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• W2085994989 abstract "Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the264CXC(X)4CXC(X)2C(X)2H279C1 motif. The lone exception was a L260S substitution. Two additionalMAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain. Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the264CXC(X)4CXC(X)2C(X)2H279C1 motif. The lone exception was a L260S substitution. Two additionalMAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain. polymerase chain reaction low copper complete medium hemagglutinin open reading frame Yeast cells respond to nutrient metal ion availability by regulating the expression of cell surface transport systems. Nutritional deprivation of copper, iron, and zinc ions inSaccharomyces cerevisiae results in up-regulation of specific high affinity transporters arising from the activation of specific transcription factors (1Dancis A. J. Pediatr. 1998; 12: 24-29Abstract Full Text Full Text PDF Google Scholar, 2Winge D.R. Jensen L.T. Srinivasan C. Curr. Opin. Chem. Biol. 1998; 2: 216-221Crossref PubMed Scopus (49) Google Scholar, 3Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Nutritional deprivation of copper ions in yeast leads initially to the activation of the Mac1 transcription factor and subsequent stimulated transcription of three genes encoding proteins involved in high affinity copper ion uptake (4Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar,5Yamaguchi-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 (151) Google Scholar). However, Mac1 lacks transcriptional activation activity in copper-replete cells (4Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Mac1 is critical for expression of the high affinity copper uptake system; therefore, cells lacking Mac1 exhibit phenotypes consistent with a copper-deficient state. Copper ions are necessary in yeast for formation of active cytochrome oxidase, superoxide dismutase, and the Fet3 ferro-oxidase. The high affinity uptake system in yeast consists of two copper ion permeases, Ctr1 and Ctr3, and a metalloreductase Fre1 capable of cupric ion reduction (6Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 269: 25660-25667Abstract Full Text PDF PubMed Google Scholar). A fourth gene activated by Mac1 is the Fre1 homolog, Fre7, the function of which remains unknown (7Martins L. Jensen L.T. Simon J.R. Keller G. Winge D.R. J. Biol. Chem. 1997; 273: 23716-23721Abstract Full Text Full Text PDF Scopus (167) Google Scholar). Reduction of extracellular cupric and ferric ions is an important step in high affinity copper and iron uptake in yeast. An important facet of copper homeostasis in yeast is the ability to restrict entry of copper ions when cells are copper-replete. This is illustrated by the redundant cellular mechanisms to block copper ion uptake and buffer cellular copper levels. The initial event is the specific inhibition of DNA binding and transactivation functions of Mac1 that occurs in cells cultured in medium containing nm Cu(II) (4Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 8Georgatsou E. Mavrogiananis L.A. Fragiadakis G.S. Alexandraki D. J. Biol. Chem. 1997; 272: 13786-13792Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 9Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (96) Google Scholar). Repression of Mac1 function inhibits transcription of CTR1and CTR3 (4Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). In response to elevated cellular copper, the Mac1 protein is degraded, ensuring that expression of the high affinity uptake system is shut down (10Zhu Z. Labbe S. Pena M.M.O. Thiele D.J. J. Biol. Chem. 1998; 273: 1277-1280Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In addition, pre-existing copper transporters are internalized and degraded (11Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (177) Google Scholar). A further, protective response occurs in cells cultured in medium containing μmCu(II) salts. Copper ions activate the nuclear transcription factor Ace1 which induces transcription of three distinct genes, products of which bind copper ions, protecting the cell from deleterious effects of elevated copper levels (12Furst P. Hu S. Hackett