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

  • Anita Kaut
    Affiliations
    Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany
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  • Heike Lange
    Affiliations
    Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany
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  • Kerstin Diekert
    Affiliations
    Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany
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  • Gyula Kispal
    Footnotes
    Affiliations
    Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany
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  • Roland Lill
    Correspondence
    To whom correspondence should be addressed. Tel.: 49-6421-286-6449; Fax: 49-6421-286-6414
    Affiliations
    Institut für Zytobiologie und Zytopathologie der Philipps-Universität Marburg, Robert-Koch-Strasse 5, 35033 Marburg, Germany
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  • Author Footnotes
    * This work was supported by grants from the Sonderforschungsbereich 286 of the Deutsche Forschungsgemeinschaft, the Volkswagen-Stiftung, the Fonds der Chemischen Industrie, and the Alexander von Humboldt-Stiftung and by Hungarian Funds OKTA Grants T6378, T020079, and T022581.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Present address: Inst. of Biochemistry, University Medical School of Pecs, Szigeti 12, 7624 Pecs, Hungary.
Open AccessPublished:May 26, 2000DOI:https://doi.org/10.1074/jbc.M909502199
      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.
      Fe/S
      iron-sulfur
      mtDNA
      mitochondrial DNA
      PCR
      polymerase chain reaction
      DAPI
      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.
      • Lill R.
      • Diekert K.
      • Kaut A.
      • Lange H.
      • Pelzer W.
      • Prohl C.
      • Kispal G.
      and
      • Craig E.A.
      • Voisine C.
      • Schilke B.
      ). First insights into this essential biosynthetic process were gained in prokaryotes (
      • 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.
      ,
      • Jacobson M.R.
      • Cash V.L.
      • Weiss M.C.
      • Laird N.F.
      • Newton W.E.
      • Dean D.R.
      ,
      • Dean D.R.
      • Bolin J.T.
      • Zheng L.
      ). Numerous genes with a direct function in the generation of the Fe/S clusters of the enzyme nitrogenase were identified in the nif operon (
      • Peters J.W.
      • Fisher K.
      • Dean D.R.
      ). Many of these genes possess close homologues in non-nitrogen-fixing bacteria (
      • Zheng L.
      • Cash V.L.
      • Flint D.H.
      • Dean D.R.
      ). 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 (
      • Zheng L.
      • Cash V.L.
      • Flint D.H.
      • Dean D.R.
      ). 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 (
      • Zheng L.
      • Cash V.L.
      • Flint D.H.
      • Dean D.R.
      ,
      • Zheng L.
      • White R.H.
      • Cash V.L.
      • Jack R.F.
      • Dean D.R.
      ). Sulfur is then assembled into an “intermediate” [2Fe-2S] cluster on NifU that carries the primary binding site for iron (
      • Yuvaniyama P.
      • Agar J.N.
      • Cash V.L.
      • Johnson M.K.
      • Dean D.R.
      ). A function of other proteins encoded by the isc operon has been implicated from their requirement for maturation of overexpressed ferredoxins inEscherichia coli (
      • Takahashi Y.
      • Nakamura M.
      ).
      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 (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ). The proteins Isu1p, Isu2p, and Nfu1p are homologues of NifU and are required for formation of mitochondrial Fe/S proteins (
      • Schilke B.
      • Voisine C.
      • Beinert H.
      • Craig E.
      ,
      • Garland S.A.
      • Hoff K.
      • Vickery L.E.
      • Culotta V.C.
      ). The mitochondrial ferredoxin homologue Yah1p is essential for Fe/S cluster formation and presumably supplies reducing equivalents to the process (
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      ). 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 (
      • Strain J.
      • Lorenz C.R.
      • Bode J.
      • Garland S.
      • Smolen G.A.
      • Ta D.T.
      • Vickery L.E.
      • Culotta V.C.
      ). 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 (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ,
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      ). 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 (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ,
      • Leighton J.
      • Schatz G.
      ,
      • Kispal G.
      • Csere P.
      • Guiard B.
      • Lill R.
      ). 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 (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ). 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.
      • Jacobson M.R.
      • Cash V.L.
      • Weiss M.C.
      • Laird N.F.
      • Newton W.E.
      • Dean D.R.
      ,
      • Zheng L.
      • Cash V.L.
      • Flint D.H.
      • Dean D.R.
      , and
      • Meijer W.G.
      • Tabita F.R.
      ,
      • Masepohl B.
      • Angermuller S.
      • Hennecke S.
      • Hubner P.
      • Moreno-Vivian C.
      • Klipp W.
      ,
      • Schrautemeier B.
      • Cassing A.
      • H B.
      ) 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 (
      • Takahashi Y.
      • Nakamura M.
      ).
      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.

