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• W2106290468 abstract "Copper is an essential trace element, yet excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. In Saccharomyces cerevisiae, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. Here we show that the copper-dependent enzyme Sod1 (Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either CCS1 or SOD1 results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. Given the presence of both Sod1 and copper-responsive transcription factors in higher eukaryotes, these studies may yield important insights into how copper deficiency is sensed and appropriate cellular responses are coordinated. Copper is an essential trace element, yet excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. In Saccharomyces cerevisiae, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. Here we show that the copper-dependent enzyme Sod1 (Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either CCS1 or SOD1 results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. Given the presence of both Sod1 and copper-responsive transcription factors in higher eukaryotes, these studies may yield important insights into how copper deficiency is sensed and appropriate cellular responses are coordinated. Unicellular organisms are constantly exposed to a plethora of changing environments and thus have developed sophisticated uptake, distribution, and storage systems that function to assimilate essential nutrients from the environment. Copper is included among these essential nutrients, and once inside cells, it is incorporated as a catalytic or structural cofactor into a variety of proteins (1Prohaska J.R. Gybina A.A. J. Nutr. 2004; 134: 1003-1006Crossref PubMed Scopus (237) Google Scholar, 2Rees E.M. Thiele D.J. Curr. Opin. Microbiol. 2004; 7: 175-184Crossref PubMed Scopus (64) Google Scholar). The redox potential that makes copper an important cofactor also allows the ion to undergo Fenton chemistry to produce the potent hydroxyl radical (OH·) (3Halliwell B. Gutteridge J.M. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4521) Google Scholar). Organisms have evolved sophisticated homeostatic systems to maintain appropriate intracellular copper levels that are below levels that could lead to cellular damage (4Balamurugan K. Schaffner W. Biochim. Biophys. Acta. 2006; 1763: 737-746Crossref PubMed Scopus (182) Google Scholar, 5Rutherford J.C. Bird A.J. Eukaryot. Cell. 2004; 3: 1-13Crossref PubMed Scopus (208) Google Scholar).In Saccharomyces cerevisiae, copper in the extracellular environment is reduced by cell surface reductases, Fre1 and Fre2, and is transported across the plasma membrane by the high affinity copper transporter Ctr1 or the functionally redundant Ctr3 protein (6Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 269: 25660-25667Abstract Full Text PDF PubMed Google Scholar, 7Knight S.A. Labbe S. Kwon L.F. Kosman D.J. Thiele D.J. Genes Dev. 1996; 10: 1917-1929Crossref PubMed Scopus (219) Google Scholar, 8Kim B.E. Nevitt T. Thiele D.J. Nat. Chem. Biol. 2008; 4: 176-185Crossref PubMed Scopus (878) Google Scholar). Inside cells, the Cox17 chaperone facilitates the delivery of copper to the cytochrome c oxidase complex in the mitochondria, and this function is required for aerobic respiration (9Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 10Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 11Srinivasan C. Posewitz M.C. George G.N. Winge D.R. Biochemistry. 1998; 37: 7572-7577Crossref PubMed Scopus (100) Google Scholar). Interestingly, recent data have demonstrated that Cox17 localized exclusively to the mitochondria is sufficient for delivery of copper to cytochrome c oxidase (12Maxfield A.B. Heaton D.N. Winge D.R. J. Biol. Chem. 2004; 279: 5072-5080Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This suggests that either an as yet unidentified chaperone or a small molecule carrier is responsible for trafficking of copper from the plasma membrane to Cox17 in the mitochondria. The Atx1 chaperone delivers copper to the Golgi, where it is pumped into the lumen of the secretory compartment by the P-type ATPase Ccc2 (13Pufahl R.A. Singer C.P. Peariso K.L. Lin S.J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V. Science. 1997; 278: 853-856Crossref PubMed Scopus (585) Google Scholar, 14Lin S.J. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3784-3788Crossref PubMed Scopus (232) Google Scholar, 15Lin 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 (351) Google Scholar). Ccs1, the copper chaperone for superoxide dismutase (16Culotta V.C. Klomp L.W. Strain J. Casareno R.L. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar), is responsible for delivery of copper to Sod1 (Cu,Zn-superoxide dismutase), an enzyme that protects cells against oxidative stress via the disproportionation of superoxide to produce hydrogen peroxide (17Bermingham-McDonogh O. Gralla E.B. Valentine J.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4789-4793Crossref PubMed Scopus (121) Google Scholar, 18McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar).In S. cerevisiae, the regulation of copper acquisition has been shown to be controlled at the level of transcription by Mac1 (19Jungmann J. Reins H.A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Crossref PubMed Scopus (229) Google Scholar). Mac1 is activated in response to copper deprivation, leading to transcription of the genes involved in high affinity copper uptake, such as CTR1, CTR3, and FRE1 (20Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 21Yamaguchi-Iwai Y. Serpe M. Haile D. Yang W. Kosman D.J. Klausner R.D. Dancis A. J. Biol. Chem. 1997; 272: 17711-17718Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 22Jamison McDaniels C.P. Jensen L.T. Srinivasan C. Winge D.R. Tullius T.D. J. Biol. Chem. 1999; 274: 26962-26967Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Mac1 is a modular protein consisting of a copper responsive trans-activation domain (TAD) 3The abbreviations used are: TAD, trans-activation domain; DBD, DNA binding domain; CuRE, copper-responsive element; GFP, green fluorescent protein; BCS, bathocuproinedisulfonic acid; SC, synthetic complete; ChIP, chromatin immunoprecipitation; hSod1, human Sod1; WT, wild type; TAP, tandem affinity purification.3The abbreviations used are: TAD, trans-activation domain; DBD, DNA binding domain; CuRE, copper-responsive element; GFP, green fluorescent protein; BCS, bathocuproinedisulfonic acid; SC, synthetic complete; ChIP, chromatin immunoprecipitation; hSod1, human Sod1; WT, wild type; TAP, tandem affinity purification. and DNA binding domain (DBD) (23Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (98) Google Scholar, 24Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (85) Google Scholar, 25Keller G. Gross C. Kelleher M. Winge D.R. J. Biol. Chem. 2000; 275: 29193-29199Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 26Serpe M. Joshi A. Kosman D.J. J. Biol. Chem. 1999; 274: 29211-29219Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Previous experiments using a fusion protein containing the Gal4 DBD and the Mac1 TAD demonstrated that the TAD is responsive to changes in bioavailable copper levels (23Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (98) Google Scholar). The carboxyl-terminal Mac1 TAD contains two cysteine- and histidine-rich domains, REP-I (C1) and REP-II (C2), that each binds four Cu1+ ions in a tetranuclear copper cluster (24Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (85) Google Scholar). Mutations in the C1 domain lead to constitutively active Mac1up proteins, whereas analogous mutations in the C2 domain decrease the trans-activation of Mac1 (19Jungmann J. Reins H.A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Crossref PubMed Scopus (229) Google Scholar, 20Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 21Yamaguchi-Iwai Y. Serpe M. Haile D. Yang W. Kosman D.J. Klausner R.D. Dancis A. J. Biol. Chem. 1997; 272: 17711-17718Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 23Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (98) Google Scholar, 25Keller G. Gross C. Kelleher M. Winge D.R. J. Biol. Chem. 2000; 275: 29193-29199Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 27Georgatsou E. Mavrogiannis L.A. Fragiadakis G.S. Alexandraki D. J. Biol. Chem. 1997; 272: 13786-13792Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Copper deprivation also results in increased DNA binding of Mac1 to copper-responsive element (CuRE) regions upstream of its target genes (20Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 21Yamaguchi-Iwai Y. Serpe M. Haile D. Yang W. Kosman D.J. Klausner