Aft2p, a Novel Iron-regulated Transcription Activator That Modulates, with Aft1p, Intracellular Iron Use and Resistance to Oxidative Stress in Yeast

The yeast, Saccharomyces cerevisiae,contains a transcription activator, Aft1p, that regulates the transcription of the high affinity iron transport system genes. This report describes the properties of Aft2p, a protein 39% homologous to Aft1p. Aft2p was found to activate transcription. Overproduction of Aft2p activates the transcription of theAFT1 target gene FET3. The doubleaft1aft2 mutant was unable to grow in iron-deprived conditions. Because a fet3 mutant does not show this deficiency, the defect is not solely caused by mis-regulation of iron transport but also involves defective iron use by the cells. Theaft1 cells were unable to grow in aerobic conditions on plates containing raffinose as the sole carbon source. The inability to grow on raffinose is not caused by the cell iron content being too low to sustain respiratory metabolism, because the oxygen consumption ofaft1 mutants showed that their respiratory activity is 2-fold higher than that of controls. The double aft1aft2mutant also has many phenotypes related to oxidative stress such as H2O2 hypersensitivity, oxygen-dependent copper toxicity, and oxygen-dependent methionine auxotrophy, which are suppressed in anaerobiosis. These results suggest that Aft2p and Aft1p have overlapping roles in the control of iron-regulated pathway(s) connected to oxidative stress resistance in yeast. The yeast, Saccharomyces cerevisiae,contains a transcription activator, Aft1p, that regulates the transcription of the high affinity iron transport system genes. This report describes the properties of Aft2p, a protein 39% homologous to Aft1p. Aft2p was found to activate transcription. Overproduction of Aft2p activates the transcription of theAFT1 target gene FET3. The doubleaft1aft2 mutant was unable to grow in iron-deprived conditions. Because a fet3 mutant does not show this deficiency, the defect is not solely caused by mis-regulation of iron transport but also involves defective iron use by the cells. Theaft1 cells were unable to grow in aerobic conditions on plates containing raffinose as the sole carbon source. The inability to grow on raffinose is not caused by the cell iron content being too low to sustain respiratory metabolism, because the oxygen consumption ofaft1 mutants showed that their respiratory activity is 2-fold higher than that of controls. The double aft1aft2mutant also has many phenotypes related to oxidative stress such as H2O2 hypersensitivity, oxygen-dependent copper toxicity, and oxygen-dependent methionine auxotrophy, which are suppressed in anaerobiosis. These results suggest that Aft2p and Aft1p have overlapping roles in the control of iron-regulated pathway(s) connected to oxidative stress resistance in yeast. Iron is required by all organisms; it is used as a cofactor in key redox enzymes involved in such diverse biological processes as cell respiration and the synthesis of metabolic intermediates. However, iron can damage cells by reacting with hydrogen peroxide to form the hydroxyl radical. This highly toxic compound damages DNA, proteins, and lipids (1Halliwell B. Gutteridge J.M. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4556) Google Scholar). Prokaryote and eukaryote cells therefore have evolved various systems for the close regulation of iron transport and its intracellular use (2Hantke K. Curr. Opin. Microbiol. 2001; 4: 172-177Crossref PubMed Scopus (580) Google Scholar, 3Rouault T. Klausner R. Curr. Top. Cell. Regul. 1997; 35: 1-19Crossref PubMed Scopus (214) Google Scholar). The yeast Saccharomyces cerevisiaehas several pathways of iron transport including a low affinity transporter encoded by FET4 (4Dix D.R. Bridgham J.T. Broderius M.A. Byersdorfer C.A. Eide D.J. J. Biol. Chem. 1994; 269: 26092-26099Abstract Full Text PDF PubMed Google Scholar) and a number of high affinity systems. One of these high affinity systems is encoded byFET3 and FTR1 (5Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (587) Google Scholar, 6Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar). It requires the reduction of Fe3+ to Fe2+ by plasma membrane reductases (FRE1 or FRE2) (7Dancis A. Klausner R.D. Hinnebusch A.G. Barriocanal J.G. Mol. Cell. Biol. 1990; 10: 2294-2301Crossref PubMed Scopus (257) Google Scholar, 8Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Crossref PubMed Scopus (197) Google Scholar). The other high affinity transport systems are siderophore-mediated and depend on several homologous transporters of the major superfacilitator (MSF) family encoded by ARN1, SIT1, TAF1, andENB1 (9Lesuisse E. Simon-Casteras M. Labbe P. Microbiology. 