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• W2046382011 abstract "Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation. Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation. Excretion and detoxification of nonphysiological metals, homeostatic absorption, and utilization of nutritional yet toxic metals are fundamental biological processes. Metal toxicity and deficiency resulting from defects in metabolism and excess accumulation through environmental contamination are implicated in a number of disorders, including failure in normal growth and development and initiation and progression of degenerative diseases (1Nelson N. EMBO J. 1999; 18: 4361-4371Crossref PubMed Scopus (248) Google Scholar, 2Pena M.M. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (602) Google Scholar, 3Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 4Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4414) Google Scholar, 5Vallee B.L. Ulmer D.D. Annu. Rev. Biochem. 1972; 41: 91-128Crossref PubMed Scopus (1240) Google Scholar, 6Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (693) Google Scholar, 7Tao T.Y. Gitlin J.D. Hepatology. 2003; 37: 1241-1247Crossref PubMed Scopus (129) Google Scholar). For instance, a genetic defect in copper excretion in the liver leads to Wilson disease exhibiting hepatic failure and neuronal dysfunctions (7Tao T.Y. Gitlin J.D. Hepatology. 2003; 37: 1241-1247Crossref PubMed Scopus (129) Google Scholar). Cadmium is a well known industrial and environmental pollutant implicated in cancer, neurological disorders, reproductive defects, and endocrine disruption (8Ercal N. Gurer-Orhan H. Aykin-Burns N. Curr. Top. Med. Chem. 2001; 1: 529-539Crossref PubMed Scopus (1439) Google Scholar, 9Henson M.C. Chedrese P.J. Exp. Biol. Med (Maywood). 2004; 229: 383-392Crossref PubMed Scopus (370) Google Scholar, 10McMurray C.T. Tainer J.A. Nat. Genet. 2003; 34: 239-241Crossref PubMed Scopus (158) Google Scholar, 11Darbre P.D. J. Appl. Toxicol. 2006; 26: 191-197Crossref PubMed Scopus (210) Google Scholar, 12Stoica A. Katzenellenbogen B.S. Martin M.B. Mol. Endocrinol. 2000; 14: 545-553Crossref PubMed Scopus (390) Google Scholar). Cadmium exerts its toxic effects by the perturbation of cellular redox balance, inhibition of DNA repair, disruption of metal ion homeostasis, and alterations in signal transduction (8Ercal N. Gurer-Orhan H. Aykin-Burns N. Curr. Top. Med. Chem. 2001; 1: 529-539Crossref PubMed Scopus (1439) Google Scholar, 9Henson M.C. Chedrese P.J. Exp. Biol. Med (Maywood). 2004; 229: 383-392Crossref PubMed Scopus (370) Google Scholar, 10McMurray C.T. Tainer J.A. Nat. Genet. 2003; 34: 239-241Crossref PubMed Scopus (158) Google Scholar, 11Darbre P.D. J. Appl. Toxicol. 2006; 26: 191-197Crossref PubMed Scopus (210) Google Scholar, 12Stoica A. Katzenellenbogen B.S. Martin M.B. Mol. Endocrinol. 2000; 14: 545-553Crossref PubMed Scopus (390) Google Scholar). Mechanistic insights into metal metabolism in living organisms would facilitate prevention and treatment of metal-related disorders and develop methods for efficient remediation of toxic metals from the environment.Organisms have evolved defense mechanisms to combat the toxic effects of heavy metal ions. The P1B-type ATPase family of heavy metal transporters that are distributed from bacteria to humans extrude toxic metal ions such as copper, silver, zinc, cobalt, lead, and/or cadmium from the cell (13Silver S. Phung L.T. Annu. Rev. Microbiol. 1996; 50: 753-789Crossref PubMed Scopus (1030) Google Scholar, 14Nies D.H. Appl. Microbiol. Biotechnol. 