Biochemical analysis of essential components involved in mitochondrial and cytosolic iron-sulfur protein biogenesis in Saccharomyces cerevisiae
Iron-sulfur (Fe/S) clusters are inorganic cofactors of many proteins found in nearly all prokaryotic and eukaryotic organisms. Fe/S proteins play important roles in different cellular processes, such as electron transport, enzyme catalysis or gene regulation. Eukaryotes contain Fe/S proteins in mito...
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|Iron-sulfur (Fe/S) clusters are inorganic cofactors of many proteins found in nearly all prokaryotic and eukaryotic organisms. Fe/S proteins play important roles in different cellular processes, such as electron transport, enzyme catalysis or gene regulation. Eukaryotes contain Fe/S proteins in mitochondria, chloroplasts, cytosol and nucleus. In S. cerevisiae 3 different machineries cooperate to synthesise Fe/S proteins. The mitochondrial ISC-assembly machinery is required for maturation of all cellular Fe/S proteins, whereas the mitochondrial ISC-export and the cytosolic CIA-machineries are specifically involved in the formation of cytosolic and nuclear Fe/S proteins. In the first part of this study Isd11 was identified as a novel component of the mitochondrial ISC-assembly machinery. Isd11 is an essential protein of 11 kDa localized in the mitochondrial matrix and conserved only in eukaryotes. Depletion of Isd11 using generegulated yeast strains resulted in impaired activities of mitochondrial (e.g., aconitase, complex II) and cytosolic (Leu1) Fe/S enzymes. Strong defects were also observed in the de novo maturation of several mitochondrial, cytosolic and nuclear Fe/S proteins indicating that Isd11 is required for the biogenesis of all cellular Fe/S proteins. Here, it was shown that cells defective in Isd11 show deregulation of iron homeostasis. All these data indicated that Isd11 represents a novel component of the ISC-assembly machinery. Isd11 forms a stable complex with the cysteine desulfurase Nfs1 and also can interact with the scaffold protein Isu1. Surprisingly, Isd11 is not required for Nfs1 activity in vitro. However, depletion of Isd11 resulted in a strong reduction of Fe/S cluster formation on Isu1 indicating a function in early steps of biogenesis in vivo. Although, Isd11 is not needed for Nfs1 desulfurase activity, it is likely that the Isd11-Nfs1 complex is the physiological cysteine desulfurase, as both proteins are required for the Fe/S cluster assembly on Isu1. The second part of the present study was focused on the Nar1 protein. Nar1 is a component of the newly identified CIA-machinery. Biogenesis of extra-mitochondrial Fe/S proteins requires the CIA-machinery, which besides Nar1 encompasses at least three other proteins, Cfd1, Nbp35 and Cia1. Nar1 is an Fe/S protein itself, most likely containing two magnetically coupled Fe/S clusters. Yeast Nar1 is a highly conserved protein with relation to Feonly hydrogenases and contains eight conserved cysteines, four of them at the N-terminus and the other four at the C-terminus. Therefore, it was important to know whether the conserved cysteine residues are involved in coordination of the two Fe/S clusters. Using site-directed mutagenesis, it was demonstrated that three of the four N-terminal cysteines (C59A, C62A and C65) are essential residues for yeast cell viability and Nar1 function in the maturation of cytosolic Fe/S proteins, such as Leu1 or Rli1. Mutation of three of the N-terminal cysteines resulted in a loss of Fe/S cluster association. Mutation of the forth N-terminal cysteine residue (C20A) showed no effect, yet the combined mutation of both C20 and C65 lead to a more severe phenotype. These results indicate that all four N-terminal cysteines are ligands of an Fe/S cluster. Moreover, the data suggest that the N-terminal Fe/S cluster is required for stable insertion of the second Fe/S cluster at the C-terminus. Surprisingly, single mutations of the C-terminal cysteines had no influence on the incorporation of the Nar1 Fe/S clusters in vivo. However, simultaneous exchange of two cysteine residues at the C-terminus resulted in the loss of the Fe/S cluster located at the C-terminus, whereas the N-terminal cluster was still bound. The data presented in this study clearly indicate that the N- and C-terminal cysteine residues coordinate two Fe/S clusters and that these clusters are essential for Nar1 function in the maturation of cytosolic and nuclear Fe/S proteins. Furthermore, the N-terminal Fe/S cluster was found to be more labile than the C-terminal one. An explanation for this observation was suggested by the structural model of Nar1 which was derived from the crystal structure of Fe-only hydrogenases. The calculated model shows that the N-terminal cluster is surface-exposed, whereas the C-terminal Fe/S cluster is buried inside the protein.