaryotic cysteine desulfurase Nfs1p from Saccharomyces cerevisiae

Previous studies have indicated that the essential protein Nfs1 performs a crucial role in cellular iron-sulfur (Fe/S) protein maturation. The protein is located predominantly in mitochondria, yet low amounts are present in cytosol and nucleus. Here we examined several aspects concerning the molecular function of yeast Nfs1p as a model protein. First, we demonstrated that purified Nfs1p facilitates the in vitro assembly of Fe/S proteins by using cysteine as its specific substrate. Thus, eukaryotic Nfs1 is a functional orthologue of the bacterial cysteine desulfurase IscS. Second, we showed that only the mitochondrial version but not the extramitochondrial version of Nfs1p is functional in generating cytosolic and nuclear Fe/S proteins. Mutation of the nuclear targeting signal of Nfs1p did not affect the maturation of cytosolic and nuclear Fe/S proteins, despite a severe growth defect under this condition. Nfs1p could not assemble an Fe/S cluster on the Isu scaffold proteins when they were located in the yeast cytosol. The lack of function of these central Fe/S cluster assembly components suggests that the maturation of extramitochondrial Fe/S protein does not involve functional copies of the mitochondrial Fe/S cluster assembly machinery in the yeast cytosol. Third, the extramitochondrial version of Nfs1p was shown to play a direct role in the thiomodification of tRNAs. Finally, we identified a highly conserved N-terminal beta-sheet of Nfs1p as a functionally essential part of the protein. The implication of these findings for the structural stability of Nfs1p and for its targeting mechanism to mitochondria and cytosol/nucleus will be discussed.     

The yeast scaffold proteins Isu1p and Isu2p are required inside mitochondria for maturation of cytosolic Fe/S proteins

Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.     

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.     

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.     

Mechanisms of iron-sulfur protein maturation in mitochondria, cytosol and nucleus of eukaryotes

Iron-sulfur (Fe/S) clusters are important cofactors of numerous proteins involved in electron transfer, metabolic and regulatory processes. In eukaryotic cells, known Fe/S proteins are located within mitochondria, the nucleus and the cytosol. Over the past years the molecular basis of Fe/S cluster synthesis and incorporation into apoproteins in a living cell has started to become elucidated. Biogenesis of these simple inorganic cofactors is surprisingly complex and, in eukaryotes such as Saccharomyces cerevisiae, is accomplished by three distinct proteinaceous machineries. The "iron-sulfur cluster (ISC) assembly machinery" of mitochondria was inherited from the bacterial ancestor of mitochondria. ISC components are conserved in eukaryotes from yeast to man. The key principle of biosynthesis is the assembly of the Fe/S cluster on a scaffold protein before it is transferred to target apoproteins. Cytosolic and nuclear Fe/S protein maturation also requires the function of the mitochondrial ISC assembly system. It is believed that mitochondria contribute a still unknown compound to biogenesis outside the organelle. This compound is exported by the mitochondrial "ISC export machinery" and utilised by the "cytosolic iron-sulfur protein assembly (CIA) machinery". Components of these two latter systems are also highly conserved in eukaryotes. Defects in the mitochondrial ISC assembly and export systems, but not in the CIA machinery have a strong impact on cellular iron uptake and intracellular iron distribution showing that mitochondria are crucial for both cellular Fe/S protein assembly and iron homeostasis.     

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.     

Depletion of thiol reducing capacity impairs cytosolic but not mitochondrial iron-sulfur protein assembly machineries

Iron‑sulfur (Fe/S) clusters are versatile inorganic cofactors that play central roles in essential cellular functions, from respiration to genome stability. >30 proteins involved in Fe/S protein biogenesis in eukaryotes are known, many of which bind clusters via cysteine residues. This opens up the possibility that the thiol-reducing glutaredoxin and thioredoxin systems are required at both the Fe/S biogenesis and target protein level to counteract thiol oxidation. To address the possible interplay of thiol redox chemistry and Fe/S protein biogenesis, we have characterized the status of the mitochondrial (ISC) and cytosolic (CIA) Fe/S protein assembly machineries in Saccharomyces cerevisiae mutants in which the three partially redundant glutathione (Glr1) and thioredoxin (Trr1 and Trr2) oxidoreductases have been inactivated in either mitochondria, cytosol, or both compartments. Cells devoid of mitochondrial oxidoreductases maintained a functional mitochondrial ISC machinery and showed no altered iron homeostasis despite a non-functional complex II of the respiratory chain due to redox-specific defects. In cells that lack either cytosolic or total cellular thiol reducing capacity, both the ISC system and iron homeostasis were normal, yet cytosolic and nuclear Fe/S target proteins were not matured. This dysfunction could be attributed to a failure in the assembly of [4Fe‑4S] clusters in the CIA factor Nar1, even though Nar1 maintained robust protein levels and stable interactions with later-acting CIA components. Overall, our analysis has uncovered a hitherto unknown thiol-dependence of the CIA machinery and has demonstrated the surprisingly varying sensitivity of Fe/S proteins to thiol oxidation.     

The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins.

