Identification of FRA1 and FRA2 as Genes Involved in Regulating the Yeast Iron Regulon in Response to Decreased Mitochondrial Iron-Sulfur Cluster Synthesis (2008)

The nature of the connection between mitochondrial Fe-S cluster synthesis and the iron-sensitive transcription factor Aft1 in regulating the expression of the iron transport system in Saccharomyces cerevisiae is not known. Using a genetic screen, we identified two novel cytosolic proteins, Fra1 and Fra2, that are part of a complex that interprets the signal derived from mitochondrial Fe-S synthesis. We found that mutations in FRA1 (YLL029W) and FRA2 (YGL220W) led to an increase in transcription of the iron regulon. In cells incubated in high iron medium, deletion of either FRA gene results in the translocation of the low iron-sensing transcription factor Aft1 into the nucleus, where it occupies the FET3 promoter. Deletion of either FRA gene has the same effect on transcription as deletion of both genes and is not additive with activation of the iron regulon due to loss of mitochondrial Fe-S cluster synthesis. These observations suggest that the FRA proteins are in the same signal transduction pathway as Fe-S cluster synthesis. We show that Fra1 and Fra2 interact in the cytosol in an iron-independent fashion. The Fra1-Fra2 complex binds to Grx3 and Grx4, two cytosolic monothiol glutaredoxins, in an iron-independent fashion. These results show that the Fra-Grx complex is an intermediate between the production of mitochondrial Fe-S clusters and transcription of the iron regulon. The nature of the connection between mitochondrial Fe-S cluster synthesis and the iron-sensitive transcription factor Aft1 in regulating the expression of the iron transport system in Saccharomyces cerevisiae is not known. Using a genetic screen, we identified two novel cytosolic proteins, Fra1 and Fra2, that are part of a complex that interprets the signal derived from mitochondrial Fe-S synthesis. We found that mutations in FRA1 (YLL029W) and FRA2 (YGL220W) led to an increase in transcription of the iron regulon. In cells incubated in high iron medium, deletion of either FRA gene results in the translocation of the low iron-sensing transcription factor Aft1 into the nucleus, where it occupies the FET3 promoter. Deletion of either FRA gene has the same effect on transcription as deletion of both genes and is not additive with activation of the iron regulon due to loss of mitochondrial Fe-S cluster synthesis. These observations suggest that the FRA proteins are in the same signal transduction pathway as Fe-S cluster synthesis. We show that Fra1 and Fra2 interact in the cytosol in an iron-independent fashion. The Fra1-Fra2 complex binds to Grx3 and Grx4, two cytosolic monothiol glutaredoxins, in an iron-independent fashion. These results show that the Fra-Grx complex is an intermediate between the production of mitochondrial Fe-S clusters and transcription of the iron regulon. Iron is an essential element required for all eukaryotes and most prokaryotes. Iron is also potentially dangerous, since it can participate in the generation of toxic oxygen molecules, such as superoxide anion and the hydroxyl radical. Iron transport is highly regulated in all species, and iron transporters are only expressed under conditions of iron need. Transcriptional and post-transcriptional regulation of iron transport systems occurs in all organisms ranging from yeast to humans. Consequently, iron acquisition in all species is tightly controlled and is coordinated with iron use. The budding yeast Saccharomyces cerevisiae expresses two different high affinity iron transport systems. One system is composed of a closely related family of four siderophore transporters. Siderophores are small organic molecules that exhibit an extremely high affinity (Kd = 10–33) for iron (1Van Ho A. Ward D.M. Kaplan J. Annu. Rev. Microbiol. 2002; 56: 237-261Crossref PubMed Scopus (181) Google Scholar). Although S. cerevisiae does not synthesize siderophores, it can accumulate siderophores produced by other organisms. The second high affinity iron transport system mediates the acquisition of ionic iron and is composed of a cell surface multicopper oxidase, Fet3, and a transmembrane permease, Ftr1. The multicopper oxidase converts Fe2+ to Fe3+, which is then transported by the transmembrane permease. The transcriptional activator Aft1 regulates both high affinity iron transport systems (2Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (318) Google Scholar). Aft1 is cytosolic when cells are iron-replete, but under conditions of iron depletion, Aft1 translocates into the nucleus, where it activates the transcription of ∼20 genes (3Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18919Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). These genes, referred to as the iron regulon, include the siderophore transporters, the high affinity iron transport system, FET3 and FTR1, and genes involved in the assembly of the high affinity iron transport system as well as genes involved in iron-based metabolism and their regulation. There is a second transcription factor, Aft2, which is a paralogue of Aft1. When overexpressed, Aft2 can also affect transcription of the iron regulon, although deletion of AFT2 alone has little effect on iron transport and metabolism (4Blaiseau P.L. Lesuisse E. Camadro J.M. J. Biol. Chem. 2001; 276: 34221-34226Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 5Rutherford J.C. Jaron S. Ray E. Brown P.O. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14322-14327Crossref PubMed Scopus (129) Google Scholar). It is clear that translocation of Aft1 into the nucleus and transcription of the iron regulon responds to cellular iron deprivation. We recently suggested that Aft1 does not respond directly to iron but rather to the production of iron-sulfur clusters within mitochondria. This conclusion was based on the observation that defects in mitochondrial Fe-S cluster synthesis induce transcription of the iron regulon even in cells that are iron-replete (6Chen O.S. Crisp R.J. Valachovic M. Bard M. Winge D.R. Kaplan J. J. Biol. Chem. 2004; 279: 29513-29518Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Aft1, however, does not appear to be an Fe-S cluster-containing protein (6Chen O.S. Crisp R.J. Valachovic M. Bard M. Winge D.R. Kaplan J. J. Biol. Chem. 2004; 279: 29513-29518Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Deletion of genes required for the assembly of cytosolic Fe-S cluster-containing proteins does not lead to activation of the iron regulon (7Rutherford J.C. Ojeda L. Balk J. Muhlenhoff U. Lill R. Winge D.R. J. Biol. Chem. 2005; 280: 10135-10140Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Thus, the mechanism by which mitochondrial Fe-S cluster synthesis affects the iron regulon is unclear. In this paper, we present a genetic test of the hypothesis that mitochondrial Fe-S cluster synthesis is involved in regulating the iron regulon by screening for mutants that would activate the iron regulon even in the presence of high media iron. Analysis of the mutants demonstrated that most were defective in genes required for mitochondrial Fe-S cluster synthesis. Two novel genes were identified that encode nonmitochondrial proteins that are required for regulation of Aft1-mediated expression of the iron regulon. Yeast Strains and Growth Media—The strains employed in this study are described in Table 1. Yeast were in yeast and were by the of the of iron in to the and by the of to the strains in the O.S. Crisp R.J. Valachovic M. Bard M. Winge D.R. Kaplan J. J. Biol. Chem. 2004; 279: 29513-29518Abstract Full Text Full Text PDF PubMed Scopus (171) Google O.S. Crisp R.J. Valachovic M. Bard M. Winge D.R. Kaplan J. J. Biol. Chem. 2004; 279: 29513-29518Abstract Full Text Full Text PDF PubMed Scopus (171) Google in E. L. M. E. PubMed Scopus Google in in in a of the the in FET3 was and into The was and into from of The was and the the with the on both was the in yeast The were by The was and to the yeast strains and and of yeast cells with the the and were on with iron and by to a in of were to were by and a for was on of the were incubated for and the of FET3 that were mutants to a gene were to a by in was for all in this were and were by the the gene was by of the of FRA1 (YLL029W) FRA2 (YGL220W) was the deletion as a and for gene were from the Deletion was into with the in the in the the of genes was by for the were and to the gene deletion in a The gene deletion was by was from a composed of of a the FRA1 and of the and of the The was and the was by The was into a for the a was from the FRA1 described The was into the and the was and of were to and into the yeast expression under the of the E. L. M. E. PubMed Scopus Google Scholar). was to a and and a in and Fra2 was to a and and a in and were by Fra2 for and for Fra2 in the and protein was as was as a from by J. M. and into with was by and then into has described L. Muhlenhoff U. J.C. Lill R. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were by and Yeast from cellular was and into E. by a to the Yeast of and the gene was for were by cells in were by and to The were with the and the of the was for a was to proteins on for were by the cells in and The proteins were by and to were with and with the as for of proteins were as described that the and the proteins were from the was as described E. Kaplan J. EMBO J. PubMed Scopus Google Scholar). The and the were described (6Chen O.S. Crisp R.J. Valachovic M. Bard M. Winge D.R. Kaplan J. J. Biol. Chem. 2004; 279: 29513-29518Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). The was as described O.S. S. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). of Aft1 was by of the protein as described R.J. A. Jaron S. Y. Kaplan J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of cells was Two were and in were and the of cells was were of Grx3 was by and mitochondrial as described PubMed Scopus Google Scholar). were with Fra1 protein expressed in E. The FRA1 with a was into the and expressed in cells production was and the protein was an were from a by Fra1 the to the the was by the to was a for and Grx3 the Grx3 the was into and expressed in cells The protein was with and then a by a The protein was to We a genetic to genes that activate the iron regulon in iron-replete of mutants be to show expression of the iron regulon. The of mutations in genes that encode the high affinity iron transport system in genes required for the assembly of the high affinity transport systems. in genes lead to cellular iron and expression of the iron transport The second mutations in genes that encode of the mitochondrial Fe-S cluster synthesis and The of mutants is to the signal between mitochondrial Fe-S clusters and Aft1. of the iron regulon by the of mutations can be by of cells in high iron medium, since transport of iron by the low affinity iron