R. Hamer D. Cell. 1988; 55: 705-717Abstract Full Text PDF PubMed Scopus (276) Google Scholar). The copper-dependent inhibition of Mac1 function appears to arise from direct interaction of Cu(I) with Mac1 (13Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (84) Google Scholar). Mac1 contains two C-terminal cysteine rich sequences with a repeating motif of CXC(X)4CXC(X)2C(X)2H (14Jungmann 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). These two Cys-rich sequences, designated C1 and C2, bind a total of 8 Cu(I) ions (13Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (84) Google Scholar). The Mac1 ortholog, Grisea from Podospora angerina, contains two related C-terminal Cys-rich motifs that are important for metalloregulation (15Borghouts C. Osiewacz H.D. Mol. Gen. Genet. 1998; 260: 492-502Crossref PubMed Scopus (52) Google Scholar). Also, theSchizosaccharomyces pombe Mac1 ortholog, Cuf1, has a single Cys-rich motif homologous to the Mac1 motif above (16Labbe S. Pena M.M.O. Fernandes A.R. Thiele D.J. J. Biol. Chem. 1999; 274: 36252-36260Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The Cys-rich motifs occur in the segment of Mac1 responsible for transactivation (9Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (96) Google Scholar). The transactivation function of Mac1 is copper-modulated (9Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (96) Google Scholar). Inhibition of transactivation is seen in chimeric molecules containing Mac1 fused to a heterologous DNA binding domain (8Georgatsou E. Mavrogiananis L.A. Fragiadakis G.S. Alexandraki D. J. Biol. Chem. 1997; 272: 13786-13792Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 9Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (96) Google Scholar). In addition, the DNA binding function of Mac1 is inhibited in copper-replete cells (4Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). DNA binding activity of Mac1 maps to the N-terminal 159 residues, but copper repression of DNA binding requires the C-terminal Cys-rich motifs (17Jensen L.T. Posewitz M.C. Srinivasan C. Winge D.R. J. Biol. Chem. 1998; 273: 23805-23811Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Likewise, full copper repression of transactivation activity is dependent on a portion of the DNA binding domain. Thus, Cu(I) binding in the C-terminal domains inhibits functions of both the N-terminal and C-terminal halves of the protein. The copper repression of Mac1 function appears to arise from a copper-induced, intramolecular interaction of the C-terminal Cys-rich motifs and the N-terminal DNA binding domain (13Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (84) Google Scholar). Mac1 was originally identified as a semidominant mutation, designatedMAC1 up1, that abrogated copper inactivation of Mac1 function (14Jungmann 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). This mutation results in constitutive expression ofCTR1 and FRE1 (4Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 5Yamaguchi-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 (151) Google Scholar). Copper ion uptake is markedly enhanced in MAC1 up1 cells, and one resulting phenotype is hypersensitivity to elevated copper levels in the growth medium (18Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Three additional constitutive mutations inMAC1 have also been identified. All four constitutive mutations map to the first cysteine-rich motif, designated C1 (Fig. 1). The constitutive mutations result in substitutions of three of the six conserved residues in the consensus,264 C XC(X)4 C XC(X)2C(X)2 H 279. The residues in bold are substituted to yield the four known constitutive mutants designated Mac1up1(H279Q), Mac1up2 (C271Y), Mac1up3(C264Y), and Mac1up4 (H279Y) (5Yamaguchi-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 (151) Google Scholar, 10Zhu Z. Labbe S. Pena M.M.O. Thiele D.J. J. Biol. Chem. 1998; 273: 1277-1280Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The copper-induced inhibitory, intramolecular interaction of the N-terminal DNA binding domain and the C-terminal Cys-rich motifs is expected to involve residues in both domains. Since mutations in the C1 motif abolish the