      DISCUSSION

      Our investigation provides evidence for a function of the protein Isa1p of S. cerevisiae mitochondria in Fe/S cluster formation. 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. Thus, the protein seems to participate in the generation of most, if not all, cellular Fe/S proteins similar to what this has been reported for the cysteine desulfurase Nfs1p and the ferredoxin Yah1p (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ,
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      ). However, in contrast to the latter components, the deficiency in Isa1p has a somewhat weaker effect on the biogenesis of the mitochondrial and cytosolic Fe/S proteins. For instance, aconitase was only slightly (2-fold) decreased in the absence of functional Isa1p, whereas succinate dehydrogenase was reduced 4-fold. These biochemical observations fit well to the observed growth phenotype of cells depleted in Isa1p. These cells grew slowly on nonfermentable carbon sources, whereas cells defective in Nfs1p or Yah1p are unable to maintain growth (
      • Kolman C.
      • Soell D.
      ,
      • Barros M.H.
      • Nobrega F.G.
      ). A possible reason for the comparatively mild consequences of the Isa1p deficiency on cellular Fe/S proteins may be the presence of a homologue, termed Isa2p, in yeast. However, simultaneous deletion of the ISA1 and ISA2 genes does not exacerbate the growth defects of single mutants (

      Jensen, L. T., and Culotta, V. C. (2000) Mol. Cell. Biol., in press

      ).
      W. Pelzer, unpublished data.
      This suggests that the Isa proteins perform nonoverlapping functions. The nonessential character of the Isa proteins points to an auxiliary function in the maturation of cellular Fe/S proteins distinguishing them from Nfs1p, the Isu proteins, Yah1p and Jac1p that perform essential functions.
      All members of the Isa/IscA protein family are characterized by two sequence motifs termed IsaI and IsaII in the middle and at the C terminus, respectively, of these proteins (Fig. 1). The motifs contain either one or two conserved cysteine residues. Our study demonstrates that each of the cysteine residues of the IsaII motif is essential for function of the protein. Mutation of the cysteines to serine residues yielded mutant proteins that did not revert the growth phenotype of cells depleted in wild-type Isa1p. Moreover, these proteins were inactive in Fe/S cluster biosynthesis for apoproteins both inside and outside mitochondria. Thus, these amino acid residues are central structural elements that might be involved in the binding of iron or an intermediate of Fe/S cluster formation to the Isa proteins. In general, cysteines appear to be important residues of proteins involved in Fe/S cluster biosynthesis. For instance, NifS contains a conserved cysteine residue that transiently carries the sulfur liberated from free cysteine as an enzyme-bound persulfide (
      • Zheng L.
      • White R.H.
      • Cash V.L.
      • Dean D.R.
      ). Similarly, bacterial NifU protein contains conserved cysteine residues at its N terminus that have been shown to be essential for the binding of the iron that is used for Fe/S cluster synthesis by NifS (
      • Yuvaniyama P.
      • Agar J.N.
      • Cash V.L.
      • Johnson M.K.
      • Dean D.R.
      ,

      Agar, J. N., Yuvaniyama, P., Jack, R. F., Cash, V. L., Smith, A. D., Dean, D. R., and Johnson, M. K. (2000) J. Biol. Inorg. Chem., in press

