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• W2148522029 abstract "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. 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. Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.Journal of Biological ChemistryVol. 292Issue 43PreviewVOLUME 286 (2011) PAGES 41205–41216 Full-Text PDF Open Access Iron-sulfur (Fe/S) proteins perform central tasks in electron transport, catalysis, and the regulation of environmental responses (1Beinert H. J. Biol. Inorg. Chem. 2000; 5: 2-15Crossref PubMed Scopus (540) Google Scholar, 2Fontecave M. Nat. Chem. Biol. 2006; 2: 171-174Crossref PubMed Scopus (171) Google Scholar, 3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar). The complex bacterial biosynthetic systems that assist in the assembly of Fe/S clusters and their transfer into apoproteins fall into three classes: the widely distributed housekeeping ISC 2The abbreviations used are: ISCiron-sulfur clusterhIRP1human IRP1HiPIPhigh potential iron-sulfur protein. assembly system; the NIF system, a dedicated assembly apparatus for the nitrogenase of nitrogen-fixing bacteria; and the SUF system, which is present in several bacterial taxa and in plastids (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 4Fontecave M. Choudens S.O. Py B. Barras F. J. Biol. Inorg Chem. 2005; 10: 713-721Crossref PubMed Scopus (95) Google Scholar, 5Johnson D.C. Dean D.R. Smith A.D. Johnson M.K. Annu. Rev. Biochem. 2005; 74: 247-281Crossref PubMed Scopus (1110) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar, 7Bandyopadhyay S. Chandramouli K. Johnson M.K. Biochem. Soc. Trans. 2008; 36: 1112-1119Crossref PubMed Scopus (134) Google Scholar). In eukaryotes, mitochondria are central for Fe/S protein biogenesis (8Rouault T.A. Tong W.H. Trends Genet. 2008; 24: 398-407Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 9Sheftel A.D. Lill R. Ann. Med. 2009; 41: 82-99Crossref PubMed Scopus (39) Google Scholar, 10Lill R. Nature. 2009; 460: 831-838Crossref PubMed Scopus (849) Google Scholar, 11Balk J. Pilon M. Trends Plant Sci. 2011; 16: 218-226Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 12Ye H. Rouault T.A. Biochemistry. 2010; 49: 4945-4956Crossref PubMed Scopus (208) Google Scholar). They harbor the ISC assembly machinery that is closely related to the bacterial ISC system and is essential for maturation of all cellular Fe/S proteins, whether located in the mitochondria, cytosol, or nucleus. Biosynthesis of extramitochondrial Fe/S proteins further depends on the mitochondrial “ISC export machinery” that exports an unknown component required for maturation of cytosolic and nuclear proteins, a step carried out by the cytosolic Fe/S protein assembly (CIA) system (9Sheftel A.D. Lill R. Ann. Med. 2009; 41: 82-99Crossref PubMed Scopus (39) Google Scholar, 10Lill R. Nature. 2009; 460: 831-838Crossref PubMed Scopus (849) Google Scholar, 13Sharma A.K. Pallesen L.J. Spang R.J. Walden W.E. J. Biol. Chem. 2010; 285: 26745-26751Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The components involved in Fe/S maturation in eukaryotes are highly conserved, and most are essential for cell viability, underscoring the importance of Fe/S protein function for the eukaryotic cell. iron-sulfur cluster human IRP1 high potential iron-sulfur protein. Fe/S cluster assembly in mitochondria and bacteria is initiated by a cysteine desulfurase (in yeast termed Nfs1-Isd11) that abstracts sulfur from cysteine and transfers it to the essential U-type ISC assembly proteins that serve as scaffolds for the de novo synthesis of the Fe/S co-factor (5Johnson D.C. Dean D.R. Smith A.D. Johnson M.K. Annu. Rev. Biochem. 2005; 74: 247-281Crossref PubMed Scopus (1110) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar, 14Zheng L. Dean D.R. J. Biol. Chem. 1994; 269: 18723-18726Abstract Full Text PDF PubMed Google Scholar, 15Lill R. Mühlenhoff U. Annu. Rev. Cell Dev. Biol. 2006; 22: 457-486Crossref PubMed Scopus (279) Google Scholar, 16Lill R. Mühlenhoff U. Annu. Rev. Biochem. 