transport systems would cellular iron and high affinity iron The other two of mutations would not be by high iron. yeast was that a the would be produced when cells are incubated in iron the of the iron is when cells are incubated in a deletion in FET3 is into this is due to defective iron The of iron to however, the production of since cells iron-replete by the transport of iron low affinity iron transporters. on this we cells an of by and for that when on iron. We to that in the presence of iron-replete was to a an and all were found to be when as a gene was with a low and that the to the in an were We identified genes that expression of Two of the genes proteins to a in Fe-S cluster Lill R. EMBO J. PubMed Scopus Google Scholar, J. M. Dancis A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). an involved in synthesis. It is that deletion of Fe-S cluster synthesis Lill R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google and it be that deletion of would also affect Fe-S cluster synthesis. The is a of the mitochondrial In the of is a in the of due to mitochondrial iron E. M. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Deletion of was also to activate the iron regulon, although the mechanism is not known. These results the hypothesis that iron-sulfur cluster synthesis in the regulation of Aft1 The gene identified which is part of the complex Y. S. R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). This complex with to in of the complex to the of transcription of Aft1 genes J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of a FRA1 (YLL029W) in the of the Iron gene identified by the was an This gene mutants that were in the same of that affects activation of the iron regulon. we from the and identified a which results in an from to in from the of and the with expression of deletion of in the deletion also expression of in high a deletion of in the in this screen, expression of and a between the deletion and on we to as FRA1 of and we to as transcriptional activation of the is on Aft1, we a an Deletion of the expression in since expression of was by This that FRA1 in the regulation of Aft1-mediated expression of the iron regulon. cells show FET3 when in high iron the expression of FET3 which was with a of FET3 in cells was on the transcription factor Aft1, since deletion of in cells in the loss of FET3 We expressed from a FET3 to transcriptional This the FET3 but the and post-transcriptional regulation of the cells show expression of in cells incubated in either with iron In cells in either expression of of cells in low iron medium, however, in a increase in that iron affects the regulation of the iron regulon even in the of of cells with for the regulon the regulated regulon expression cells not This that deletion of FRA1 is for transcriptional activation of Deletion of FRA2 of the Iron study that Fra1 with Grx4, and a protein by an Y. A. A. L. A. L. S. J. M. J. M. A. E. J. J. M. M. 2002; PubMed Scopus Google Scholar). We the effect of deletion of on expression of the since two that deletion of and to of the iron regulon L. Muhlenhoff U. J.C. Lill R. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. J. Sci. PubMed Scopus Google Scholar). cells with a deletion in are and show expression of FET3 as by not the between FRA1 and we a and the of the gene under iron Deletion of FRA2 led to an increase in expression that was to that in Deletion of FRA2 in a not lead to increase in expression that Fra1 and Fra2 are part of the same of cells in low iron in a increase in transcriptional We then deletion of in with deletion of FRA1 and FRA2 expression of We the that deletion of little effect on L. Muhlenhoff U. J.C. Lill R. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. J. Sci. PubMed Scopus Google Scholar). Deletion of either gene alone in with in FRA1 FRA2 not an additive effect in the iron regulon not that deletion of both and in of the iron regulon that was of iron L. Muhlenhoff U. J.C. Lill R. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. J. Sci. PubMed Scopus Google Scholar). In we that iron activation of the iron regulon in and There is in however, is a in It is that the expression of the iron regulon in cells is a to conditions that lead to Aft1 the FET3 in are two required for Aft1-mediated transcriptional Aft1 from cytosol to nucleus, and within the nucleus it the Aft1 and then We a Aft1 to Aft1 in and high iron conditions in is with of cells in the nucleus, under low iron is in the nucleus of of the cells and In is in the of and cells under both high and low iron We that the of in the nucleus of cells in low iron is not as as in although we this We Aft1 occupies the FET3 in cells by Aft1. were in high and low iron and was an to the FET3 and the gene were for the iron regulon is in and cells in low iron medium, and the of Aft1 to the FET3 was in and In and high iron medium, however, was to the FET3 in cells in cells that of the iron regulon in cells is the of Aft1 Fra1 and Fra2 in the as Fe-S that mitochondrial Fe-S cluster synthesis activates the iron regulon (6Chen O.S. Crisp R.J. Valachovic M. Bard M. Winge D.R. Kaplan J. J. Biol. Chem. 2004; 279: 29513-29518Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Fra1 and Fra2 are part of the Fe-S cluster we the effect of Fe-S cluster synthesis in We cells with a deletion in the which is essential for Fe-S cluster synthesis. The strains are by expression of under the of a promoter. of cells in low iron led to of of cells in high iron led to a in of In the of is although the of expression is that when cells are in low iron When was expressed in cells in high iron medium, the of was in cells In the of was increase in expression in of genes involved in Fe-S cluster synthesis to expression of the iron regulon and high of mitochondrial iron. In the of is an increase in cellular and mitochondrial iron In cells a small increase in cellular iron with cells and show increase in mitochondrial iron. 