      ). Further, cysteines in the central domain of NifU and in the ferredoxin proteins (e.g. in bacterial Fdx or in yeast Yah1p) are known to co-ordinate the [2Fe-2S] cluster present in these proteins (
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      ,
      • Barros M.H.
      • Nobrega F.G.
      ,
      • Fu W.
      • Jack R.F.
      • Morgan T.V.
      • Dean D.R.
      • Johnson M.K.
      ).
      The accumulation of iron within mitochondria has emerged to be a rather common consequence of defects in components with a function in Fe/S cluster assembly. 10–30-fold elevated levels of free (i.e.non-heme, non-Fe/S) iron within mitochondria have been observed for mutants defective in ATM1, NFS1, SSQ1, and YAH1 genes (
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      ,
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      ,
      • Kispal G.
      • Csere P.
      • Guiard B.
      • Lill R.
      ,
      • Knight S.A.
      • Sepuri N.B.
      • Pain D.
      • Dancis A.
      ,
      • Li J.
      • Kogan M.
      • Knight S.A.
      • Pain D.
      • Dancis A.
      ). A 4-fold iron increase was found in a mutant lacking both ISU1 and NFU1(
      • Schilke B.
      • Voisine C.
      • Beinert H.
      • Craig E.
      ). Gal-ISA1 cells deficient in Isa1p contained only 2-fold higher amounts of mitochondrial iron as compared with wild-type organelles (not shown). This result is compatible with the auxiliary function of Isa1p in Fe/S cluster biosynthesis.
      Our study is of importance for the proposed role of mitochondria in initiating the maturation of cytosolic Fe/S proteins (
      • Lill R.
      • Diekert K.
      • Kaut A.
      • Lange H.
      • Pelzer W.
      • Prohl C.
      • Kispal G.
      ). To date, a function in this process has been reported for the mitochondrial proteins Nfs1p, Yah1p, Atm1p, and Isa1p (Refs.
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      and
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      and this study). The requirement of these proteins for maturation of Fe/S proteins outside the mitochondria strongly suggests that the entire mitochondrial Fe/S cluster biosynthesis machinery participates in this pathway. This makes it likely that an Fe/S cluster is preassembled in the matrix and, after proper stabilization, becomes exported from the mitochondria, presumably by the transport function of Atm1p. It should be noted that the requirement of Isa1p for Fe/S protein maturation in the cytosol does not necessarily indicate a direct function in the production of these Fe/S clusters. Because the mitochondrial ferredoxin Yah1p itself is an Fe/S protein, the rather strong defect in the maturation of cytosolic Leu1p upon depletion of Isa1p may be due to defective assembly of functional Yah1p, which then causes the defects in the incorporation of Fe/S clusters into cytosolic apoproteins (
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      ).
      Currently available protein sequence data suggest the existence of a universal apparatus for the biosynthesis of Fe/S clusters in eukaryotes, prokaryotes, and archaea. Many of the central components of Fe/S cluster biosynthesis are conserved in organisms representing these evolutionary kingdoms. These proteins include homologues of NifS, NifU, and the [2Fe-2S] ferredoxins. Interestingly, Isa/IscA homologues are present in higher and lower eukaryotes and in prokaryotes but have not been identified yet in archaebacteria. Presumably, the prokaryotic and eukaryotic proteins use similar molecular mechanisms for the assembly of the Fe/S clusters. Nevertheless, some variance in the pathways in different species might be expected, e.g. from the absence of IscU homologues in some bacteria.
      Mitochondria have been shown to represent the primary site of Fe/S cluster synthesis in a eukaryotic cell and may be involved in the assembly of virtually all cellular Fe/S proteins (Refs.
      • Kispal G.
      • Csere P.
      • Prohl C.
      • Lill R.
      and
      • Lange H.
      • Kispal G.
      • Kaut A.
      • Lill R.
      and this study). To date, Fe/S protein biosynthesis is the only known process that renders mitochondria essential for a eukaryotic cell (
      • Lill R.
      • Diekert K.
      • Kaut A.
      • Lange H.
      • Pelzer W.
      • Prohl C.
      • Kispal G.
      ). Yeast provides an excellent experimental system to study this fundamental process and to dissect its molecular mechanisms by facilitating the combined use of genetic, cell biological, and biochemical methodologies.

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