1998; 144: 3455-3462Crossref PubMed Scopus (129) Google Scholar, 10Lesuisse E. Blaiseau P.L. Dancis A. Camadro J.M. Microbiology. 2001; 147: 289-298Crossref PubMed Scopus (94) Google Scholar, 11Yun C.W. Tiedeman J.S. Moore R.E. Philpott C.C. J. Biol. Chem. 2000; 275: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). All the genes involved in the high affinity iron transport systems are under the control of the iron-dependent transcription activator Aft1p (12Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 13Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar, 14Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (289) Google Scholar). However, Aft1p does not affect the regulation of FET4 (15Dix D. Bridgham J. Broderius M. Eide D. J. Biol. Chem. 1997; 272: 11770-11777Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In addition to the high affinity transport genes, Aft1p regulates the transcription of other iron-responsive genes such as those encoding Ccc2p (the intracellular copper transporter responsible for delivering copper to Fet3p) (16Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Crossref PubMed Scopus (393) Google Scholar), Fth1p (which forms an iron transport complex on the vacuole with Fet5p) (17Urbanowski J.L. Piper R.C. J. Biol. Chem. 1999; 274: 38061-38070Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), Fre3p/Fre4p (which are potential siderophore-iron reductases) (18Yun C.W. Bauler M. Moore R.E. Klebba P.E. Philpott C.C. J. Biol. Chem. 2001; 276: 10218-10223Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and Fre5p/Fre6p of unknown function (19Martins L.J. Jensen L.T. Simon J.R. Keller G.L. Winge D.R. Simons J.R. J. Biol. Chem. 1998; 273: 23716-23721Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Finally, Aft1p seems to be involved in, but is not required for, the transcription of genes such as ATX1, which encodes the copper chaperone that delivers copper to Ccc2p, andISU1/ISU2, the products of which are involved in the mitochondrial assembly of iron-sulfur clusters (20Lin S.J. Pufahl R.A. Dancis A. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1997; 272: 9215-9220Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 21Garland S.A. Hoff K. Vickery L.E. Culotta V.C. J. Mol. Biol. 1999; 294: 897-907Crossref PubMed Scopus (167) Google Scholar). In conditions of iron deprivation, Aft1p actives the transcription of target genes by binding directly to the consensus sequence T/CG/ACACCC, a motif in its 5′-upstream regions. Although the functional DNA binding and activation domains of Aft1p have not yet been characterized, its similarities to other transcription factors suggest that the N-terminal region rich in basic residues is involved in recognizing DNA, and the C-terminal glutamine-rich domains may be required for the transcriptional activation function. The dominant allele AFT1up, which leads to Phe-291 in place of Cys-291 in the N-terminal region of the protein, constitutively binds to DNA (13Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar, 14Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (289) Google Scholar). Just how Aft1p senses iron and activates gene expression is not understood, and recent data indicate that the Aft1p-mediated response is not restricted to iron deprivation conditions but can be induced by the concentrations of molecular oxygen or copper in the culture medium (22Gross C. Kelleher M. Iyer V.R. Brown P.O. Winge D.R. J. Biol. Chem. 2000; 275: 32310-32316Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 23Hassett R.F. Romeo A.M. Kosman D.J. J. Biol. Chem. 1998; 273: 7628-7636Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We have now attempted to obtain further information about the regulation of iron homeostasis by inspecting the yeast genome by blast analysis. We found a 416-amino acid protein encoded by the YPL202c gene that presents 39% overall identity to Aft1p, which we designated Aft2p (Fig. 1). This paper describes some of the characteristics of Aft2p. It is a second iron-regulated transcriptional activator in yeast. Aft2p is required for iron homeostasis and resistance to oxidative stress when Aft1p is absent. Molecular data and phenotypic analysis suggest that Aft1p and Aft2p have overlapping functions. The strains used in this study were CM3260 (MATα, trp1-63, leu2-3, 112 gcn4-101, his3-609), Y18 (MATα, trp1-63, leu2-3, 112 gcn4-101, his3-609, aft1::TRP1), and Y19 (MATα, trp1-63, leu2-3, 112 gcn4-101, his3-609, fet3::URA3). The CM3260aft2Δ (MATα, trp1-63, leu2-3, 112 gcn4-101, his3-609, aft2:: kanMX4) and Y18aft2Δ (MATα, trp1-63, leu2-3, 112 gcn4-101, his3-609, aft1::TRP1,aft2:: kanMX4) strains were constructed by integrating kanMX4 at the AFT2 locus in strains CM3260 and Y18 as described in Ref. 24Wach A. Yeast. 