inhibitory interaction, we sought to map the interactive region in the N-terminal DNA binding domain of Mac1 by isolating additional constitutive Mac1 mutants. The question was whether any mutations within the DNA binding domain would abolish the repressive intramolecular interaction, without perturbing DNA binding activity. The second motive for the present work was to determine whether the repeated Cys-rich sequence motifs function separately or as a single domain in mediating copper metalloregulation of Mac1. Conceivably, the binding of 8 Cu(I) ions in a polypeptide containing both C1 and C2 sequences may occur due to 4 Cu(I) ions binding in each motif. Alternatively, the two Cys-rich motifs may come together creating a single octa-copper center. The clustering of constitutive mutations in the C1 motif is consistent with C1 being an independent copper regulatory domain. The presence of two Cys-rich motifs in Mac1 and Grisea leaves open the possibility that both motifs function in copper metalloregulation. We show in the current work that the C1 motif is indeed an independent copper regulatory domain. The yeast strains used, CM66J (MATα, ino1-13, gcn4-101, his3-609, ura3-52, FRE1-HIS3::LEU2) and CM66E (MATa, trp1-63, gcn4-101, his3-609, ura3-52, FRE1-HIS3:: LEU2), were derived from strains 66 (MATα, trp1-63, gcn4-101, his3-609, ura3-52, FRE1-HIS3::LEU2) and CM3262 (MATa, ino1-13, gcn4-101, his3-609, ura3-52, leu2-3, 112, ino1-13). Strains 66 and CM3262 were generously provided by Andrew Dancis (19Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (310) Google Scholar). CM66Δmac1 was constructed by integratingURA3 at the MAC1 locus in strain CM66E by one-step gene replacement as described (20Baker Brachmann C. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2538) Google Scholar). The disruption was verified by diagnostic PCR1and sequencing. CM66Δmac1 was then crossed with YJJ1 (MATα, his3-Δ200, leu2-3, 112, lys2-801, trp1-1, ura3-52, mac1-1) (14Jungmann 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), sporulated, and dissected. A resulting spore that was ura3, and thusmac1-1, was selected and designated CMΔmac1. Two-hybrid experiments were conducted in strain Y190 (MATa, ade2-101, cyh2, his3, leu2-3, 112, trp1-901, ura3-52, gal4, gal80, LYS2:pGAL-HIS3, URA3:pGAL-lacZ) (21Bai C. Elledge S.J. Methods Enzymol. 1997; 283: 141-156Crossref PubMed Scopus (75) Google Scholar). One-hybrid experiments were carried out in strain DY2042 (MATa, ade2-101, his3, leu2-3, trp1, ura3-52, gal4Δ, gal80Δ, MEL1, GAL1, GAL1-10:lacZ). Yeast strains were cultured in YPD, complete medium (SC), or low copper complete medium (LCCM) using copper and iron-limiting yeast nitrogen base (Bio 101, Inc.), and lacking the appropriate amino acids to ensure maintenance of plasmid and reporters. Strain CM66J was grown overnight in LCCM lacking leucine and plated to a density of 108 cells/plate on 40 LCCM plates, lacking leucine and histidine. Mutants were isolated following UV irradiation. Plates were exposed to 50 J/m2 UV light, corresponding to approximately 50% survival rate, followed by incubation at 30 °C for 3 days while wrapped in aluminum foil to exclude light. The resulting colonies were plated onto LCCM lacking leucine and histidine but containing 30 μm Cu(II). Mutants were confirmed by re-plating on the same medium and screened for copper sensitivity on SC medium containing 1.75 mmCuSO4. In addition, CTR1 expression was assessed by S1 nuclease assay. The affected mutant allele was determined to be a single nuclear, recessive gene by crossing candidate mutants with the isogenic wild-type strain, CM66E. The resulting diploids were sporulated and dissected, and mutant status was assessed by growth on LCCM lacking histidine and containing 30 μm Cu(II). A 1.7-kilobase pair Sau3A genomic fragment containing MAC1 was subcloned from pYACu1 (generously provided by D. Hamer) into pBluescript (Stratagene) at theBamHI site, generating pBSMAC1. pBSMAC1 was then subjected to XL1-Red mutagenesis according to manufacturer's instructions (Stratagene). The resulting plasmids were rescued from XL1-RedEscherichia coli, inserts cleaved byXbaI/BamHI digest, cloned into pRS316 (22Sikorski R.S. Boeke J.D. Methods Enzymol. 