1999; 51: 730-750Crossref PubMed Scopus (1748) Google Scholar, 15Gatti D. Mitra B. Rosen B.P. J. Biol. Chem. 2000; 275: 34009-34012Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 16Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 17Axelsen K.B. Palmgren M.G. Plant. Physiol. 2001; 126: 696-706Crossref PubMed Scopus (338) Google Scholar, 18Williams L.E. Mills R.F. Trends Plant. Sci. 2005; 10: 491-502Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Functional characterization of this family of transporters has supported the conclusion that efflux mechanisms play critical roles in metal detoxification in bacterial cells (13Silver S. Phung L.T. Annu. Rev. Microbiol. 1996; 50: 753-789Crossref PubMed Scopus (1030) Google Scholar, 14Nies D.H. Appl. Microbiol. Biotechnol. 1999; 51: 730-750Crossref PubMed Scopus (1748) Google Scholar, 15Gatti D. Mitra B. Rosen B.P. J. Biol. Chem. 2000; 275: 34009-34012Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Copper-specific P1B-type ATPases have been identified in eukaryotes as well (2Pena M.M. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (602) Google Scholar, 7Tao T.Y. Gitlin J.D. Hepatology. 2003; 37: 1241-1247Crossref PubMed Scopus (129) Google Scholar, 16Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 17Axelsen K.B. Palmgren M.G. Plant. Physiol. 2001; 126: 696-706Crossref PubMed Scopus (338) Google Scholar). Cloning of genes defective in Menkes and Wilson disease have revealed that two P1B-type ATPases (ATP7a and ATP7b) play essential roles in copper acquisition and excretion in humans (2Pena M.M. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (602) Google Scholar, 7Tao T.Y. Gitlin J.D. Hepatology. 2003; 37: 1241-1247Crossref PubMed Scopus (129) Google Scholar, 16Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Plants express copper-, zinc-, cobalt-, lead-, and/or cadmium-translocating ATPases that appear to be involved in distribution and compartmentalization of these metal ions (17Axelsen K.B. Palmgren M.G. Plant. Physiol. 2001; 126: 696-706Crossref PubMed Scopus (338) Google Scholar, 18Williams L.E. Mills R.F. Trends Plant. Sci. 2005; 10: 491-502Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar).Although metal efflux systems have begun to be characterized (16Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 17Axelsen K.B. Palmgren M.G. Plant. Physiol. 2001; 126: 696-706Crossref PubMed Scopus (338) Google Scholar, 18Williams L.E. Mills R.F. Trends Plant. Sci. 2005; 10: 491-502Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 19Papoyan A. Kochian L.V. Plant Physiol. 2004; 136: 3814-3823Crossref PubMed Scopus (253) Google Scholar, 20Hussain D. Haydon M.J. Wang Y. Wong E. Sherson S.M. Young J. Camakaris J. Harper J.F. Cobbett C.S. Plant Cell. 2004; 16: 1327-1339Crossref PubMed Scopus (499) Google Scholar, 21Eren E. Argüello J.M. 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Pharmacol. 2003; 186: 163-188Crossref PubMed Scopus (336) Google Scholar), it has been suggested that in eukaryotes metallothionine (MT), 2The abbreviations used are: MT, metallothionine; GFP, green fluorescent protein; PGK, phosphoglycerate kinase; ORF, open reading frame; ICPMS, inductively coupled plasma mass spectrometry; PBS, phosphate-buffered saline; GPD, glyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are: MT, metallothionine; GFP, green fluorescent protein; PGK, phosphoglycerate kinase; ORF, open reading frame; ICPMS, inductively coupled plasma mass spectrometry; PBS, phosphate-buffered saline; GPD, glyceraldehyde-3-phosphate dehydrogenase. a Cys-rich low molecular weight protein, and GSH-mediated sequestration appear to be the major mechanism in neutralizing toxic metals (25Hamer D.H. Annu. Rev. Biochem. 1986; 55: 913-951Crossref PubMed Google Scholar, 26Klaassen C.D. Liu J. Choudhuri S. