Isa1p Is a Component of the Mitochondrial Machinery for Maturation of Cellular Iron-Sulfur Proteins and Requires Conserved Cysteine Residues for Function

Abstract

In eukaryotes, mitochondria execute a central task in the assembly of cellular iron-sulfur (Fe/S) proteins. The organelles synthesize their own set of Fe/S proteins, and they initiate the generation of extramitochondrial Fe/S proteins. In the present study, we identify the mitochondrial matrix protein Isa1p ofSaccharomyces cerevisiae as a new member of the Fe/S cluster biosynthesis machinery. Isa1p belongs to a family of homologous proteins present in prokaryotes and eukaryotes. Deletion of theISA1 gene results in the loss of mitochondrial DNA precluding the use of the Δisa1 strain for functional analysis. Cells in which Isa1p was depleted by regulated gene expression maintained the mitochondrial DNA, yet the cells displayed retarded growth on nonfermentable carbon sources. This finding indicates the importance of Isa1p for mitochondrial function. Deficiency of Isa1p caused a defect in mitochondrial Fe/S protein assembly. Moreover, Isa1p was required for maturation of cytosolic Fe/S proteins. Two cysteine residues in a conserved sequence motif characterizing the Isa1p protein family were found to be essential for Isa1p function in the biogenesis of both intra- and extramitochondrial Fe/S proteins. Our findings suggest a function for Isa1p in the binding of iron or an intermediate of Fe/S cluster assembly. In eukaryotes, mitochondria execute a central task in the assembly of cellular iron-sulfur (Fe/S) proteins. The organelles synthesize their own set of Fe/S proteins, and they initiate the generation of extramitochondrial Fe/S proteins. In the present study, we identify the mitochondrial matrix protein Isa1p ofSaccharomyces cerevisiae as a new member of the Fe/S cluster biosynthesis machinery. Isa1p belongs to a family of homologous proteins present in prokaryotes and eukaryotes. Deletion of theISA1 gene results in the loss of mitochondrial DNA precluding the use of the Δisa1 strain for functional analysis. Cells in which Isa1p was depleted by regulated gene expression maintained the mitochondrial DNA, yet the cells displayed retarded growth on nonfermentable carbon sources. This finding indicates the importance of Isa1p for mitochondrial function. Deficiency of Isa1p caused a defect in mitochondrial Fe/S protein assembly. Moreover, Isa1p was required for maturation of cytosolic Fe/S proteins. Two cysteine residues in a conserved sequence motif characterizing the Isa1p protein family were found to be essential for Isa1p function in the biogenesis of both intra- and extramitochondrial Fe/S proteins. Our findings suggest a function for Isa1p in the binding of iron or an intermediate of Fe/S cluster assembly. Isa1p is a component of the mitochondrial machinery for maturation of cellular iron-sulfur proteins and requires conserved cysteine residues for function.Journal of Biological ChemistryVol. 292Issue 43PreviewVOLUME 275 (2000) PAGES 15955–15961 Full-Text PDF Open Access iron-sulfur mitochondrial DNA polymerase chain reaction 4′,6-diamidino-2-phenylindole dihydrochloride Investigations during the past few years have demonstrated the complexity of the assembly of iron-sulfur (Fe/S)1 proteins in a living cell (reviewed by Refs. 1.Lill R. Diekert K. Kaut A. Lange H. Pelzer W. Prohl C. Kispal G. Biol. Chem. Hoppe-Seyler. 1999; 380: 1157-1166Crossref PubMed Scopus (124) Google Scholar and 2.Craig E.A. Voisine C. Schilke B. Biol. Chem. Hoppe-Seyler. 1999; 380: 1167-1173Crossref PubMed Scopus (42) Google Scholar). First insights into this essential biosynthetic process were gained in prokaryotes (3.Jacobson M.R. Brigle K.E. Bennett L.T. Setterquist R.A. Wilson M.S. Cash V.L. Beynon J. Newton W.E. Dean D.R. J. Bacteriol. 1989; 171: 1017-1027Crossref PubMed Google Scholar, 4.Jacobson M.R. Cash V.L. Weiss M.C. Laird N.F. Newton W.E. Dean D.R. Mol. Gen. Genet. 1989; 219: 49-57Crossref PubMed Scopus (247) Google Scholar, 5.Dean D.R. Bolin J.T. Zheng L. J. Bacteriol. 1993; 175: 6737-6744Crossref PubMed Google Scholar). Numerous genes with a direct function in the generation of the Fe/S clusters of the enzyme nitrogenase were identified in the nif operon (6.Peters J.W. Fisher K. Dean D.R. Annu. Rev. Microbiol. 1995; 49: 335-366Crossref PubMed Scopus (140) Google Scholar). Many of these genes possess close homologues in non-nitrogen-fixing bacteria (7.Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). In some bacteria, these genes are clustered in operons termed isc (iron sulfurcluster assembly). The encoded proteins were suggested to perform crucial tasks in the biogenesis of the normal equipment of Fe/S proteins within a prokaryotic cell (7.Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). However, only a few proteins have been functionally investigated in detail. The best studied examples are the cysteine desulfurases NifS and IscS that produce elemental sulfur from cysteine for biogenesis (7.Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 8.Zheng L. White R.H. Cash V.L. Jack R.F. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2754-2758Crossref PubMed Scopus (503) Google Scholar). Sulfur is then assembled into an “intermediate” [2Fe-2S] cluster on NifU that carries the primary binding site for iron (9.Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (277) Google Scholar). A function of other proteins encoded by the isc operon has been implicated from their requirement for maturation of overexpressed ferredoxins inEscherichia coli (10.Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (227) Google Scholar). Recently, eukaryotic homologues of some of the Nif and Isc proteins have been identified. All of these factors have been localized to mitochondria, and an involvement in the biosynthesis of mitochondrial Fe/S proteins has been demonstrated in yeast. Nfs1p has been shown to represent the functional orthologue of the cysteine desulfurase NifS/IscS (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar). The proteins Isu1p, Isu2p, and Nfu1p are homologues of NifU and are required for formation of mitochondrial Fe/S proteins (12.Schilke B. Voisine C. Beinert H. Craig E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10206-10211Crossref PubMed Scopus (268) Google Scholar,13.Garland S.A. Hoff K. Vickery L.E. Culotta V.C. J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (167) Google Scholar). The mitochondrial ferredoxin homologue Yah1p is essential for Fe/S cluster formation and presumably supplies reducing equivalents to the process (14.Lange H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar). The two chaperones Ssq1p and Jac1p, mitochondrial homologues of the heat shock proteins Hsp70/DnaK and Hsp40/DnaJ, respectively, are necessary for normal activity of mitochondrial Fe/S proteins (15.Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The target of these proteins is not yet known. At least some of these factors appear to perform an additional role in the generation of Fe/S proteins outside the organelle. Direct evidence for a function in the production of Fe/S clusters for incorporation into cytosolic apoproteins has been reported for Nfs1p and Yah1p (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar,14.Lange H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar). Apparently, mitochondria produce a component required for the maturation of Fe/S proteins in the cytosol. Export of this component, presumably an Fe/S cluster, to the cytosol is mediated by the ATP-binding cassette transporter Atm1p of the mitochondrial inner membrane (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar, 16.Leighton J. Schatz G. EMBO J. 1995; 14: 188-195Crossref PubMed Scopus (170) Google Scholar, 17.Kispal G. Csere P. Guiard B. Lill R. FEBS Lett. 1997; 418: 346-350Crossref PubMed Scopus (248) Google Scholar). The participation of cytosolic proteins in the incorporation of Fe/S clusters into extramitochondrial apoproteins seems likely, but none of these factors have been identified hitherto. A number of additional eukaryotic proteins have been predicted to participate in Fe/S cluster biosynthesis (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar). This expectation is based on sequence similarity to bacterial proteins encoded by thenif and isc operons. One of these proteins is Isa1p of the yeast Saccharomyces cerevisiae. It displays sequence homology to bacterial proteins termed IscA (also coined HesB; Refs. 4.Jacobson M.R. Cash V.L. Weiss M.C. Laird N.F. Newton W.E. Dean D.R. Mol. Gen. Genet. 1989; 219: 49-57Crossref PubMed Scopus (247) Google Scholar, 7.Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, and 18.Meijer W.G. Tabita F.R. J. Bacteriol. 1992; 174: 3855-3866Crossref PubMed Google Scholar, 19.Masepohl B. Angermuller S. Hennecke S. Hubner P. Moreno-Vivian C. Klipp W. Mol. Gen. Genet. 1993; 238: 369-382Crossref PubMed Scopus (38) Google Scholar, 20.Schrautemeier B. Cassing A. H B. J. Bacteriol. 1994; 176: 1037-1046Crossref PubMed Google Scholar) and to a few eukaryotic proteins including Isa2p of yeast. Recently, E. coli IscA was shown to improve the efficiency of the maturation of an overexpressed [2Fe-2S] ferredoxin, suggesting a function of this protein in Fe/S cluster biosynthesis (10.Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (227) Google Scholar). Here, we present a study on the subcellular localization and functional characterization of Isa1p. The protein was shown to reside in the mitochondrial matrix, where it performs a crucial role in the generation of Fe/S clusters for both mitochondrial and cytosolic apoproteins. Two conserved cysteine residues located in a sequence motif characteristic for the Isa/IscA proteins were shown to be essential for Isa1p function. Our study adds Isa1p, and by extrapolation the eukaryotic and bacterial homologues, to the growing list of components involved in cellular Fe/S cluster biogenesis. The following strains ofS. cerevisiae were used: strain W303 (MATα,ura3-1, ade2-1, trp1-1, his3-11, 15, leu2-3, 112) in diploid and haploid form served as wild type. Strain W303ρ0 is derived from W303 and lacks mitochondrial DNA (mtDNA). Deletion of the ISA1 gene in W303 diploid cells was performed by a PCR-based method using the kanamycin resistance marker (21.Wach A. Brachat A. Poehlmann R. Phillipsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2237) Google Scholar). The haploid Δisa1 strain was obtained after sporulation and tetrad dissection. Strain Δisa1/ISA1 was generated by transforming Δisa1 cells with the yeast expression plasmid pRS416MET25 carrying the ISA1 gene (22.Mumberg D. Müller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (803) Google Scholar). Exchange of the endogenous promoter of the ISA1 gene for a galactose-inducible promoter (strain Gal-ISA1) was performed as described (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar, C. Guiard B. W. M. PubMed Scopus Google Scholar). to the to and the to of ISA1 were into respectively, of the carrying the Cells were as using or G. Csere P. Guiard B. Lill R. FEBS Lett. 1997; 418: 346-350Crossref PubMed Scopus (248) Google Scholar, PubMed Scopus Google G. H. B. Lill R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and G. Schatz G. J. Biol. Chem. Full Text PDF PubMed Google Scholar) the required carbon sources. cell with iron was to the The following were used: of DNA and J. A of yeast cells D. A. R.A. R.H. Nucleic Acids Res. 1992; PubMed Scopus Google of from yeast J. A of yeast mitochondria G. Schatz G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 1995; PubMed Scopus Google of proteins into mitochondria Biol. PubMed Scopus (42) Google Scholar, H. A. A. W. Lill R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google cell by cells with M. J. Biol. Chem. Full Text PDF PubMed Google using to E. D. A enzyme of G. Csere P. Guiard B. Lill R. FEBS Lett. 1997; 418: 346-350Crossref PubMed Scopus (248) Google 1995; PubMed Scopus Google PubMed Scopus (42) Google Scholar). The of the of enzyme and The of yeast cells with iron and the of the incorporation of into the cytosolic protein by and was described (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar). The for of with cytosolic in a cell was the cellular of by The of the two conserved cysteine residues and of Isa1p to was by PCR-based of the ISA1 gene using and and were the ISA1 genes were into the of the yeast expression pRS416MET25 (22.Mumberg D. Müller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (803) Google Scholar). and the a DNA were into the DNA was to the of ISA1 The protein Isa1p of S. cerevisiae residues to a of A identified homologous proteins in and but not in In some as S. cerevisiae and E. two of the Isa/IscA protein family have been However, we were to the family into two by sequence The Isa/IscA proteins sequence for two in the and the coined and or respectively, conserved cysteine residues Isa2p of S. cerevisiae an sequence of residues that are not present in other Isa/IscA proteins. The and proteins that are predicted to function as mitochondrial P. J. Biochem. PubMed Scopus Google Scholar). the proteins only as with the bacterial their mitochondrial The cellular localization of Isa1p was by using Isa1p was in mitochondria and was in the that the of Isa1p direct the protein into mitochondria and to Isa1p was in and for in into The a of in with mitochondria, the protein was to a form of This protein was by in both mitochondria and in that were generated by a of the organelles by of Isa1p was by the was for the endogenous Isa1p and for marker proteins of the mitochondrial matrix both and endogenous Isa1p are located in the matrix and of Isa1p were not the reaction was performed in the of the the of Isa1p was on the of a membrane This is characteristic for proteins of the mitochondrial from these that Isa1p is a of the mitochondrial matrix and a sequence for into the initiate the functional of Isa1p, the ISA1 gene was The yeast strain Δisa1 on and not to on nonfermentable carbon as The growth was not by the ISA1 gene into Δisa1 cells on a yeast expression plasmid not suggesting that the of ISA1 was by the loss of This expectation was by which in Δisa1 cells Apparently, Isa1p, other mitochondrial proteins R.A. EMBO J. 1995; 14: PubMed Scopus Google