The Yeast Iron Regulon Is Induced upon Cobalt Stress and Crucial for Cobalt Tolerance

To identify yeast genes involved in cobalt detoxification, we performed RNA expression profiling experiments and followed changes in gene activity upon cobalt stress on a genome-wide scale. We found that cobalt stress specifically results in an immediate and dramatic induction of genes involved in iron uptake. This response is dependent on the Aft1 protein, a transcriptional factor known to regulate a set of genes involved in iron uptake and homeostasis (iron regulon). Like iron starvation, cobalt stress induces accumulation of the Aft1 protein in the nucleus to activate transcription of its target genes. Cells lacking the AFT1 gene (aft1) are hypersensitive to cobalt as well as to other transition metals, whereas expression of the dominant AFT1–1 up allele, which results in up-regulation of AFT1-controlled genes, confers resistance. Cobalt resistance correlates with an increase in intracellular iron in AFT1–1 up cells, and sensitivity of aft1 cells is associated with a lack of iron accumulation. Furthermore, elevated iron levels in the growth medium suppress the cobalt sensitivity of the aft1 mutant cells, even though they increase cellular cobalt. Results presented indicate that yeast cells acquire cobalt tolerance by activating the Aft1p-dependent iron regulon and thereby increasing intracellular iron levels. To identify yeast genes involved in cobalt detoxification, we performed RNA expression profiling experiments and followed changes in gene activity upon cobalt stress on a genome-wide scale. We found that cobalt stress specifically results in an immediate and dramatic induction of genes involved in iron uptake. This response is dependent on the Aft1 protein, a transcriptional factor known to regulate a set of genes involved in iron uptake and homeostasis (iron regulon). Like iron starvation, cobalt stress induces accumulation of the Aft1 protein in the nucleus to activate transcription of its target genes. Cells lacking the AFT1 gene (aft1) are hypersensitive to cobalt as well as to other transition metals, whereas expression of the dominant AFT1–1 up allele, which results in up-regulation of AFT1-controlled genes, confers resistance. Cobalt resistance correlates with an increase in intracellular iron in AFT1–1 up cells, and sensitivity of aft1 cells is associated with a lack of iron accumulation. Furthermore, elevated iron levels in the growth medium suppress the cobalt sensitivity of the aft1 mutant cells, even though they increase cellular cobalt. Results presented indicate that yeast cells acquire cobalt tolerance by activating the Aft1p-dependent iron regulon and thereby increasing intracellular iron levels. hemagglutinin bathophenanthroline-disulfonic acid yeast extract/peptone/dextrose Although cobalt is an essential micronutrient as a cofactor of vitamin B12 (reviewed in Ref. 1Battersby A.R. Science. 1994; 264: 1551-1557Crossref PubMed Scopus (136) Google Scholar) and various other enzymes in animals, yeasts, bacteria, Archaea, and plants (2Kobayashi M. Shimizu S. Eur. J. Biochem. 1999; 261: 1-9Crossref PubMed Scopus (374) Google Scholar), exposure to inorganic cobalt is associated with various human diseases such as contact dermitis (3Barceloux D.G. J. Toxicol. Clin. Toxicol. 1999; 37: 201-206Crossref PubMed Scopus (447) Google Scholar), allergic asthma leading to subsequent interstitial fibrosis (4Lauwerys R. Lison D. Sci. 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Cobalt stimulates, as does hypoxia, the production of erythropoietin, a glycoprotein hormone essential for the differentiation of red blood cells in response to hypoxia. There is evidence that the cellular oxygen-sensing mechanism, present in most if not all tissues, utilizes a heme protein where cobalt might substitute for the iron in the porphyrin ring, thereby decreasing its affinity to oxygen and mimicking a hypoxic environment (13Goldberg M.A. Dunning S.P. Bunn H.F. Science. 1988; 242: 1412-1415Crossref PubMed Scopus (877) Google Scholar, 14Bunn H.F., Gu, J. Huang L.E. Park J.W. Zhu H. J. Exp. Biol. 1998; 8: 1197-1201Google Scholar). The notion that iron and cobalt compete for this heme protein is supported by the fact that cells grown in low-iron medium require less cobalt for erythropoietin stimulation than those exposed to iron-rich medium (15Ho V.T. Bunn H.F. Biochem. Biophys. Res. Commun. 1996; 223: 175-180Crossref PubMed Scopus (119) Google Scholar). To understand the cellular response to cobalt stress and screen for genes involved in cobalt detoxification, we determined the transcriptional profile of Saccharomyces cerevisiae upon cobalt exposure. We found that the iron regulon, in particular, genes dependent on the transcriptional factor Aft1p, plays a vital role in cellular cobalt tolerance. The 77.7-kDa protein Aft1p has been shown to be transported into the nucleus upon iron starvation to mediate iron-regulated transcription by binding to the consensus motif PyPuCACCCPu in the 5′-upstream region of a number of genes involved in iron transport and homeostasis (16Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (292) Google Scholar, 17Casas C. Aldea M. Espinet C. Gallego C. Gil R. Herrero E. Yeast. 