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 267-294Crossref PubMed Scopus (980) Google Scholar, 27Cobbett C. Goldsbrough P. Annu. Rev. Plant. Biol. 2002; 53: 159-182Crossref PubMed Scopus (1915) Google Scholar, 28Singhal R.K. Anderson M.E. Meister A. FASEB J. 1987; 1: 220-223Crossref PubMed Scopus (367) Google Scholar, 29Wimmer U. Wang Y. Georgiev O. Schaffner W. Nucleic Acids Res. 2005; 33: 5715-5727Crossref PubMed Scopus (124) Google Scholar). Ycf1 in Saccharomyces cerevisiae, a vacuolar membrane ATP-binding cassette (ABC) transporter, sequesters glutathione-conjugated cadmium (30Li Z.S. Lu Y.P. Zhen R.G. Szczypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (495) Google Scholar). Phytochelatin, a GSH polymer synthesized in plants and yeast Schizosaccharomyces pombe, also detoxifies heavy metals (27Cobbett C. Goldsbrough P. Annu. Rev. Plant. Biol. 2002; 53: 159-182Crossref PubMed Scopus (1915) Google Scholar). Metal-responsive transcription factor 1 (MTF-1) regulates basal and metal-inducible expression of MTs and other genes in mammals, fruit fly, and fish (31Lichtlen P. Schaffner W. BioEssays. 2001; 23: 1010-1017Crossref PubMed Scopus (114) Google Scholar, 32Wang Y. Wimmer U. Lichtlen P. Inderbitzin D. Stieger B. Meier P.J. Hunziker L. Stallmach T. Forrer R. Rulicke T. Georgiev O. Schaffner W. FASEB J. 2004; 18: 1071-1079Crossref PubMed Scopus (82) Google Scholar, 33Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2870-2875Crossref PubMed Scopus (403) Google Scholar, 34Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1219-1223Crossref PubMed Scopus (327) Google Scholar). In contrast to mammalian genes encoding MT, copper but not cadmium and zinc induces the Cup1 gene in S. cerevisiae through the ACE1 transcription regulator (35Thiele D.J. Mol. Cell. Biol. 1988; 8: 2745-2752Crossref PubMed Scopus (238) Google Scholar, 36Zhou P. Thiele D.J. Biofactors. 1993; 4: 105-115PubMed Google Scholar). Consistent with their critical roles in induction of the genes involved in metal detoxification, deletion of MTF1 in mice and ACE1 in S. cerevisiae results in enhanced metal sensitivity (32Wang Y. Wimmer U. Lichtlen P. Inderbitzin D. Stieger B. Meier P.J. Hunziker L. Stallmach T. Forrer R. Rulicke T. Georgiev O. Schaffner W. FASEB J. 2004; 18: 1071-1079Crossref PubMed Scopus (82) Google Scholar, 35Thiele D.J. Mol. Cell. Biol. 1988; 8: 2745-2752Crossref PubMed Scopus (238) Google Scholar).To gain better insights into the mechanisms of heavy metal metabolism, we have selected S. cerevisiae cDNAs that suppress copper sensitivity of the ace1Δ yeast strain. One of the cDNAs identified encodes the putative P1B-type ATPase, PCA1, which has been suggested to be involved in copper and/or iron homeostasis (37Rad M.R. Kirchrath L. Hollenberg C.P. Yeast. 1994; 9: 1217-1225Crossref Scopus (41) Google Scholar, 38De Freitas J.M. Kim J.H. Poynton H. Su T. Wintz H. Fox T. Holman P. Loguinov A. Keles S. van der Laan M. Vulpe C. J. Biol. Chem. 2004; 279: 4450-4458Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Previously, the cadmium resistance of a mutant yeast strain selected on toxic concentrations of cadmium was mapped to the PCA1 gene that contained multiple mutations (39Shiraishi E. Inouhe M. Joho M. Tohoyama H. Curr. Genet. 2000; 37: 79-86Crossref PubMed Scopus (53) Google Scholar). However, the role and mechanisms of action of PCA1 in metal metabolism have remained elusive. Here we show that PCA1-mediated copper resistance is dependent on its Cys-rich N-terminal domain. The PCA1 allele in all laboratory yeast strains examined carries a missense mutation in a conserved residue resulting in loss of function. The wild-type PCA1 allele confers cadmium resistance by an efflux mechanism accompanied by a novel mode of metal-dependent post-transcriptional regulation.EXPERIMENTAL PROCEDURESYeast Strains, Media, and Phenotypic Tests—Strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and null mutants ace1Δ::KanMX6, pca1Δ::KanMX6 were purchased from Open Biosystems. Strain RM11-1a (40Brem R.B. Yvert R. Clinton R. Kruglyak L. Science. 