is required for the of The of Δisa1 cells the of Isa1p function in these cells and it necessary to a strain in which ISA1 gene expression be this the ISA1 promoter was for a galactose-inducible In the cells the of Isa1p was after of growth in the of the of other mitochondrial proteins were not cells depleted in Isa1p were by loss of was after growth for in the of It from these that the of Isa1p in cells not the that were in the strain The and of Isa1p the functional of this the growth of cells was The cells on the nonfermentable carbon in the of but displayed retarded growth in the of this to cells was The growth defect of cells on nonfermentable carbon indicates an role of Isa1p in the generation of functional the role of Isa1p in Fe/S cluster we Δisa1 cells and the of the Fe/S and of were in mitochondria of Δisa1 a defect in these Fe/S proteins However, this be as evidence for a function of Isa1p in the maturation of mitochondrial Fe/S proteins. found a with in the of the Fe/S proteins of and Δisa1/ISA1 cells that both to a the of Δisa1 cells for the defect in mitochondrial Fe/S proteins. a displayed activity in these cells the of Δisa1 cells the of the role of Isa1p in the maturation of cellular Fe/S proteins. these mitochondria were from cells that not growth in the of of Isa1p caused an to in the of and a of In the activity of of Isa1p. The of both Fe/S were by the of Isa1p as by from these that Isa1p is required for maturation of mitochondrial Fe/S proteins. have a primary function in the maturation of Fe/S proteins located in the cytosol (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar, H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar). Isa1p participate in the generation of extramitochondrial Fe/S The of Isa1p on the activity of the cytosolic Fe/S enzyme was were from cells and from cells and after of Isa1p and for of In with derived from cells and from cells in the of cells depleted in Isa1p displayed an to in activity This was to the results obtained for cells in the proteins and Yah1p that are essential for the generation of cytosolic Fe/S proteins (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar, H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar). by the of were only suggesting that the for the of activity is not the of the the requirement of Isa1p for the assembly of the Fe/S cluster in cells were depleted in Isa1p and for with iron A cell was and the of the Fe/S cluster in was from the of that be with G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar). of Isa1p in a in the assembly of with as with cells or with cells in the of performed with not of iron with the and G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar). the into the cells was of the cellular of Isa1p, that cells were not in iron these a crucial function of the mitochondrial protein Isa1p in the assembly of a cytosolic Fe/S The functional importance of two conserved cysteine residues in the sequence motif of Isa1p was investigated we generated cells Isa1p proteins in which the cysteine residues and were or to this genes were by and into the yeast expression plasmid cells were with these or with carrying the gene or DNA then the of the Isa1p proteins by the expression of the Isa1p by of the cells in the of The cells the proteins and displayed a growth from cells Isa1p the expression of growth of cells in the of This suggested an role of the conserved cysteine residues for Isa1p function. the function of the Isa1p proteins in Fe/S cluster the enzyme of the mitochondrial Fe/S proteins and and the cytosolic were of the wild and expression of the Isa1p proteins caused a in the of both the mitochondrial Fe/S proteins and the cytosolic of the ISA1 gene of these proteins. the incorporation of Fe/S clusters into cytosolic was in cells the Isa1p proteins In the in Fe/S protein maturation were to in cells Isa1p. results the essential of of the cysteine residues of the motif for Isa1p function. an these Isa1p to be Our evidence for a function of the protein Isa1p of S. cerevisiae mitochondria in Fe/S cluster Isa1p is a new component of the mitochondrial Fe/S cluster biosynthesis machinery and is required for the maturation of both mitochondrial and cytosolic Fe/S proteins. the protein seems to participate in the generation of not cellular Fe/S proteins to this has been reported for the cysteine desulfurase Nfs1p and the ferredoxin Yah1p (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar, H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar). However, in to the the in Isa1p has a on the biogenesis of the mitochondrial and cytosolic Fe/S proteins. was only in the of functional Isa1p, was to the growth of cells depleted in Isa1p. cells on nonfermentable carbon cells in Nfs1p or Yah1p are to growth C. D. J. Bacteriol. 