2008; 77: 669-700Crossref PubMed Scopus (495) Google Scholar). In yeast, de novo Fe/S cluster synthesis on the U-type proteins Isu1 and Isu2 involves an electron transfer chain consisting of the ferredoxin reductase Arh1 and the ferredoxin Yah1. In addition, the desulfurase and the Isu proteins interact with frataxin (yeast Yfh1), which may serve as an iron donor and/or a regulator of desulfurase activity (17Stemmler T.L. Lesuisse E. Pain D. Dancis A. J. Biol. Chem. 2010; 285: 26737-26743Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 18Tsai C.L. Barondeau D.P. Biochemistry. 2010; 49: 9132-9139Crossref PubMed Scopus (244) Google Scholar). The subsequent cluster transfer to recipient apoproteins is facilitated by the Hsp70 chaperone Ssq1 (bacterial HscA), its cognate J-type co-chaperone Jac1 (bacterial HscB), and the monothiol glutaredoxin Grx5 (7Bandyopadhyay S. Chandramouli K. Johnson M.K. Biochem. Soc. Trans. 2008; 36: 1112-1119Crossref PubMed Scopus (134) Google Scholar, 19Mühlenhoff U. Gerber J. Richhardt N. Lill R. EMBO J. 2003; 22: 4815-4825Crossref PubMed Scopus (342) Google Scholar, 20Vickery L.E. Cupp-Vickery J.R. Crit. Rev. Biochem. Mol. Biol. 2007; 42: 95-111Crossref PubMed Scopus (153) Google Scholar). The function of two additional members of the yeast mitochondrial ISC assembly machinery, the matrix proteins Isa1 and Isa2, is poorly understood. They are related to the ubiquitous “A-type” ISC assembly factors from bacteria and other eukaryotes (21Vinella D. Brochier-Armanet C. Loiseau L. Talla E. Barras F. PLoS Genet. 2009; 5e1000497Crossref PubMed Scopus (149) Google Scholar) and interact with the ISC assembly protein Iba57 (22Gelling C. Dawes I.W. Richhardt N. Lill R. Mühlenhoff U. Mol. Cell Biol. 2008; 28: 1851-1861Crossref PubMed Scopus (152) Google Scholar). In contrast to most other ISC components, Isa1 and Isa2 are not required for cell viability, but low levels of either protein induce virtually identical phenotypes, including respiratory incompetence and glutamate-lysine auxotrophy, demonstrating their involvement in cellular ISC assembly (23Jensen L.T. Culotta V.C. Mol. Cell Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar, 24Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar, 25Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Deletion of Iba57 elicits similar phenotypes as the lack of either Isa1 or Isa2 (22Gelling C. Dawes I.W. Richhardt N. Lill R. Mühlenhoff U. Mol. Cell Biol. 2008; 28: 1851-1861Crossref PubMed Scopus (152) Google Scholar). In bacteria, A-type ISC proteins are diverse and widespread (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar, 21Vinella D. Brochier-Armanet C. Loiseau L. Talla E. Barras F. PLoS Genet. 2009; 5e1000497Crossref PubMed Scopus (149) Google Scholar). Escherichia coli harbors three canonical A-type proteins, IscA, SufA, and ErpA, and the non-canonical NfuA. Deletion of iscA is not associated with a clear phenotype, whereas that of ErpA or a double deletion of iscA and sufA results in strong growth defects (26Loiseau L. Gerez C. Bekker M. Ollagnier-de Choudens S. Py B. Sanakis Y. Teixeira de Mattos J. Fontecave M. Barras F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13626-13631Crossref PubMed Scopus (119) Google Scholar, 27Tan G. Lu J. Bitoun J.P. Huang H. Ding H. Biochem. J. 2009; 420: 463-472Crossref PubMed Scopus (72) Google Scholar). These phenotypes frequently aggravate under special growth conditions, such as iron limitation or oxidative stress, and are linked to the loss of function of crucial cellular Fe/S proteins (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 21Vinella D. Brochier-Armanet C. Loiseau L. Talla E. Barras F. PLoS Genet. 2009; 5e1000497Crossref PubMed Scopus (149) Google Scholar, 26Loiseau L. Gerez C. Bekker M. Ollagnier-de Choudens S. Py B. Sanakis Y. Teixeira de Mattos J. Fontecave M. Barras F. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 13626-13631Crossref PubMed Scopus (119) Google Scholar, 27Tan G. Lu J. Bitoun J.P. Huang H. Ding H. Biochem. J. 2009; 420: 463-472Crossref PubMed Scopus (72) Google Scholar, 28Johnson D.C. Unciuleac M.C. Dean D.R. J. Bacteriol. 2006; 188: 7551-7561Crossref PubMed Scopus (102) Google Scholar, 29Balasubramanian R. Shen G. Bryant D.A. Golbeck J.H. J. Bacteriol. 