R.D. Dancis A. J. Biol. Chem. 1997; 272: 17711-17718Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 22Jamison McDaniels C.P. Jensen L.T. Srinivasan C. Winge D.R. Tullius T.D. J. Biol. Chem. 1999; 274: 26962-26967Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 28Joshi A. Serpe M. Kosman D.J. J. Biol. Chem. 1999; 274: 218-226Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 29Keller G. Bird A. Winge D.R. Eukaryot. Cell. 2005; 4: 1863-1871Crossref PubMed Scopus (59) Google Scholar), and there is evidence that a constitutively active Mac1up1 protein binds fewer copper ions per molecule than the wild type protein (24Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (85) Google Scholar). Moreover, Mac1up1 is constitutively bound to the promoter of its target genes (20Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). These observations suggest that loss of copper ions from Mac1 may be important for its activation. Studies using yeast two-hybrid analysis also indicate that copper starvation results in release of an intramolecular interaction between the Mac1 DBD and TAD (24Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (85) Google Scholar).Although it has previously been demonstrated that Mac1 protein fragments bind copper ions and that this binding is important for its regulation (24Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (85) Google Scholar), it is unclear how copper binding may be regulated. Previous studies have shown that Mac1 is a nuclear resident protein, suggesting that copper is either assembled co-translationally or is delivered to the nucleus in order to regulate Mac1 (19Jungmann J. Reins H.A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Crossref PubMed Scopus (229) Google Scholar, 24Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (85) Google Scholar). However, there is virtually no free copper in the cell, and it has been demonstrated that copper is associated almost exclusively with either chaperones or the copper-containing proteins that are targets of these chaperones (30Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1346) Google Scholar).We began with the hypothesis that one of the three known copper chaperone proteins, Atx1, Cox17, or Ccs1, might be responsible for copper delivery to or removal from Mac1. Here we find that both Ccs1 and its target, Sod1, are necessary for robust activation of Mac1 in response to low copper conditions. We found that the requirement for Ccs1 during Mac1 activation is due to its role in delivery of copper to Sod1 and that the disproportionation of superoxide is necessary for Mac1 activation. However, the role of Sod1 in Mac1 activation appears to be more complex than simply a global protection against oxidative stress, since both genetic and chemical suppression of oxidative stress in sod1Δ cells failed to restore Mac1 activity to wild type levels. Moreover, we demonstrated that Sod1 and the Ccs1 copper chaperone partially localize to the yeast nucleus and that deletion of SOD1 reduces the ability of Mac1 to bind to CuRE elements in the genome upstream of the CTR1 gene in response to low copper bioavailability. Taken together, these results suggest that in Saccharomyces cerevisiae the Cu,Zn-superoxide dismutase enzyme plays a role in the sensing or responding to copper deficiency to activate gene transcription.EXPERIMENTAL PROCEDURESYeast Strains and Plasmids—All isogenic S. cerevisiae deletion strains were created by replacement of the endogenous locus with a floxed kanamycin resistance cassette and subsequent removal of this cassette (31Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1341) Google Scholar). The sod1Δ pmr1Δ double mutant was created by deletion of PMR1 in an sod1Δ strain, and the ccs1Δ sod1Δ double mutant was created by deletion of SOD1 in a ccs1Δ strain. The SOD1-GFP and the MAC1-TAP strains were obtained from the GFP- and TAP-tagged collections (32Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Nature. 2003; 425: 737-741Crossref PubMed Scopus (2972) Google Scholar, 33Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Nature. 