1996; 12: 259-265Crossref PubMed Scopus (703) Google Scholar. The following primers were used to amplify the open reading frame replacement cassette containing the kanMX4 marker with long flanking homology regions of the AFT2 promoter and terminator: 5′-GGGTATAAGGAGTGTCAAAG-3′, 5′-GGGGATCCGTCGACCTGCAGCGTACCATTTCTTGGGGTCGCTTTC-3′, 5′-AACGAGCTCGAATTCATCGATGATATAATTATTTAGTTTTCAACTC-3′, and 5′-GATGCCTTATTTGTGGTCTG-3′. The sequences underlined are homologous to the kanMX4 marker. Kanamycin-resistant clones were selected on YPD plates containing G418 (200 μg/ml). Deletions were confirmed by polymerase chain reaction using primers flanking the insertion region. The plasmid pEG202 and the derivative plasmids pEG202-AFT1 and pEG202-AFT2 contained the DNA binding domain of LexA. Plasmid pEG202-AFT1 expressing LexA-Aft1p and plasmid pEG202-AFT2 encoding LexA-Aft2p were constructed by integrating the open reading frames ofAFT1 or AFT2 in frame with the DNA binding domain of LexA. Open reading frames were amplified with the following oligonucleotides: AFT1, 5′-CAGAAGAATTCACGACAATGGAAGGCTTC-3′ and 5′-TTCATCTCGACTAATCTTCTGGCTTCAC-3′; and AFT2, 5′-AAGCGGGATCCAAGAAATGAAAGCAAAGTCGA-3′ and 5′-GAAAACTCGAGAATTAATATTTTGATATTAAGGC-3′. The amplified fragments were digested by EcoRI and XhoI for AFT1and by BamHI and XhoI for AFT2 and ligated to the DNA binding domain of LexA derived from the plasmid pEG202 (25Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar) digested with the appropriate restriction enzymes. The plasmid pSH18–34 contained the GAL1-lexop-lacZ reporter gene from which the upstream activation sequence GAL1 had been deleted and replaced by Brent R. Cell. Full Text PDF PubMed Scopus Google Scholar). Plasmid by contained a cassette of the upstream activation sequence to to Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (289) Google Scholar). strains were in rich medium yeast containing or or in copper and iron yeast the required and was to the medium to the was to the medium to the medium in copper was However, because of the of the double mutant to this copper was from the were in a The cells were in in on a at the cells were in at in and at for to was by the K. H. PubMed Scopus Google Scholar). was as described P.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The DNA fragments used as to the open reading frames of gene were as for a for a and a that was from a polymerase chain to the open reading frame of by using the primers and was as described L. PubMed Scopus Google Scholar). The protein was by the Biochem. PubMed Scopus Google Scholar). The designated yeast strains were in the iron and copper yeast medium with iron for by and in and in The iron was by plasma at the of the on cells in The designated yeast strains were in the YPD medium or with iron The respiratory activity of cells was by an The of oxygen consumption was using a cell with a The respiratory medium was a with containing The cells were as in The open reading frame of AFT2 a protein with a molecular of and a of The N-terminal of Aft2p and Aft1p were the regions in the This region is rich in basic and which are potential iron at residues and in Aft1p and at and in Aft2p. we found the protein sequences their Aft2p contained or C-terminal these have been to iron in Aft1p (13Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar). Aft2p the C-terminal glutamine-rich domain of Aft1p, which may be required for the function of Aft1p C. M. C. C. R. E. Yeast. 