Iron-sulfur (Fe/S) cluster-containing proteins catalyse a number of electron transfer and metabolic reactions. Little is known about the biogenesis of Fe/S clusters in the eukaryotic cell. Here, we demonstrate that mitochondria perform an essential role in the synthesis of both intra- and extra-mitochondrial Fe/S proteins. Nfs1p represents the yeast orthologue of the bacterial cysteine desulfurase NifS that initiates biogenesis by producing elemental sulfur. The matrix-localized protein is required for synthesis of both mitochondrial and cytosolic Fe/S proteins. The ATP-binding cassette (ABC) transporter Atm1p of the mitochondrial inner membrane performs an essential function only in the generation of cytosolic Fe/S proteins by mediating export of Fe/S cluster precursors synthesized by Nfs1p and other mitochondrial proteins. Assembly of cellular Fe/S clusters constitutes an indispensable biosynthetic task of mitochondria with potential relevance for an iron-storage disease and the control of cellular iron uptake.     

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.     

The hydrogenase-like Nar1p is essential for maturation of cytosolic and nuclear iron-sulphur proteins

The genome of the yeast Saccharomyces cerevisiae encodes the essential protein Nar1p that is conserved in virtually all eukaryotes and exhibits striking sequence similarity to bacterial iron-only hydrogenases. A human homologue of Nar1p was shown previously to bind prenylated prelamin A in the nucleus. However, yeast neither exhibits hydrogenase activity nor contains nuclear lamins. Here, we demonstrate that Nar1p is predominantly located in the cytosol and contains two adjacent iron-sulphur (Fe/S) clusters. Assembly of its Fe/S clusters crucially depends on components of the mitochondrial Fe/S cluster biosynthesis apparatus such as the cysteine desulphurase Nfs1p, the ferredoxin Yah1p and the ABC transporter Atm1p. Using functional studies in vivo, we show that Nar1p is required for maturation of cytosolic and nuclear, but not of mitochondrial, Fe/S proteins. Nar1p-depleted cells do not accumulate iron in mitochondria, distinguishing these cells from mutants in components of the mitochondrial Fe/S cluster biosynthesis apparatus. In conclusion, Nar1p represents a crucial, novel component of the emerging cytosolic Fe/S protein assembly machinery that catalyses an essential and ancient process in eukaryotes.     

The essential WD40 protein Cia1 is involved in a late step of cytosolic and nuclear iron-sulfur protein assembly

The assembly of cytosolic and nuclear iron-sulfur (Fe/S) proteins in yeast is dependent on the iron-sulfur cluster assembly and export machineries in mitochondria and three recently identified extramitochondrial proteins, the P-loop NTPases Cfd1 and Nbp35 and the hydrogenase-like Nar1. However, the molecular mechanism of Fe/S protein assembly in the cytosol is far from being understood, and more components are anticipated to take part in this process. Here, we have identified and functionally characterized a novel WD40 repeat protein, designated Cia1, as an essential component required for Fe/S cluster assembly in vivo on cytosolic and nuclear, but not mitochondrial, Fe/S proteins. Surprisingly, Nbp35 and Nar1, themselves Fe/S proteins, could assemble their Fe/S clusters in the absence of Cia1, demonstrating that these components act before Cia1. Consequently, Cia1 is involved in a late step of Fe/S cluster incorporation into target proteins. Coimmunoprecipitation assays demonstrated a specific interaction between Cia1 and Nar1. In contrast to the mostly cytosolic Nar1, Cia1 is preferentially localized to the nucleus, suggesting an additional function of Cia1. Taken together, our results indicate that Cia1 is a new member of the cytosolic Fe/S protein assembly (CIA) machinery participating in a step after Nbp35 and Nar1.     

Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe–S cluster biogenesis regulation

Iron–sulfur (Fe–S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe–S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe–S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe–S cluster biogenesis does not meet cellular demands.     

Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins

Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins.     

The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation

The mitochondrial Hsp70 chaperone Ssq1 plays a dedicated role in the maturation of iron–sulfur (Fe/S) proteins, an essential process of mitochondria. Similar to its bacterial orthologue HscA, Ssq1 binds to the scaffold protein Isu1, thereby facilitating dissociation of the newly synthesized Fe/S cluster on Isu1 and its transfer to target apoproteins. Here we use in vivo and in vitro approaches to show that Ssq1 also interacts with the monothiol glutaredoxin 5 (Grx5) at a binding site different from that of Isu1. Grx5 binding does not stimulate the ATPase activity of Ssq1 and is most pronounced for the ADP-bound form of Ssq1, which interacts with Isu1 most tightly. The vicinity of Isu1 and Grx5 on the Hsp70 chaperone facilitates rapid Fe/S cluster transfer from Isu1 to Grx5. Grx5 and its bound Fe/S cluster are required for maturation of all cellular Fe/S proteins, regardless of the type of bound Fe/S cofactor and subcellular localization. Hence Grx5 functions as a late-acting component of the core Fe/S cluster (ISC) assembly machinery linking the Fe/S cluster synthesis reaction on Isu1 with late assembly steps involving Fe/S cluster targeting to dedicated apoproteins.     

Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin,a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA.     

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.