1991; 194: 302-318Crossref PubMed Scopus (489) Google Scholar), and transformed into E. coli DH5α. The resulting library of mutagenized MAC1 was transformed into CMΔmac1. Transformants were replica-plated onto LCCM containing 30 μm Cu(II), lacking histidine and uracil and medium containing 1.75 mm CuSO4. Positive growth on the former and negative growth on the later was indicative of a plasmid-borne MAC1 up allele and was verified by loss of histidine prototrophy following shedding of plasmid by growth on media containing 5-fluoroorotic acid. The ability of each mutagenized plasmid to confer elevated CTR1 transcript levels was determined by S1 nuclease assay. Each plasmid conferring aMAC1 up phenotype was rescued to E. coli DH5α and sequenced. DNA sequencing was performed on an ABI Prism 377 Sequencer using Taq FS DNA polymerase. The 1.7-kilobase pairXbaI/BamHI MAC1 fragment from pBSMAC1 was subcloned into pAlter (Promega). Mutagenesis was carried out according to manufacturer's instructions. The mutations were verified by sequencing and subsequently cloned into either pRS316 or pRS426. Phenotypes were determined by transforming into CMΔmac1. MAC1 and MAC1 up1truncates encoding codons 1–311 were generated by PCR amplification using PstI and SalI cleavage sites for subcloning into vector pVT102U. pVT102U is a YEp-based vector containing theADH1 promoter and terminator (23Vernet T. Dignard D. Thomas D.Y. Gene ( Amst. ). 1987; 52: 225-233Crossref PubMed Scopus (461) Google Scholar). The HA epitope tag encoding the sequence YPYDVPDYA was inserted at the 3′ end of codon 311 of MAC1. GAL4/MAC1 fusions, engineered previously (9Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (96) Google Scholar), were excised as HindIII/SalI fragments and inserted into pRS425, a YEp based vector containing the MET25promoter and CYC1 terminator (24Mumberg D. Muller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (792) Google Scholar). GAL4/MAC1constructs were transformed into DY2042 that contains theGAL1–10/lacZ fusion reporter gene. The two hybridMAC1 vectors were described previously (13Jensen L.T. Winge D.R. EMBO J. 1998; 17: 5400-5408Crossref PubMed Scopus (84) Google Scholar). DNA sequences coding for residues 240–417 of Mac1 and containing the double Cys → Ser substitutions (residues 264, 266; 271, 273; 322, 324; and 329, 331) previously engineered (9Graden J.A. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5550-5555Crossref PubMed Scopus (96) Google Scholar) were PCR-amplified and subcloned into the vector pVT-VP16 at BamHI/ClaI sites, generating vectors fused to the minimal activation domain from the herpes simplex virus VP16 protein (25Sadowski I. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (964) Google Scholar). Sequences encoding residues 1–159 were subcloned into pBG4D-1 creating vector Mac1-(1–159)-pBG4D-1. Two-hybrid vectors were co-transformed into strain Y190. Total RNA isolated from mid-logarithmic cells by the hot acid phenol method was hybridized with a 32P-labeled single-stranded DNA oligonucleotide and digested with S1 nuclease. The samples were electrophoresed through an 8% polyacrylamide, 5 m urea gel, and data were quantified by PhosphorImager analysis. S1 probes used included 60 nucleotides of the 5′ CTR1 ORF, 40 nucleotides of the calmodulin (CDM1) 5′ ORF, and 60 nucleotides from the lacZ ORF. Cellular lysates for immunoprecipitation analysis were prepared by glass beading in 50 mm Tris-Cl, pH 7.5, 150 mm sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate and a protease inhibitor mixture. The supernatant was incubated with either a rabbit polyclonal anti-HA antibody or a rabbit polyclonal anti-Gal4 DBD antibody for 1 h at 4 °C. Protein A-agarose was added and incubated overnight at 4 °C. The protein A-agarose was collected by centrifugation, washed six times, and boiled in SDS sample buffer. The immunoprecipitated protein was resolved on SDS-polyacrylamide (15%) gel electrophoresis and transferred to nitrocellulose. Membranes were blocked and probed with either mouse monoclonal anti-HA or mouse monoclonal anti-Gal4 DBD. Detection was by enhanced chemiluminescence after incubation