2003; 425: 686-691Crossref PubMed Scopus (3256) Google Scholar). The MAC1-TAP sod1Δ strain was created by deletion of SOD1 in the MAC1-TAP background. The MAC1up1 strain and its wild type parental strain have been previously described (19Jungmann J. Reins H.A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Crossref PubMed Scopus (229) Google Scholar), and the MAC1up1 sod1Δ isogenic variant was created by deletion of SOD1 in this strain. The Y190 yeast strain was used in the yeast one/two-hybrid experiments, and the Y190 sod1Δ strain was created by deletion of the SOD1 gene in the Y190 background (34Bai C. Elledge S.J. Methods Enzymol. 1997; 283: 141-156Crossref PubMed Scopus (75) Google Scholar).The GAL1–10-LacZ reporter plasmid was a generous gift from Dr. Alan Hinnebusch. The Caenorhabditis elegans SOD1 plasmid was previously described by Jensen and Culotta (35Jensen L.T. Culotta V.C. J. Biol. Chem. 2005; 280: 41373-41379Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The ySOD1 plasmid was created by cloning a PCR fragment containing the SOD1 gene and its endogenous promoter and terminator as an XbaI/XhoI fragment into the pRS416 vector. The SOD1 plasmid was created by subcloning the SOD1 sequence from ySOD1 as an XbaI/Xho1 fragment into the pRS415 vector. The SOD1R143D and the SOD1G85R alleles encoding catalytically inactive SOD1 mutants were created by site-directed mutagenesis of SOD1 using overlap PCR and then cloned as BamHI/XhoI fragments into pRS415 (36Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar, 37Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6797) Google Scholar). A DNA fragment with the coding sequence for the first 105 amino acids of SCO2 as an in-frame amino-terminal fusion with the SOD1 gene under the control of the SOD1 promoter was created using overlap PCR and cloned by gap repair into pRS415 to create the SCO2-SOD1 plasmid. The pGB4D1-Trp MAC1 1–159, pVT102-Leu VP16, pVT102-leu MAC1 240–417, and pVT102-leu MAC1up1 240–417 plasmids were a generous gift from Dr. Dennis Winge (24Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (85) Google Scholar). For the yeast one-hybrid experiment, a PCR product containing codons 42–417 of the MAC1 gene was cloned by gap repair as an in frame fusion with the GAL4 DNA binding domain of the pGBKT7 plasmid backbone (Clontech).β-Galactosidase Activity Assay for Mac1 Function—Cells were transformed with the previously described Mac1 reporter plasmid pCm64CTR3-LacZ or pRSCTR3-LacZ (20Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and grown to mid-log phase in synthetic complete (SC) selective media with or without 10 μm or 100 μm bathocuproinedisulfonic acid (BCS). β-Galactosidase assays were performed as described by Liu et al. (38Liu X.D. Morano K.A. Thiele D.J. J. Biol. Chem. 1999; 274: 26654-26660Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).RNA Blot Analysis—RNA was extracted from cells grown to mid-log phase using a modified hot phenol method (39Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1147) Google Scholar). CTR1 or ACT1 gene fragments were radiolabeled with [32P]dCTP to be used as probes. Quantification of the RNA blot was performed using ImageQuant TL version 2003.02 software (Amersham Biosciences) and processed using Adobe Photoshop version 7.0 (Adobe Systems).Immunoblotting—Protein extracts were prepared either using a glass bead/Triton X-100 method (6Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 269: 25660-25667Abstract Full Text PDF PubMed Google Scholar) or by alkali extraction (40Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (178) Google Scholar). Mitochondria were isolated using the Yeast Mitochondria Isolation Kit (Sigma) and then resuspended in buffer containing 2% Triton X-100, 10 mm Tris-HCl (pH 7.5), 500 mm NaCl, and 0.5 mm EDTA and solubilized on ice for 30 min. SDS-PAGE was performed, and samples were probed with anti-Sod1 antibody (a generous gift from Dr. Thomas O’Halloran), anti-TAP antibody (Open Biosystems), anti-Pgk1 antibody (Invitrogen), or anti-Por1 antibody (Molecular Probes).Functional Assays for Sod1 and Mac1—For phenotypic analysis, wild type and mutants were spotted on SC plates, SC -lysine plates, SC -methionine -lysine plates, or media containing ethanol (2%) and glycerol (3%) as the sole carbon sources (YPEG). To test superoxide dismutase catalytic activity, protein extracts were obtained using the glass bead/Triton X-100 method, and samples were subjected to nondenaturing gel electrophoresis followed by nitro blue tetrazolium staining (41Flohe L. Otting F. Methods Enzymol. 