1997; 13: 621-637Crossref PubMed Scopus (76) Google Scholar, 18Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Whereas aft1 mutant cells are unable to switch on the iron regulon upon cobalt stress and appear sensitive to cobalt, permanent induction of these genes (via theAFT1–1 up allele) leads to cobalt resistance. Cellular iron levels coincide with cobalt tolerance; iron is decreased in aft1 disrupted cells and increased inAFT1–1 up cells, and in addition, the wild-type shows elevated iron concentrations upon cobalt stress. Furthermore, high external iron can restore cobalt tolerance of the aft1disrupted cells. Escherichia coli strain DH5α (Stratagene) and the following plasmids were used: YEp351HA (19Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), pVT102-U (20Vernet T. Dignard D. Thomas Y.T. Gene (Amst.). 1987; 52: 225-233Crossref PubMed Scopus (465) Google Scholar), YIplac211 (21Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar), pRS416-AFT1-HA (18Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and pT14 (22Yamaguchi-Iwai Y. Dancis A. Klausner A.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (318) Google Scholar). S. cerevisiae strain JS034-4C (relevant genotypeMATa, ura3, his3, leu2,trp1) is a haploid derivative of FY1679 (23Winston F. Dollard C. Ricupero-Hovasse S.L. Yeast. 1995; 11: 53-55Crossref PubMed Scopus (788) Google Scholar). To create theaft1::TRP1 mutant JS018, JS034-4C cells were transformed with the 4.0-kb XhoI/SacI fragment of plasmid pT20 (22Yamaguchi-Iwai Y. Dancis A. Klausner A.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (318) Google Scholar) carrying the aft1 gene withTRP1 insertion. The disruption was verified using analytical PCR (data not shown). To overexpress C-terminally HA1-tagged Cot1p, theCOT1 gene was PCR-amplified from FY1679 chromosomal DNA using the mutagenic oligonucleotide primers COT1-PstI/XbaI (5′-CCCCCTGCAGTCTAGACTCAGCACTTTCTACATT-3′) and COT1-PstI (5′-CCCCCTGCAGGATGATCCTCTAAGCAATC-3′), starting at positions −425 and +2008, respectively, relative to the COT1 coding sequence, and introducing PstI and XbaI sites (underlined). The product was digested with PstI and XbaI and then ligated to the PstI and XbaI linearized vector YEp351HA to obtain the 7.5-kb vector YEpCOT1HA. To clone pVTUCOT1–1HA,the HA-tagged COT1 was amplified from YEpCOT1HA using the primers COT1-X (5′-CTAGTCTAGAATAGTTCTGCATAGC-3′) and COT1-H (5′-CAGTGCCAAGCTTTCAGC-3′) located at −61 and +1432, respectively, relative to the COT1 open reading frame and featuring a XbaI and a HindIII site, respectively (underlined). The PCR product was digested withXbaI and HindIII and cloned into pVT102-U, placing the COT1 gene behind the constitutive pADH1promoter. Cells were fixed and stained with antibodies as described previously (24Nasmith K. Adolf G. Lydall D. Seddon G. Cell. 1990; 62: 631-647Abstract Full Text PDF PubMed Scopus (156) Google Scholar). Antibodies used were mouse anti-HA (19Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and goat anti-mouse antibody conjugated to rhodamine (SC-2092; Santa Cruz Biotechnology). Nuclear and mitochondrial DNA was visualized by staining with 4′,6-diamidino-2-phenylindole (Molecular Probes). Fluorescence microscopy was done with a Zeiss Axioplan 2 microscope and a Visitron Systems Imaging system. Cells grown in YPD medium at 28 °C to A 600 = 0.5–0.6 were treated with 100 μm to 2 mmCoCl2 or with 80 μm BPS (Sigma) for 30 or 90 min at 28 °C, and then the cells were harvested, and RNA was prepared essentially according to the hot acidic phenol method (25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. J. Wiley and Sons, New York1997: 13.12.1-13.12.5Google Scholar); the only difference was that three chloroform extractions were performed instead of one. The yeast DNA chips were obtained from the Ontario Cancer Institute Microarray Centre. Reverse transcription, probe cleanup, and microarray were performed according to the were using an and using The and for were used to on genes. Cells grown in YPD medium at 28 °C 600 = 0.5–0.6 and then with 2 or for min at 28 °C were harvested, and RNA was prepared (25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. J. Wiley and Sons, New York1997: 13.12.1-13.12.5Google Scholar). and of RNA were performed T. J. Molecular A Scholar) and using an system. were by PCR from chromosomal DNA using and oligonucleotide primers and Cells grown in YPD medium at 28 °C to A 600 = 0.5–0.6 were with or for 90 Cells were harvested, with high or and then at °C for of the the was digested in at 90 °C for 30 were using a or To the response to yeast wild-type cells grown in YPD medium were with 100 μm for 30 min or with 2 for 30 and 90 Whereas 100 μm stress that does not the growth of yeast wild-type cells, 2 a of growth (data not shown). profiling experiments were three using grown expression was or at are shown in The set of is at expression upon cobalt μm 30 30 90 30 min μm BPS 90 consensus Aft1p binding sites are in to heme cobalt acid acid and and stress stress protein protein transcription mitochondrial consensus Aft1p binding sites are in in a concentrations of 100 μm were found to a set of genes known to be involved in iron transport and iron homeostasis the of all the genes of this are known to be by the transcription factor Aft1p (22Yamaguchi-Iwai Y. 