1997; PubMed Scopus (76) Google Scholar). The homologous domains in the N-terminal region were by sequences in Aft1p. the highly of Aft1p was from Aft2p Finally, the that are several such as basic clusters and a sequence in the N-terminal region of Aft1p. such be in the Aft2p sequence (Fig. 1). is a that Aft1p is but a that Aft2p is a is also a that Aft2p is a mitochondrial The open reading frame of the Aft2p protein was to that of the DNA binding This was in the CM3260 a reporter gene with the upstream activation sequence The protein was also in these cells. The protein the transcription of the reporter that Aft2p has a function were in medium with or cells and cells the of activity in the of cells as as cells in the of The results were with iron as a of iron not as a transcription CM3260 was with the plasmid and plasmids or in the of were in copper and iron medium or with activity was as described under The of are with Open in a CM3260 was with the plasmid and plasmids or in the of were in copper and iron medium or with activity was as described under The of are with We the of AFT2 in the transcription of the genes by and mutant strains We also constructed a Aft2p by a high number plasmid the the double We the of and in the mutants under or conditions. The and strains contained of The in the was and the low of transcription was by was in the double mutant in iron-deprived conditions. The of Aft2p in the aft1aft2mutant the iron-regulated transcription of FET3. were in conditions than in conditions in the or in and However, the of by the iron in the double of Aft2p in the cells was by a in We the of AFT2 to activate the transcription of by at the Aft1p DNA binding of the strains were with a plasmid containing the of in of a reporter This activity by Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (289) Google Scholar). the activity a of the activation of transcription at the and strains under iron-deprived conditions had In activity was in the and of Aft2p by the cells the which is in with the results in A. transcription activity was also by The and the and fet3 mutants on plates with the iron the under these (Fig. The of the mutant was by the of iron from to The of the doubleaft1aft2 mutant was under iron-deprived conditions. It was by iron to the the was to iron deprivation, and this was by the of Aft2p. The inability of the and strains to grow on medium was not caused by a in the high affinity iron transport because the of the fet3 mutant was not under these conditions. We the of iron by and mutants was because of a intracellular iron or a defect in the iron in the mutant cells. We the intracellular iron content of the cells in The had iron to those of the the (Fig. However, the in the medium iron by was with the strains (Fig. The defect was for the The of was by a of iron (Fig. The of oxygen consumption of the mutants were higher than those of the and strains The oxygen consumption was to and was the with or high iron concentrations in the We this further by the phenotypes of cells in a medium containing raffinose as the sole carbon source. raffinose is a not a carbon source. We the of the cells in the or of The of the fet3 mutant on raffinose under aerobic conditions was with and strains In mutant strains were unable to use this carbon under aerobic conditions. The was suppressed by Aft2p. The mutant also be by iron and also copper to the mutant was not by the addition of iron or results were when was used of raffinose not Finally, the of and the strains was under conditions iron or of and designated yeast strains were in the YPD medium or with iron for and by The oxygen consumption was as described under All were on at protein In all the the respiratory activity was by Open in a The designated yeast strains were in the YPD medium or with iron for and by The oxygen consumption was as described under All were on at protein In all the the respiratory activity was by We the of the to oxygen by their in a medium containing hydrogen peroxide The of hydrogen peroxide by such as iron toxic hydroxyl by the reaction (1Halliwell B. Gutteridge J.M. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4556) Google Scholar). The on YPD medium containing than the and fet3 strains The aft1aft2mutant was unable to grow under the conditions. The of Aft2p by the double mutant this to grow in the of The of the was by the medium with iron or the of the was by These results indicate that and the mutants are to oxidative We confirmed these results by analysis for stress phenotypes related to redox The mutant was to copper in YPD medium (Fig. This was also unable to grow in medium we methionine or It also had a (Fig. and data not All these oxidative phenotypes were suppressed under conditions and suppressed by iron to the aerobic the transport of iron the and the siderophore-mediated high affinity iron systems in response to iron deprivation conditions. We have now that the protein, Aft2p, is a second of iron-regulated control of transcription in yeast. The domains of Aft1p and Aft2p are in the basic N-terminal regions of the This region has residues including the sequence to iron in Aft1p (13Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar). Aft2p was found to a activity that is to that of Aft1p The activity of Aft2p was by when was iron in the the activity of Aft1p was not This a report that that the transcriptional activation function of Aft1p is of iron C. M. C. C. R. E. Yeast. 