1984; 105: 93-104Crossref PubMed Scopus (1276) Google Scholar). Mac1-TAP protein function was tested by spotting 10-fold serial dilutions of cells on YPD, YPEG, and YPEG with 100 μm CuSO4.Fluorescence Microscopy—A BY4742-derived yeast strain with a functional genomic fusion of GFP at the carboxyl terminus of the SOD1 or CCS1 open reading frame was used for localization of Sod1 and Ccs1, respectively (33Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Nature. 2003; 425: 686-691Crossref PubMed Scopus (3256) Google Scholar).ChIP PCR Analysis—Chromatin immunoprecipitation was carried out as previously described (42Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (325) Google Scholar). Cells were grown for 3 h to mid-log phase in YPD medium, 100 μm CuSO4 or 500 μm BCS was added, and the incubation continued for an additional 15 min before cross-linking with formaldehyde. After cell lysis by vortexing with glass beads and ultrasonication to shear DNA, 250 μg of protein was immunoprecipitated with IgG-Sepharose beads (GE Healthcare). The precipitated DNA was used for PCR with primers for either the CTR1 promoter region or the CMD1 promoter region. The primers used to amplify the CTR1 promoter region were 5′-TAA GGA TCG AAA CTG CAC CTC AAC-3′ and 5′-ACA TAC AAG ACC CTC TCG AGA TGA CA-3′. The primers used to amplify the CMD1 promoter region were 5′-CGCTTCCTCTCAATTCCCAAAGT-3′ and 5′-GTG ATG TAG GAC ACT CTC CAA GG-3′. PCRs were performed using serial dilutions of the output DNA to be sure that the reaction was in the linear range, and the ChIP experiment was repeated three times with similar results. The data presented are representative of three independent experiments. Digital images of ChIP results were quantitated using ImageQuant software and processed using Adobe Photoshop.RESULTSThe Ccs1 Copper Chaperone Is Required for Robust Activation of Mac1—In S. cerevisiae, expression of the high affinity copper uptake system is regulated by the copper-responsive transcription factor Mac1. Mac1 has been demonstrated to be a nuclear resident protein, and protein fragments have been shown to directly bind copper atoms, suggesting that the copper status of Mac1 could be important to its regulation. The mechanism by which copper is incorporated into this protein remains unknown, and it is unclear how copper might enter or leave the nucleus. We began by testing whether one of the three known copper chaperones, Atx1, Ccs1, or Cox17, is involved in the regulation of Mac1. A CTR3-LacZ reporter plasmid that contains two copies of the CuRE from the CTR3 promoter upstream of the LacZ gene was used to quantitate Mac1 activity. We found that the yeasts lacking CCS1 display a severe defect in the activation of Mac1 in response to decreased copper availability induced by supplementation of the growth medium with the copper-specific chelator BCS. Cells harboring an atx1Δ allele do not show defects in activation of the Mac1 reporter, and, as expected, mac1Δ cells are completely defective in CTR3-LacZ activity in response to copper deficiency (Fig. 1A). The cox17Δ mutant also displays a defect in activation of the Mac1 reporter. However, these same cells show a significant reduction in activation of a reporter gene for the unfolded protein response (supplemental Fig. 1). These results suggest that the COX17-dependent defect in activation of the Mac1 reporter is due to a more general loss of transcriptional regulation. RNA blotting analysis of the CTR1 transcript confirmed that ccs1Δ mutants show decreased induction of this Mac1 target gene in response to copper deprivation as compared with wild type cells. As expected, mac1Δ cells show nearly undetectable levels of CTR1 mRNA (Fig. 1, B and C). Similar results were observed with the transcript of a second Mac1 target, FRE1, indicating that Ccs1 plays a more general, rather than a CTR1-specific, role in Mac1 activation. 4L. K. Wood and D. J. Thiele, unpublished data.Cu,Zn-superoxide Dismutase Functions in the Activation of Mac1—The copper chaperone Ccs1 delivers copper to the Sod1 enzyme in a series of steps that are critical for Cu,Zn-superoxide dismutase activation in yeast (16Culotta V.C. Klomp L.W. Strain J. Casareno R.L. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 30Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1346) Google Scholar, 43Schmidt P.J. Kunst C. Culotta V.C. J. Biol. Chem. 2000; 275: 33771-33776Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 44Lamb A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 751-755Crossref PubMed Scopus (247) Google Scholar, 45Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Two possibilities could explain the diminished ability of ccs1Δ mutants to activate Mac1 in response to low copper. First, it is possible that Ccs1 functions directly in the activation of Mac1. Second, it is possible that the defect of ccs1Δ cells is an indirect effect due to an inability to deliver copper to, and thus activate, Sod1. To distinguish between these two possibilities, cells harboring a wild type CCS1 gene but lacking SOD1 were tested for the ability to activate the CTR3-LacZ reporter plasmid in response to copper deprivation. sod1Δ mutants display a Mac1 regulation phenotype that phenocopies ccs1Δ cells, consistent with the observation that yeast Sod1 is largely dependent on Ccs1 for its activation (Fig. 2A) (46Carroll M.C. Girouard J.B. Ulloa J.L. Subramaniam J.R. Wong P.C. Valentine J.S. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5964-5969Crossref PubMed Scopus (150) Google Scholar). As expected, mac1Δ cells are completely defective in activation of CTR1 expression under conditions of copper deficiency. This reduced ability to activate Mac1 is also evident at the level of Mac1 target mRNA, as shown by RNA blotting analysis of the CTR1 transcript (Fig. 2, B and C). The poor activation of Mac1 in an sod1Δ strain is not due to a general defect in gene transcription, since the induction of the galactose-inducible reporter plasmid is unaffected in sod1Δ cells (Fig. 2D). However, deletion of SOD1 in a strain expressing the constitutively active MAC1up1 allele does not affect the regulation of Mac1 protein (Fig. 2E). These results suggest that the Sod1 protein is also required for physiological Mac1 activation in the same pathway as Ccs1, yet this requirement can be bypassed by a constitutively active variant of the Mac1 protein.FIGURE 2Cu,Zn-superoxide dismutase functions in the activation of Mac1. A, sod1Δ cells exhibit defects in the induction of a Mac1 reporter plasmid upon copper depletion. WT, ccs1Δ, and sod1Δ cells transformed with the CTR3-LacZ reporter plasmid were grown to mid-log phase in complete media or media with 10 μm or 100 μm BCS and β-galactosidase assays were performed. ccs1Δ cells and sod1Δ display similar defects in the ability to activate the Mac1 reporter in response to limiting copper. Samples were analyzed in triplicate and data are representative of at least three independent experiments. B, the induction of Mac1 target mRNA upon copper depletion is decreased in sod1Δ cells. RNA blotting analysis for the CTR1 transcript also indicates that sod1Δ mutants show decreased activation of Mac1 in response to copper deprivation. C, quantification of mRNA blots from B. D, sod1Δ cells WT and sod1Δ cells transformed with a galactose-inducible reporter plasmid were grown for 0, 1, or 2 h in media containing galactose as the sole carbon source. β-Galactosidase activity assays demonstrate that β-galactosidase is transcribed/translated at similar levels to WT in sod1Δ mutants. E, WT, MAC1up1, and isogenic MAC1up1 sod1Δ cells transformed with the CTR3-LacZ reporter plasmid were grown to mid-log phase in synthetic complete media supplemented with 1 μm CuSO4 or 100 μm BCS. MAC1up1 and MAC1up1 sod1Δ cells show similar levels of Mac1 reporter activity which is higher than WT control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Activation of Mac1 Requires Catalytically Active Sod1—Since deletion of either CCS1 or SOD1 leads to a similar defect in Mac1 target gene activation in r" @default.
• W2106290468 created "2016-06-24" @default.
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• W2106290468 creator A5074848042 @default.
• W2106290468 date "2009-01-01" @default.
• W2106290468 modified "2023-09-29" @default.
• W2106290468 title "Transcriptional Activation in Yeast in Response to Copper Deficiency Involves Copper-Zinc Superoxide Dismutase" @default.
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• W2106290468 doi "https://doi.org/10.1074/jbc.m807027200" @default.
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