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M. 2002; PubMed Scopus Google a between Fra1 and We the effect of iron on the of Fra2, and were with a and either a a FRA2 the were from cells in either iron-replete was and the was by We Fra1 and in the from both high and low that the of Fra2, and is iron-independent a for affinity Fra1 was in from cells with an We that was an of with in high iron This however, was not We also Grx3 with in both iron-replete and cells We of to Fra2 and Grx3 would a complex when expressed of Fra2 in the of Grx3 from both iron-replete and cells The of both proteins were however, was clear of a complex was in the of the We the iron-independent Fra-Grx complex a Y. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). were with and E. L. M. E. PubMed Scopus Google Scholar). was in cells with alone in the presence of not was in cells with both but only in the of The to be and the and of were These results suggest that Fra2 and Grx3 a complex that is not by cytosolic iron We not that Aft1 to the Fra1-Fra2 complex in either high low iron medium, although an between Aft1 and Grx3 and was Aft1 and Grx3 and L. Muhlenhoff U. J.C. Lill R. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. J. Sci. PubMed Scopus Google Scholar). that Aft1 and interact under expression we cells an gene the of We that this Aft1 regulated the expression of FET3 in an not were incubated in iron-replete medium, and Grx3 was The Grx3 was and the was by with Grx3 under both low and high iron conditions These results the by that Aft1 and Grx3 an iron-independent complex even in the of Fra2, and Grx3 of Fe-S and Fra2 were found to be cytosolic proteins by a study PubMed Scopus Google Scholar). 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We a Grx3 which for of Grx3 in cells of yeast was in and was cytosolic in cells a high The cytosolic in cells was to that in cells for the cytosolic which is with a cytosolic of from a in cells led to but not affect The cytosolic in cells in high and low iron of cells that most Grx3 is show a in although protein can be with in the we a which to mitochondrial in which the of the is in the cytosol This protein was to the of activation of the iron regulon, that the protein is affects Aft1 cells protein were to and for and by and The with the mitochondrial protein that the protein is to the to the with were to and were for FET3 and an and are expressed We that the high affinity iron transport system is regulated by the of the Fe-S cluster of the iron regulon occurs in the of genes that encode of the Fe-S cluster pathway even when cells are not (6Chen O.S. Crisp R.J. Valachovic M. Bard M. Winge D.R. Kaplan J. J. Biol. Chem. 2004; 279: 29513-29518Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In Fe-S clusters are from the and are on cytosolic by a cytosolic complex R. Muhlenhoff U. Annu. Rev. Biol. PubMed Scopus Google Scholar). Deletion of genes that encode proteins that participate in that complex led to defective cytosolic Fe-S protein assembly but not induce the iron regulon (7Rutherford J.C. Ojeda L. Balk J. Muhlenhoff U. Lill R. Winge D.R. J. Biol. Chem. 2005; 280: 10135-10140Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). These that Aft1 a to mitochondrial Fe-S cluster synthesis but not to cytosolic Fe-S protein Thus, the of Fe-S cluster synthesis in regulation of the iron regulon is unclear. We a genetic to the of mitochondrial Fe-S cluster synthesis in iron-sensing and to of the iron-sensing identified of the Fe-S cluster pathway and as well as two genes and to affect expression of the iron regulon. 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Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The to proteins, the is to proteins participate in Fe-S assembly their to between proteins S. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). FRA1 an proteins are that can with an but the of Fra1 in this has not The transcription factor also has an which is to in protein We not this is for of Fra1 to results suggest that Fra1 and Fra2 are cytosolic proteins that the Fe-S cluster and in their the iron regulon is The proteins are of the and we that deletion of either protein affects mitochondrial that of the iron regulon by deletion of the FRA genes is not additive with due to the of Fe-S cluster synthesis. Iron to a increase in FET3 transcription in both cells and in cells with defects in Fe-S cluster synthesis. 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These are with a in which Aft1 between cytosol and nucleus, and the of Aft1 a in the of One is that Aft1 is the This that Aft1 between nucleus and cytosol even in high also show that within the nucleus, of cells in media the of Aft1 to the FET3 effect on Fe-S cluster as by mitochondrial These observations lead to an of all Aft1 is with iron can affect These results suggest that iron affects the transcriptional of Aft1 of the Fe-S cluster We of the Kaplan for the