2006; 188: 3182-3191Crossref PubMed Scopus (76) Google Scholar, 30Lu J. Yang J. Tan G. Ding H. Biochem. J. 2008; 409: 535-543Crossref PubMed Scopus (70) Google Scholar, 31Angelini S. Gerez C. Ollagnier-de Choudens S. Sanakis Y. Fontecave M. Barras F. Py B. J. Biol. Chem. 2008; 283: 14084-14091Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 32Wang W. Huang H. Tan G. Si F. Liu M. Landry A.P. Lu J. Ding H. Biochem. J. 2010; 432: 429-436Crossref PubMed Scopus (35) Google Scholar, 33Py B. Moreau P.L. Barras F. Curr. Opin. Microbiol. 2011; 14: 218-223Crossref PubMed Scopus (61) Google Scholar). Canonical A-type ISC proteins contain three highly conserved cysteine residues in two segments close to the terminus that are essential for function in vivo (21Vinella D. Brochier-Armanet C. Loiseau L. Talla E. Barras F. PLoS Genet. 2009; 5e1000497Crossref PubMed Scopus (149) Google Scholar, 23Jensen L.T. Culotta V.C. Mol. Cell Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar, 25Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 30Lu J. Yang J. Tan G. Ding H. Biochem. J. 2008; 409: 535-543Crossref PubMed Scopus (70) Google Scholar). These residues are part of a flexible structure that was predicted to bind iron or an Fe/S cluster (34Cupp-Vickery J.R. Silberg J.J. Ta D.T. Vickery L.E. J. Mol. Biol. 2004; 338: 127-137Crossref PubMed Scopus (76) Google Scholar, 35Morimoto K. Yamashita E. Kondou Y. Lee S.J. Arisaka F. Tsukihara T. Nakai M. J. Mol. Biol. 2006; 360: 117-132Crossref PubMed Scopus (63) Google Scholar). In fact, bacterial and eukaryotic A-type proteins were shown to bind Fe/S clusters upon chemical reconstitution in vitro, and in vivo Fe/S co-factor binding has been demonstrated for SufA (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar, 36Gupta V. Sendra M. Naik S.G. Chahal H.K. Huynh B.H. Outten F.W. Fontecave M. Ollagnier de Choudens S. J. Am. Chem. Soc. 2009; 131: 6149-6153Crossref PubMed Scopus (84) Google Scholar). The Fe/S cluster assembled on A-type proteins by can be transferred to recipient Fe/S apoproteins in vitro. Collectively, these observations may indicate that bacterial A-type ISC proteins play a scaffold role for de novo synthesis of Fe/S clusters before their transfer to apoproteins (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar, 37Wu G. Mansy S.S. Hemann C. Hille R. Surerus K.K. Cowan J.A. J. Biol. Inorg. Chem. 2002; 7: 526-532Crossref PubMed Scopus (70) Google Scholar, 38Ollagnier-de Choudens S. Nachin L. Sanakis Y. Loiseau L. Barras F. Fontecave M. J. Biol. Chem. 2003; 278: 17993-18001Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 39Ollagnier-de-Choudens S. Sanakis Y. Fontecave M. J. Biol. Inorg. Chem. 2004; 9: 828-838Crossref PubMed Scopus (89) Google Scholar). In addition, E. coli ISCA and SufA and human ISCA have been demonstrated to bind mononuclear iron (27Tan G. Lu J. Bitoun J.P. Huang H. Ding H. Biochem. J. 2009; 420: 463-472Crossref PubMed Scopus (72) Google Scholar, 30Lu J. Yang J. Tan G. Ding H. Biochem. J. 2008; 409: 535-543Crossref PubMed Scopus (70) Google Scholar, 32Wang W. Huang H. Tan G. Si F. Liu M. Landry A.P. Lu J. Ding H. Biochem. J. 2010; 432: 429-436Crossref PubMed Scopus (35) Google Scholar, 40Yang J. Bitoun J.P. Ding H. J. Biol. Chem. 2006; 281: 27956-27963Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 41Ding H. Yang J. Coleman L.C. Yeung S. J. Biol. Chem. 2007; 282: 7997-8004Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 42Lu J. Bitoun J.P. Tan G. Wang W. Min W. Ding H. Biochem. J. 2010; 428: 125-131Crossref PubMed Scopus (33) Google Scholar). Based on in vitro studies, this iron moiety was proposed to be used for the de novo synthesis of Fe/S co-factors on U-type ISC assembly proteins, suggesting an iron chaperone function for the A-type ISC proteins. Here, we address several key questions concerning the physiological roles of the Isa proteins in Fe/S protein assembly in yeast. For instance, we asked by yeast complementation experiments whether the two yeast Isa proteins and the functionally diverse bacterial A-type ISC proteins perform orthologous or distinct functions. We then comprehensively