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PubMed Scopus Google Scholar). of the genes known to be or AFT1 in and found to be upon in the wild-type were upon stress to mutant cells not the concentration of 100 was found to expression of a of other genes of are involved in stress acid and and a genes were upon stress 90 min The genes for To iron the BPS was to wild-type cells to a concentration of 80 μm 90 min The response of yeast upon iron was to the immediate response upon stress 100 by BPS were the and and the induction (data not shown). To the cellular iron is in response to cells were grown in YPD medium to and with 2 for 90 Total cellular iron of the cells and cells were determined by In wild-type cells iron was increased upon with up-regulation of the iron transport genes as in the microarray mutant cells cellular iron than wild-type cells and to iron with This is with that the mutant was found to a iron uptake than wild-type cells E. M. P. 1998; PubMed Scopus Google Scholar). Cells the dominant AFT1–1 up which permanent transcription genes, iron than wild-type cells. increase in cellular iron was AFT1–1 up cells were with stress induces of iron an the of the Aft1p transcriptional factor is by its in cells and to the nucleus in cells (18Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), we were in high was of Aft1p to the Cells carrying HA-tagged Aft1p grown in YPD medium or exposed to 2 for 90 were to with the of Aft1p target genes upon stress Aft1p was in the at and to the nucleus by To the role of the AFT1 gene product in various were for to on elevated levels. shown in mutant was hypersensitive to whereas the dominant AFT1–1 up resistance to elevated levels. mutant cells were sensitive to iron increased levels of and and elevated the AFT1–1 up resistance to iron and increased and concentrations in the expression of the protein, which is known to be for cellular tolerance J.A. C. Cell. Biol. 1992; PubMed Scopus Google and J. J. Biol. 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Cell. 1994; Full Text PDF PubMed Scopus Google Scholar) upon exposure to these expression was by not by or whereas to be by only and not by or with microarray (data not not or specifically induces the iron To of the aft1 mutant cells be a of intracellular iron and of to increase cellular iron upon exposure to we changes in growth as well as cellular iron and cobalt concentrations of cells were with 90 in the or of 2 90 μm growth cells was and cellular cobalt increased 2 in the growth medium increased cellular iron not growth 90 μm was in to 2 the aft1 mutant was by cellular cobalt was even increased as with of 90 μm This that high iron levels in the medium can for the of induction of iron (aft1) in cobalt tolerance and that high iron does not cobalt by in cellular cobalt and iron of aft1 cells upon of cobalt and cells were grown in YPD medium to A 600 = and then and μm and 2 were at was for 90 and then the cells were harvested, and cellular iron and cobalt were determined are of at three in a cells were grown in YPD medium to A 600 = and then and μm and 2 were at was for 90 and then the cells were harvested, and cellular iron and cobalt were determined are of at three This at the that in the yeast S. cobalt stress induces a number of genes known as the iron regulon and coding for iron transport a response to upon iron a of this cellular iron is stress response genes and appear to be by cobalt, only at cobalt whereas the iron regulon is even by cobalt iron the response to cobalt stress in Aft1p, the transcription factor of the iron regulon genes Y. Dancis A. Klausner A.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (318) Google and T. J. D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. J. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. L.E. J. Biol. 1999; PubMed Scopus Google Scholar, F. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), plays a vital role in this cobalt response cobalt Aft1p from the to the as in the of iron starvation (18Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The role of Aft1p in the cellular response to cobalt has been by an aft1 disrupted strain and theAFT1–1 up in which the iron regulon is (18Yamaguchi-Iwai Y. Ueta R. Fukunaka A. Sasaki R. J. Biol. Chem. 2002; 277: 18914-18918Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, Y. Dancis A. Klausner A.D. 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of these transition is increased the high affinity iron is or to be the that Aft1p is with the accumulation of transition by of J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). to be determined the sensitivity mutant cells to transition correlates with the intracellular iron or is by expression of of the many Aft1p target genes as a of the of In cobalt to be the ions in that Aft1p or a factor associated with this transcription factor and results in a response to that to iron by iron this of the iron cells appear to the of high levels of cobalt by iron transport and increasing intracellular iron This response is by the and to cobalt is to an mechanism, which might not be to the yeast S. to with cobalt cobalt by increasing cellular iron is tolerance is with to or the cellular iron not be as as iron are cobalt, the the affinity iron uptake M.A. J. Biol. 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