with a horseradish peroxidase-conjugated secondary antibody. Cellular lysates for VP16 Western analysis were prepared by glass beading in 30 mm Tris-Cl, pH 7.5, 500 mm sodium chloride, 5 mm EDTA, 0.1% Triton X-100, 12% (v/v) glycerol, and a protease inhibitor mixture. The supernatant was collected and treated with SDS. Thirty micrograms of protein were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The membrane was probed with a mouse monoclonal anti-VP16 antibody. Detection was by enhanced chemiluminescence. FRE1 is one of the four known Mac1-regulated genes in S. cerevisiae (14Jungmann 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 activates FRE1 expression in copper-deficient cells. Cells harboring aMAC1 up1 allele show elevated FRE1expression even in copper-replete conditions (14Jungmann 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). In addition,FRE1 expression is also enhanced in iron-deficient cells due to the iron-regulated Aft1 transcriptional activator (19Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (310) Google Scholar). The dual regulation of FRE1 by the copper and iron status of cells enabled a genetic screen utilizing a FRE1/HIS3 fusion gene to identify a variety of genes whose products were involved in copper and iron uptake (26Dancis A. Yuan D.S. Haile D. Askwith C. Elde D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (557) Google Scholar, 27Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (569) Google Scholar). This elegant screen also identified Aft1 as the primary iron regulator in S. cerevisiae and uncovered a second Mac1 constitutive mutant designatedMAC1 up2 (5Yamaguchi-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 (151) Google Scholar, 19Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (310) Google Scholar). The successful use of the FRE1/HIS3 fusion gene to identify a Mac1 mutant that was not repressed in copper-replete cells validated its use in mapping other constitutive MAC1 alleles. The starting strain (CM66J) exhibited histidine (His) prototrophy only when cultured in low copper medium containing a Cu(I)-specific chelator, bathocuproine disulfonate. The cells were His-auxotrophic when cultured in medium containing 10 μm Cu(II). Following ultraviolet-induced mutagenesis, mutants exhibiting His prototrophy were selected on medium containing 30 μm Cu(II). Subsequently, His+ colonies were screened for growth sensitivity to 1.75 mm CuSO4. Wild-type cells were resistant to elevated copper levels, whereas cells containing a constitutively active mutant Mac1 failed to grow due to unrestricted uptake of copper ions. Colonies conforming to both selection criteria, growth on −His+Cu medium and copper sensitivity (Cus), were analyzed for constitutive expression of chromosomalCTR1 by quantifying CTR1 mRNA levels. Eight mutants exhibited high, constitutive CTR1 expression. The eight mutants were determined to carry a single nuclear, recessive mutation by crossing with the isogenic wild-type strain followed by tetrad analysis. Diploids of the mutants crossed with CMΔmac1 showed His prototrophy in medium containing 30 μm CuSO4. These experiments suggested that the mutations were in MAC1. The MAC1 locus was PCR-amplified and sequenced in multiple reactions. All eight clones showed mutations in the C1 region of MAC1 with the following substitutions (number in parentheses indicate number of independent isolates): L260S (2Winge D.R. Jensen L.T. Srinivasan C. Curr. Opin. Chem. Biol. 1998; 2: 216-221Crossref PubMed Scopus (49) Google Scholar), C264Y, C271Y, C271G, H279Q (3Radisky D. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) (Fig.1). The C264Y, C271Y, and H279Q substitutions represent three of the four known constitutive Mac1 mutants (5Yamaguchi-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 (151) Google Scholar, 10Zhu Z. Labbe S. Pena M.M.O. Thiele D.J. J. Biol. Chem. 1998; 273: 1277-1280Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Two new constitutive alleles were L260S and C271G. The involvement of Leu260 in copper regulation of Mac1 function has not been previously reported. To verify that the L260S substitution abolished copper inhibition of Mac1 function, the mutation was engineered in MAC1, and the mutant gene was subcloned into a low copy YCp vector for transformation into CMΔmac1cells containing the FRE1/HIS3 fusion gene. ThisMAC1 mutant and all subsequent mutants were tested in the context of the MAC1 promoter and terminator on a low copy vector. Cells harboring L260S Mac1 were His prototrophs on medium containing 30 μm CuSO4 and were Cus, confirming the constitutive nature of the L260S substitution (Fig. 2, line 3). A second approach, taken simultaneously, to identify constitutive Mac1 mutations was to mutate randomly MAC1 in a mutator E. coli strain (XL1-Red), defective in three primary DNA repair genes. After propagation of episomal MAC1 in XL1-Red cells, plasmid DNA was recovered and the MAC1 locus excised and subcloned into a yeast YCp vector. Eleven transformants of CMΔmac1 cells were His prototrophs on medium containing 30 μm CuSO4 and failed to grow on medium containing 1.75 mm CuSO4. These phenotypes were reversed in cells in which the URA3-based plasmid was shed following selection on medium containing 5-fluoroorotic acid. The 11 transformants also exhibited high, constitutive expression of chromosomal CTR1. Sequencing of the MAC1 locus in rescued plasmids revealed eight distinct MAC1 mutations. Three such mutations were isolated in two independent colonies. All eight mutations occurred within the C1 region of Mac1. Six missense mutations were found with the following codon substitutions: C264F, C266G, C271W, C271F, H279N, and H279Y (Fig. 1). The phenotypes of these mutants are shown in Fig. 2 (lines 5, 7, 9, 10, 15, and16). Only the H279Y substitution was observed previously, but other substitutions at residues Cys264 and Cys271 have been shown to yield constitutive Mac1 function (5Yamaguchi-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 (151) Google Scholar, 10Zhu Z. Labbe S. Pena M.M.O. Thiele D.J. J. Biol. Chem. 1998; 273: 1277-1280Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The remaining two mutations were deletions encompassing portions of C1 (Fig. 2, lines 19 and 20). A six-base deletion of codons 262–264 and a 120-base deletion removing codons 226–267 yielded in-frame Mac1 truncates with constitutive Mac1 activity. The conserved residues in the C1 motif are as follows:264CXC(X)4CXC(X)2C(X)2H279. Substitutions at four of the six conserved residues yielded constitutive Mac1 activity. No mutations were isolated at the remaining codons, Cys273 and Cys276. To determine whether substitutions at these codons yielded a similar constitutive phenotype, Cys → Tyr substitutions were engineered at codons 273 and 276.MAC1 mutants with these changes were evaluated in CMΔmac1 cells containing the FRE1/HIS3 reporter gene on a low copy YCp vector. Unexpectedly, cells harboring either the C273Y or the C276Y Mac1 were His auxotrophic and were copper-resistant (Fig. 2, lines 12 and 14). Expression of either C273Y or the C276Y mutant Mac1 on a high copy YEp vector imparted a weak His prototrophy to cells (Fig. 3,lines 6 and 8). Quantitation of CTR1expression in cells expressing these mutant Mac1 molecules revealed constitutive mRNA levels (Fig.4 A).Figure 4A, expression of CTR1 in cells harboring MAC1 mutants expressed on high copy YEp vectors. Cells were cultured in low copper medium containing 100 μm bathocuproine sulfonate (−Cu) or 100 μmCuSO4. RNA was extracted from cells atA600 nm= 0.5, incubated with theCTR1 and calmodulin (CMD1) DNA probes, and digested with S1 nuclease. B, expression of CTR1in cells harboring MAC1 mutants on low copy vectors as detected by S1 analysis of mRNA. Cells harboring MAC1 ormac1 variants on low copy vectors were cultured in low copper medium containing 100 μm bathocuproine sulfonate or 100 μm CuSO4. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Serine substitutions were engineered at each of the six conserved residues. Surprisingly, cells containing Mac1 with Cys → Ser substitutions at any of the conserved Cys residues in C1 failed to show constitutive Mac1 activity when mutant genes were expressed on low copy vectors. In each case, FRE1/HIS3 expression was reduced in c" @default.
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