investigated numerous cellular Fe/S proteins for their in vivo dependence on Isa1 and Isa2 function to gain insights into the substrate specificity of the Isa proteins. A major goal of our study was the understanding of the functional relation between the Isa proteins and the major Fe/S scaffold proteins Isu1-Isu2. We asked whether de novo Fe/S cluster synthesis on Isu proteins depends on Isa proteins, as proposed for bacterial A-type ISC proteins (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar). We also investigated what kind of metal co-factor is bound to the Isa proteins and what role this co-factor might play. Because we found in vivo evidence for iron rather than Fe/S cluster binding to the Isa proteins, we investigated its potential physiological role. In this respect, we were interested in the particular task of Iba57. Together, our findings show that the Isa proteins, in close cooperation with Iba57, form a specialized ISC assembly subcomplex that is dedicated to the maturation of virtually all mitochondrial proteins carrying a [4Fe-4S] cluster, presumably by transferring iron from the Isa proteins in an Iba57-facilitated fashion to recipient apoproteins. Yeast strains used in this study are listed in supplemental Table SI. Cells were cultivated in rich medium (YP) or minimal medium containing a minimal set of supplements (SC) and 2% (w/v) glucose or galactose (43Sherman F. Methods Enzymol. 2002; 350: 3-41Crossref PubMed Scopus (992) Google Scholar). Iron-depleted minimal media were prepared using yeast nitrogen base lacking FeCl3 (ForMedium). Media for anaerobic cell growth were supplemented with 0.2% Tween 80, 25 μg/ml ergosterol, and 20 μg/ml methionine. Gal-ISA1 and Gal-ISA2 and derivatives of these strains were grown in SD minimal medium for 4 days to deplete the Isa proteins to critical levels. Repression of other Gal strains was performed as described in the corresponding literature (see supplemental Table SI). Plasmids used in this study are listed in supplemental Table SII. Constructs were verified by DNA sequencing and/or functional complementation of corresponding yeast mutant. In vivo radiolabeling of yeast cells with 55FeCl3 (ICN) and measurement of 55Fe incorporation into Fe/S proteins by immunoprecipitation and scintillation counting were carried out as described previously (44Stehling O. Smith P.M. Biederbick A. Balk J. Lill R. Mühlenhoff U. Methods Mol. Biol. 2007; 372: 325-342Crossref PubMed Scopus (30) Google Scholar, 45Molik S. Lill R. Mühlenhoff U. Methods Cell Biol. 2007; 80: 261-280Crossref PubMed Scopus (33) Google Scholar). Antibodies were raised in rabbits against recombinant proteins expressed in E. coli. Labeling of Fe/S proteins in mitochondrial extracts with 35S sulfur was performed using the protocol described in Ref. 46Mühlenhoff U. Richhardt N. Gerber J. Lill R. J. Biol. Chem. 2002; 277: 29810-29816Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, except that 10 μCi of [35S]cysteine and 0.3 mm FeCl2 were used. The following published methods were used: manipulation of DNA and PCR (47Sambrook J. Russel D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar); transformation of yeast cells (48Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2101) Google Scholar); PCR-mediated gene replacement in yeast (49Gueldener U. Heinisch J. Koehler G.J. Voss D. Hegemann J.H. Nucleic Acids Res. 2002; 30: e23Crossref PubMed Scopus (758) Google Scholar); preparation of yeast mitochondria (50Diekert K. de Kroon A.I. Kispal G. Lill R. Methods Cell Biol. 2001; 65: 37-51Crossref PubMed Google Scholar); immunological techniques (51Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar); and enzyme activities of succinate dehydrogenase, malate dehydrogenase, and cytochrome c oxidase (44Stehling O. Smith P.M. Biederbick A. Balk J. Lill R. Mühlenhoff U. Methods Mol. Biol. 2007; 372: 325-342Crossref PubMed Scopus (30) Google Scholar, 45Molik S. Lill R. Mühlenhoff U. Methods Cell Biol. 2007; 80: 261-280Crossref PubMed Scopus (33) Google Scholar), aconitase (52Kennedy M.C. Emptage M.H. Dreyer J.L. Beinert H. J. Biol. Chem. 1983; 258: 11098-11105Abstract Full