2002; 296: 752-755Crossref PubMed Scopus (1045) Google Scholar) (MATa leu2Δ0 ura3Δ0 HO::KanMX6) was kindly provided by Leonid Kruglyak (Fred Hutchison Cancer Research Center, Seattle, WA). RM11-1a pca1Δ::URA3 and BY4741 ace1Δ::KanMX6 Δpca1:HIS3 were generated by PCR-based homologous recombination (41Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Phillippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4108) Google Scholar). For consistency and simultaneous comparisons of copper and cadmium sensitivity, all experiments unless indicated otherwise were performed using the BY4741 ace1Δ strain. Plasmids were transformed into yeast using the lithium acetate procedure (42Geitz D.R. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Crossref PubMed Scopus (1679) Google Scholar). Yeast cells were grown on synthetic complete (SC) media (2% dextrose or galactose, 0.67% yeast nitrogen base, and 0.2% dropout mixture for plasmid selection). For phenotypical analysis, ∼5 μl of yeast cells (A600 1.0) were spotted on selective media (1.5% agar) supplemented with the indicated concentrations of cadmium (CdCl2) or copper (CuCl2) (Sigma) and incubated at 30 °C for 2 days.Selection of cDNAs Conferring Copper Resistance—Strain BY4741 ace1Δ strain was transformed with a yeast cDNA library (43Liu H. Krizek J. Bretscher A. Genetics. 1992; 132: 665-673Crossref PubMed Google Scholar). Transformants (∼1 × 106 colonies) growing on SC-Ura media were collected by resuspending in sterilized distilled water, diluted, and plated on galactose-containing SC-Ura media supplemented with copper (0.1 mm CuCl2). Plasmids containing cDNA were isolated from yeast colonies growing on selection media and identified by sequencing. cDNA-dependent copper resistance was confirmed by retransformation of the isolated plasmid into ace1Δ yeast cells.Plasmid Construction—The PCA1 open reading frame (ORF) was PCR-amplified from yeast genomic DNA and cloned into BamHI and XhoI restriction sites of a single-copy yeast expression vector for GPD (p413 GPD) (44Mumberg D. Müller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1571) Google Scholar) or GAL1 (p413 GAL1) (45Johnston M. Davis R.W. Mol. Cell. Biol. 1984; 4: 1440-1448Crossref PubMed Scopus (632) Google Scholar) gene promoter-mediated expression. The coding sequences of the PCA1 N-terminal domains (amino acids 1–392 and 1–452) were PCR-amplified with a primer set containing a start codon and an artificially generated stop codon and were subcloned into BamHI and EcoRI sites of p413-GPD. The PCA1 truncation mutant (amino acids 393–1216) was generated by PCR amplification of the sequences, including start and stop codons, and was subcloned into p413 GPD at BamHI and XhoI restriction sites. PCA1-GFP and PCA1-FLAG were constructed by generation of a NotI restriction site after the start codon for insertion of NotI-flanked green fluorescent protein (GFP) without start and stop codons or two tandem FLAG epitopes, respectively. For natural promoter-driven expression, the PCA1 ORF, including 810 bp upstream of the start position, was cloned into SacI and XhoI restriction sites of plasmid pRS413 (46Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Site-directed mutagenesis was conducted by the primer overlap extension method (47Ho 