1993; 175: PubMed Google Scholar, 1999; PubMed Scopus Google Scholar). A for the of the Isa1p on cellular Fe/S proteins be the of a termed in yeast. However, of the ISA1 and genes not the growth of L. and C. (2000) Mol. in Scholar). This that the proteins perform The of the proteins to an function in the maturation of cellular Fe/S proteins from the proteins, Yah1p and that perform essential All of the Isa/IscA protein family are by two sequence termed and in the and the respectively, of these proteins The or two conserved cysteine Our study that of the cysteine residues of the motif is essential for function of the of the to residues proteins that not the growth of cells depleted in Isa1p. Moreover, these proteins were in Fe/S cluster biosynthesis for apoproteins both and outside these residues are central that be involved in the binding of iron or an intermediate of Fe/S cluster formation to the proteins. In appear to be residues of proteins involved in Fe/S cluster NifS a conserved cysteine that carries the sulfur from cysteine as an L. White R.H. Cash V.L. Dean D.R. 1994; PubMed Scopus Google Scholar). bacterial NifU protein conserved cysteine residues that have been shown to be essential for the binding of the iron that is for Fe/S cluster by NifS (9.Yuvaniyama P. Agar J.N. Cash V.L. Johnson M.K. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 599-604Crossref PubMed Scopus (277) Google Scholar, J. R. A. D. and M. K. (2000) J. Biol. in Scholar). in the central of NifU and in the ferredoxin proteins in bacterial or in yeast are to the [2Fe-2S] cluster present in these proteins (14.Lange H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google 1999; PubMed Scopus Google Scholar, W. Jack R.F. Dean D.R. Johnson M.K. 1994; PubMed Scopus Google Scholar). The of iron within mitochondria has to be a of in components with a function in Fe/S cluster assembly. of iron within mitochondria have been for in and genes (11.Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar, H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar, 17.Kispal G. Csere P. Guiard B. Lill R. FEBS Lett. 1997; 418: 346-350Crossref PubMed Scopus (248) Google Scholar, S.A. D. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. S.A. D. A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A iron was found in a both and B. Voisine C. Beinert H. Craig E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10206-10211Crossref PubMed Scopus (268) Google Scholar). cells in Isa1p only of mitochondrial iron as with organelles This is with the function of Isa1p in Fe/S cluster Our study is of importance for the role of mitochondria in the maturation of cytosolic Fe/S proteins R. Diekert K. Kaut A. Lange H. Pelzer W. Prohl C. Kispal G. Biol. Chem. Hoppe-Seyler. 1999; 380: 1157-1166Crossref PubMed Scopus (124) Google Scholar). a function in this process has been reported for the mitochondrial proteins and Isa1p G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar and H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar and this The requirement of these proteins for maturation of Fe/S proteins outside the mitochondria that the mitochondrial Fe/S cluster biosynthesis machinery in this This it that an Fe/S cluster is in the matrix after from the mitochondria, presumably by the function of It be that the requirement of Isa1p for Fe/S protein maturation in the cytosol not a direct function in the production of these Fe/S the mitochondrial ferredoxin Yah1p is an Fe/S the defect in the maturation of cytosolic of Isa1p be to assembly of functional which then the in the incorporation of Fe/S clusters into cytosolic apoproteins (14.Lange H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar). protein sequence suggest the of a for the biosynthesis of Fe/S clusters in eukaryotes, and Many of the central components of Fe/S cluster biosynthesis are conserved in these proteins homologues of and the [2Fe-2S] Isa/IscA homologues are present in and and in prokaryotes but have not been identified yet in the prokaryotic and eukaryotic proteins use for the assembly of the Fe/S some in the in be from the of homologues in some have been shown to represent the primary site of Fe/S cluster in a eukaryotic cell and be involved in the assembly of cellular Fe/S proteins G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar and H. Kispal G. Kaut A. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1050-1055Crossref PubMed Scopus (245) Google Scholar and this Fe/S protein biosynthesis is the only process that mitochondria essential for a eukaryotic cell R. Diekert K. Kaut A. Lange H. Pelzer W. Prohl C. Kispal G. Biol. Chem. Hoppe-Seyler. 1999; 380: 1157-1166Crossref PubMed Scopus (124) Google Scholar). an to study this process and to by the use of cell and