Text PDF PubMed Google Scholar), and homoaconitase (53Wallace M.A. Liou L.L. Martins J. Clement M.H. Bailey S. Longo V.D. Valentine J.S. Gralla E.B. J. Biol. Chem. 2004; 279: 32055-32062Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Error bars represent the standard error of the mean (n > 4). Most of the recent work on A-type ISC proteins has been carried out with bacterial members (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar). Whether the bacterial and eukaryotic A-type proteins are functional orthologues has not been established. We therefore tested if the bacterial homologues can complement the glutamate and lysine auxotrophies of Saccharomyces cerevisiae isa1Δ and isa2Δ cells (23Jensen L.T. Culotta V.C. Mol. Cell Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar, 24Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar, 25Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). When fused to a mitochondrial targeting sequence, all three A-type members from E. coli, IscA, SufA, and ErpA, rescued the lysine and glutamate auxotrophies of isa1Δ cells (Fig. 1A). This identifies Isa1 as a functional orthologue of bacterial A-type ISC proteins. In contrast, neither bacterial protein was able to rescue isa2Δ cells. Apparently, S. cerevisiae Isa2, which carries a unique sequence insertion that is not found in other A-type ISC proteins, cannot be functionally replaced by the bacterial proteins, indicating that it performs a eukaryote-specific function. Bacterial A-type proteins form oligomers. In order to test the complex formation between yeast Isa1 and Isa2, we immunoprecipitated Isa1 from cell lysates with specific antibodies. Indeed, upon overproduction, Isa2 co-purified with anti-Isa1 immunobeads (Fig. 1B). This association was specific, because it was not detected with antibodies that do not recognize Isa1 (Fig. 1B). When Isa1 was immunoprecipitated from cells overproducing both Isa proteins, the amount of co-immunoprecipitated Isa2 was largely increased, indicating a concentration-dependent complex formation. The inverse analysis was not possible, because Isa1 co-migrates with the small IgG subunits (data not shown). We have shown previously that Isa1 and Isa2 interact with Iba57 in S. cerevisiae (22Gelling C. Dawes I.W. Richhardt N. Lill R. Mühlenhoff U. Mol. Cell Biol. 2008; 28: 1851-1861Crossref PubMed Scopus (152) Google Scholar). Deletions of either ISA1, ISA2, or IBA57 elicit virtually identical phenotypes (22Gelling C. Dawes I.W. Richhardt N. Lill R. Mühlenhoff U. Mol. Cell Biol. 2008; 28: 1851-1861Crossref PubMed Scopus (152) Google Scholar, 23Jensen L.T. Culotta V.C. Mol. Cell Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar, 24Pelzer W. Mühlenhoff U. Diekert K. Siegmund K. Kispal G. Lill R. FEBS Lett. 2000; 476: 134-139Crossref PubMed Scopus (77) Google Scholar, 25Kaut A. Lange H. Diekert K. Kispal G. Lill R. J. Biol. Chem. 2000; 275: 15955-15961Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Therefore, it is likely that the hetero-oligomeric complex of Isa1, Isa2, and Iba57 is the functional unit in S. cerevisiae. The fact that this complex includes a component, Isa2, that cannot be functionally replaced by bacterial A-type ISC proteins, raises the interesting question of whether the physiological roles of bacterial A-type proteins are conserved in eukaryotes. Recent work on bacterial A-type proteins suggests two different models for the function of this protein class. Bacterial IscA and SufA were shown to bind a transient Fe/S co-factor that can be transferred to recipient Fe/S apoferredoxins. Hence, it was suggested that the A-type ISC proteins may perform a role as an Fe/S scaffold or Fe/S transfer protein that operates in parallel or downstream, respectively, of U-type ISC proteins (3Py B. Barras F. Nat. Rev. Microbiol. 2010; 8: 436-446Crossref PubMed Scopus (262) Google Scholar, 5Johnson D.C. Dean D.R. Smith A.D. Johnson M.K. Annu. Rev. Biochem. 2005; 74: 247-281Crossref PubMed Scopus (1110) Google Scholar, 6Fontecave M. Ollagnier-de-Choudens S. Arch. Biochem. Biophys. 