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). All constructs were confirmed by sequencing.Fluorescence Microscopy—Yeast cells were transformed with PCA1-GFP or PCA1-WT-GFP expression plasmids and cultured in SC-His media at 30 °C with agitation until mid-log phase. Metals were added to the culture media for 15 min to 2 h prior to imaging. Cells were collected, washed in phosphate-buffered saline (PBS), and imaged on a confocal microscope (Olympus FV500).Metal Measurements—Yeast cells were cultured until mid-log phase, and metal ions were added to the culture media for 6 h. Cells were collected in 2-ml aliquots and washed two times in PBS containing 10 mm EDTA. Cell pellets were dissolved in 70% nitric acid at 60 °C for 30 min and diluted to 10% nitric acid. To measure cadmium excretion, cells were cultured with 5 mm CdCl2 for 30 min, washed two times in PBS containing 10 mm EDTA, and resuspended in fresh media prior to sample collection at the indicated time points. Total cellular metal levels were measured by inductively coupled plasma mass spectrometry (ICPMS) at the Department of Geological Sciences, University of Michigan.Immunoblotting—Total protein extracts were prepared from yeast cells using glass beads and Triton X-100 (1%) as described previously (48Pena M.M. Puig S. Thiele D.J. J. Biol. Chem. 2000; 275: 33244-33251Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Protein concentrations were measured by the BCA method (Pierce) according to the manufacturer's specifications. Protein samples were resolved by reducing SDS-PAGE and transferred to a nitrocellulose membrane. PCA1-GFP was detected by chemiluminescence using a primary rabbit anti-GFP polyclonal antibody (1:2000) (Santa Cruz Biotechnology) and secondary goat anti-rabbit horseradish peroxidase-conjugated antibody (1:5000) (Santa Cruz Biotechnology). PCA1-FLAG was probed with primary mouse anti-FLAG M2 monoclonal antibody (1:1000) (Sigma) and secondary sheep anti-mouse IgG horseradish peroxidase-conjugated antibody (Amersham Biosciences) (1:5000). Loading control, phosphoglycerate kinase (PGK), was detected by chemiluminescence using mouse monoclonal anti-PGK antibodies (1:4000) (Molecular Probes) and secondary sheep anti-mouse IgG horseradish peroxidase-conjugated antibody (1:5000) (Amersham Biosciences).Northern Blotting—Yeast cells were cultured until mid-log phase and supplemented with either 50 μm CdCl2 or 50 μm CuCl2 for 15 and 60 min. Total RNA was extracted from cells, and 25 μg was separated on an RNA gel (0.75% agarose and 2% formaldehyde) and transferred to a nitrocellulose membrane (Protran). Gene-specific DNA probes labeled with [α-32P]dCTP (Amersham Biosciences) were generated using the random primer labeling system (Invitrogen). Hybridization of radiolabeled probes with RNA transcripts was performed by methods described previously (49Lee J. Petris M.J. Thiele D.J. J. Biol. Chem. 2002; 277: 40253-40259Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Relative mRNA levels were detected by autoradiography.RESULTSPCA1 N-terminal Domain Confers Copper Resistance in Yeast—To identify new factors involved in heavy metal defense, we carried out a selection of S. cerevisiae cDNAs that suppress lethality of the ace1Δ yeast strain on toxic copper media. A cDNA encoding PCA1, one of two P1B-type ATPases in the S. cerevisiae genome (37Rad M.R. Kirchrath L. Hollenberg C.P. Yeast. 