1997; PubMed Scopus (76) Google Scholar). In the transcriptional activation function of Aft2p may be data that indicate that a iron transcription of is in deleted C. M. C. C. R. E. Yeast. 1997; PubMed Scopus (76) Google Scholar). We show that Aft2p may be responsible for the transcription of FET3. We have also that of Aft2p in an iron-regulated the transcription of the and the reporter gene under the control of the The by iron of regulation in seems to be than that iron the and DNA binding of Aft2p. These results suggest that Aft2p is to a sequence or to the and may in the transcription of genes that target genes. show that the of is by This regulation to be in the double the of the is with the of Aft2p. These results a report that that is in strains containing an but is in the allele (20Lin S.J. Pufahl R.A. Dancis A. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1997; 272: 9215-9220Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). It that the in addition to other transcription factors may be involved in the iron-regulated transcription of We also the of the strains with those of the fet3 and strains under iron-deprived conditions to the of Aft2p in iron have that the mutant is to iron deprivation than is the fet3 mutant their iron It has been that Aft1p some intracellular iron use in addition to its in the regulation of iron transport (13Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar). phenotypic analysis of on plates containing an medium that the of the mutant is by iron that of the fet3 mutant is The double mutant is unable to grow under these conditions. We have also that cells have iron to those of cells. the inability of mutants to grow in an medium is not solely caused by defective iron It the for in the control of intracellular iron use and that Aft2p is with Aft1p, for the of iron homeostasis in yeast. report that the mutant is unable to grow in medium with as a respiratory carbon C. M. C. C. R. E. Yeast. 1997; PubMed Scopus (76) Google Scholar). The this to a defective high affinity iron This defect the intracellular iron content too low to sustain respiratory data not mutants all have functional mitochondrial respiration that is 2-fold higher than in the and strains the iron in the medium This is with the of and not that indicate that the and mutants have a to and a respiratory the is unable to grow under aerobic conditions on plates containing raffinose as carbon the mutant does this oxygen-dependent is not to an iron deficiency, because copper the of mutant to the as does iron (Fig. iron or copper does not the mutant phenotypes in the of Aft2p. This that iron or copper in the medium some other pathway(s) to for the of and that this requires the of Aft2p. these results not the that the inability of mutants to grow on respiratory medium is to a defective high affinity iron The and the mutants are to and mutant has several oxygen-dependent and iron-dependent phenotypes such as a to copper and a methionine the copper of which is such as the and are to copper under aerobic conditions D.J. Mol. Cell. Biol. PubMed Scopus Google Science. PubMed Scopus Google Scholar). methionine has been for the mutant and for the of which are for the redox of cells Kosman D.J. Culotta V.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). We suggest therefore that the of iron the cells to a of redox This with recent data a stress by a mutant and the of iron homeostasis J.M. A. R. J.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. J.S. J. Biol. 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The phenotypic analysis that mutant phenotypes are by the of AFT2 and are suppressed by the of Aft2p. some phenotypes related to oxidative stress not in the and mutants but are in the double These results suggest that Aft2p has that those of Aft1p in the regulation of iron We are a analysis to the target genes that are involved in these We Yamaguchi-Iwai for the of the plasmid The was by Sci.