2008; 474: 226-237Crossref PubMed Scopus (148) Google Scholar, 36Gupta V. Sendra M. Naik S.G. Chahal H.K. Huynh B.H. Outten F.W. Fontecave M. Ollagnier de Choudens S. J. Am. Chem. Soc. 2009; 131: 6149-6153Crossref PubMed Scopus (84) Google Scholar). Alternatively, the proteins were demonstrated to bind mononuclear iron, and thus they were suggested to serve as iron chaperones promoting Fe/S cluster assembly on the U-type ISC scaffold proteins (30Lu J. Yang J. Tan G. Ding H. Biochem. J. 2008; 409: 535-543Crossref PubMed Scopus (70) Google Scholar, 32Wang W. Huang H. Tan G. Si F. Liu M. Landry A.P. Lu J. Ding H. Biochem. J. 2010; 432: 429-436Crossref PubMed Scopus (35) Google Scholar, 40Yang J. Bitoun J.P. Ding H. J. Biol. Chem. 2006; 281: 27956-27963Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 41Ding H. Yang J. Coleman L.C. Yeung S. J. Biol. Chem. 2007; 282: 7997-8004Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 42Lu J. Bitoun J.P. Tan G. Wang W. Min W. Ding H. Biochem. J. 2010; 428: 125-131Crossref PubMed Scopus (33) Google Scholar). In order to test the relevance of these observations for the yeast Isa proteins, we analyzed the de novo synthesis of the Fe/S cluster of Isu1 by 55Fe radiolabeling of Gal-ISA1 and Gal-ISA2 cells overproducing Isu1. In these Gal strains, the expression of ISA1 and ISA2 can be repressed upon cultivation in glucose (see supplemental Table SII). Following the radiolabeling, Isu1 was immunoprecipitated from cell lysates with specific antibodies. 55Fe bound to the immunobeads reflects the amount of de novo Fe/S cluster assembly on Isu1 and was quantified by scintillation counting (19Mühlenhoff U. Gerber J. Richhardt N. Lill R. EMBO J. 2003; 22: 4815-4825Crossref PubMed Scopus (342) Google Scholar). Remarkably, the amount of 55Fe co-immunoprecipitated with Isu1 from these cells slightly increased in both Gal-ISA1 and Gal-ISA2 under depleting conditions, indicating that the maturation of Isu1 can occur independently of the Isa proteins (Fig. 2A). Because Isu1 binds a [2Fe-2S] cluster, we compared these findings for 55Fe binding to the mitochondrial [2Fe-2S] ferredoxin Yah1 by in vivo radiolabeling of wild-type, isa1Δ, isa2Δ, and isa1/2Δ cells upon overexpression of Yah1. The amounts of 55Fe associated with Yah1 were 1.5-fold higher in cells lacking the Isa proteins compared with wild-type cells (Fig. 2B). As judged by immunostaining, levels of Yah1 and Isu1 remained unaltered in all strains analyzed. Similar results were obtained with Gal-ISA1 cells (not shown). Collectively, these findings demonstrate that the Isa proteins are not involved in the formation of the [2Fe-2S] clusters on Isu1 and Yah1 in vivo and thus do not serve as iron chaperones for mitochondrial Fe/S protein biogenesis. Moreover, these findings are compatible with the non-essential character of the ISA genes in yeast, because Yah1 and Isu1 are the only known essential mitochondrial Fe/S proteins. The data above suggest that the Isa proteins are specialized ISC assembly factors that are required only for a subset of Fe/S proteins. In order to comprehensively define this subset, we next analyzed the de novo maturation of the Rieske Fe/S protein (Rip1) and of dihydroacid dehydratase Ilv3, the two other known mitochondrial [2Fe-2S] proteins in S. cerevisiae (see supplemental Fig. S1 for data indicating that Ilv3 is a [2Fe-2S] protein). In order to distinguish the insertion of 55Fe into the Fe/S cluster of Rip1 from that into cytochromes of complex III, we constructed vector p426-ΔN-RIP1, which allows the synthesis of a soluble Rieske protein in the mitochondrial matrix (54Link T.A. Saynovits M. Assmann C. Iwata S. Ohnishi T. von Jagow G. Eur. J. Biochem. 1996; 237: 71-75Crossref PubMed Scopus (49) Google Scholar). Wild-type levels of 55Fe were immunoprecipitated with anti-Rip1 antibodies from extracts of depleted Gal-ISA1 cells, whereas only background levels of 55Fe were found after depletion of Isu1 in Gal-ISU1" @default.
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• W2148522029 title "Specialized Function of Yeast Isa1 and Isa2 Proteins in the Maturation of Mitochondrial [4Fe-4S] Proteins" @default.
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