1994; 9: 1217-1225Crossref Scopus (41) Google Scholar, 50Yuan 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 (390) Google Scholar), was identified. Yeast GPD gene promoter-driven constitutive expression of PCA1 confers resistance in the ace1Δ strain to copper (0.1 mm CuCl2) compared with empty vector transformed control cells (Fig. 1A). Deletion of PCA1 in the ace1Δ strain resulted in a slight increase in sensitivity to copper toxicity (Fig. 1A). However, in a wild-type strain, copper resistance was not easily discernible upon either overexpression or deletion of PCA1 (data not shown).PCA1 contains all of the conserved features of the P1B-type ATPase family, which is specific for heavy metal transport (16Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 17Axelsen K.B. Palmgren M.G. Plant. Physiol. 2001; 126: 696-706Crossref PubMed Scopus (338) Google Scholar, 18Williams L.E. Mills R.F. Trends Plant. Sci. 2005; 10: 491-502Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 51Kuhlbrandt W. Nat. Rev. Mol. Cell Biol. 2004; 5: 282-295Crossref PubMed Scopus (440) Google Scholar) (Fig. 1B). These features include eight predicted transmembrane domains, where an intramembranous metal-transporting CPX motif is located in the sixth transmembrane domain (Fig. 1B). The catalytic domains reside within cytosolic loops that include the nucleotide binding domain (N-domain) and the phosphorylation domain (P-domain) (Fig. 1B). PCA1 contains a single CXXC heavy metal-binding motif located within the N-terminal region. An intriguing feature of PCA1 compared with other P1B-type ATPases is a Cys-rich N-terminal extension ∼550 amino acids before the first transmembrane domain (Fig. 1B). To ascertain whether the observed copper resistance by PCA1 is dependent on ATPase function, we carried out site-directed mutagenesis of conserved residues of this family of proteins and assayed for copper sensitivity. However, copper resistance remained unchanged in all mutants (Fig. 1C), suggesting that copper resistance is not dependent on metal translocation. Given that the N terminus contains several cysteine residues (33 Cys), which could serve as copper ligands, we tested whether this domain could independently confer resistance. A peptide corresponding to amino acid residues 1–452, which includes the CXXC motif (Fig. 1B), was expressed in the ace1Δ strain. Expression of this peptide resulted in copper resistance above that of full-length PCA1, presumably by chelating copper ions in a manner analogous to MT (Fig. 1D). Copper resistance is maintained even after mutation of the conserved CXXC motif (C421A, C423A) (Fig. 1D) implying the existence of other copper coordination site(s) in addition to this well characterized metal-binding motif.Common Laboratory Yeast Strains Possess a G970R Mutation in PCA1—S288c is a commonly used S. cerevisiae haploid strain in which its entire genome has been sequenced (52Goffeau A. Barrell B.G. Bussey H. Davis R.W. Dujon B. Feldmann H. Galibert F. Hoheisel J.D. Jacq C. Johnson M. Louis E.J. Mewes H.W. Murakami Y. Philippsen P. Tettelin Oliver S.G. Science. 1996; 274: 563-567Crossref Scopus (3208) Google Scholar). Comparisons of the PCA1 sequence of S288c with natural isolates, RM11-1a (40Brem R.B. Yvert R. Clinton R. Kruglyak L. Science. 2002; 296: 752-755Crossref PubMed Scopus (1045) Google Scholar) and YJM789 (53Tawfik O.W. Papasian C.J. Dixon A.Y. Potter L.M. J. Clin. Microbiol. 1989; 27: 1689-1691Crossref PubMed Google Scholar), using BLAST (NCBI, www.ncbi.nlm.nih.gov) revealed that the S288c strain carries a single nucleotide change in the PCA1 gene, which results in a G970R substitution. (Fig. 2). Our sequencing results of PCA1 cloned from the RM11-1a strain as well as an industrial bakers' yeast strain (Fleischmann's) confirmed the G970R substitution. To determine whether this mutation was unique to the S288c strain, we sequenced the PCA1 ORF of several laboratory yeast strains, BY4741 (Open Biosystems), DTY1 (54Rymond B.C. Zitomer R.S. Schumperli D. Rosenberg M. Gene (Amst.). 1983; 25: 249-262Crossref PubMed Scopus (37) Google Scholar), EG103 (55Gralla E.B. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar), SKY9 (56Knight S.A. Labbé S. Kwon L.F. Kosman D.J. Thiele D.J. Genes Dev. 1996; 10: 1917-1929Crossref PubMed Scopus (219) Google Scholar), and YPH98 (46Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). All contained the G970R mutation (data not shown), which suggests a common lineage among theses strains. Furthermore, sequence alignments of prokaryotic, yeast, plant, and human P1B-type ATPases show that this Gly residue is invariantly conserved among this family of proteins (Fig. 2). Recently, structural studies of the nucleotide binding domain (N-domain) of two P1B-type ATPases (CopA and ATP7b) places this Gly residue within an ATP-binding pocket (57Sazinsky M.H. Mandal A.K. Argüello J.M. Rosenwieg A.C. J. Biol. Chem. 2006; 281: 11161-11166Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 58Dmitriev O. Tsivkovskii R. Abildgaard F. Morgan C.T. Markley J.L. Lutsenko S. Proc. Nat. Acad. Sci. U. S. A. 2006; 103: 5302-5307Crossref PubMed Scopus (100) Google Scholar). The functional importance of this residue is further underscored by an equivalent G1101R substitution in ATP7b previously reported in patients with Wilson disease (59Thomas G.R. Forbes J.R. Roberts E.A. Walshe J.M. Cox D.W. Nat. Genet. 1995; 9: 210-217Crossref PubMed Scopus (487) Google Scholar). Together, our data along with recent reports strongly support the conclusion that many laboratory S. cerevisiae strains contain a mutation of a critical residue in PCA1 that may play essential roles in function, expression, and/or regulation.FIGURE 2Yeast laboratory strains contain a G970R mutation in PCA1. A region of the predicted nucleotide binding domain (N-domain) of PCA1 from strains S288c, RM11-1a, and YJM789 were aligned with the corresponding region of other P1B-type ATPases, including human copper-transporting ATP7a and ATP7b, S. cerevisiae CCC2, A. thaliana HMA4, S. aureus CadA, and Archaeoglobus fulgidus CopA. Boxes highlight conserved residues. Asterisk marks G970R substitution identified in the S288C and other laboratory S. cerevisiae strains. Sequences were obtained from data bases at The National Center for Biotechnological Information (NCBI), and alignments were performed with ClustalX (1.81).View Large Image Figure ViewerDownload Hi-res image Download (PPT)PCA1 Natural Allele Confers Hyper-resistance to Cadmium—To characterize the function of PCA1, we expressed the natural allele (PCA1-WT) and the G970R mutant allele (PCA1) in a laboratory yeast strain and examined metal tolerance. Indeed, expression of PCA1-WT allows cells to grow on media containing a cadmium concentration over 10-fold higher compared with vector and PCA1 carrying the G970R mutation (Fig. 3A). However, growth on media supplemented with copper was indistinguishable, which is consistent for copper resistance being dependent on metal binding rather than ATPase activity (Fig. 3A). No growth advantages in cells expressing PCA1-WT were observed when toxic concentrations of zinc, manganese, cobalt, or iron were added to growth media (data not shown), suggesting specificity in metal binding and translocation by PCA1-WT.FIGURE 3PCA1 natural allele (PCA1-WT) plays a critical role in cadmium defense. A, PCA1-WT confers resistance to cadmium toxicity. BY4741 ace1Δ cells expre" @default.
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• W2046382011 title "A Cadmium-transporting P1B-type ATPase in Yeast Saccharomyces cerevisiae" @default.
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