Mobilization of Intracellular Copper Stores by the Ctr2 Vacuolar Copper Transporter
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m411669200
ISSN1083-351X
AutoresErin M. Rees, Jaekwon Lee, Dennis J. Thiele,
Tópico(s)Heavy Metal Exposure and Toxicity
ResumoCopper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development. Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores. We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores. Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane. We demonstrate that Ctr2 mobilizes vacuolar copper stores in a manner dependent on amino acid residues conserved between the Ctr1 and Ctr2 copper transport family and that ctr2Δ mutants hyper-accumulate vacuolar copper. Furthermore, a Ctr2 mutant that is mislocalized to the plasma membrane stimulates extracellular copper uptake, supporting a direct role for Ctr2 in copper transport across membranes. These studies identify a novel mechanism for copper mobilization and suggest that organisms cope with copper deprivation via the use of intracellular vesicular stores. Copper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development. Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores. We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores. Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane. We demonstrate that Ctr2 mobilizes vacuolar copper stores in a manner dependent on amino acid residues conserved between the Ctr1 and Ctr2 copper transport family and that ctr2Δ mutants hyper-accumulate vacuolar copper. Furthermore, a Ctr2 mutant that is mislocalized to the plasma membrane stimulates extracellular copper uptake, supporting a direct role for Ctr2 in copper transport across membranes. These studies identify a novel mechanism for copper mobilization and suggest that organisms cope with copper deprivation via the use of intracellular vesicular stores. Metal ions such as calcium, copper, iron, and zinc play numerous and diverse roles in cellular physiology. 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Eukaryotes from yeast to humans utilize Ctr1 1The abbreviations used are: Ctr, copper transporter; GFP, green fluorescent protein; ICP-MS, inductively coupled plasma mass spectrometry; PIPES, 1,4-piperazinediethanesulfonic acid. 1The abbreviations used are: Ctr, copper transporter; GFP, green fluorescent protein; ICP-MS, inductively coupled plasma mass spectrometry; PIPES, 1,4-piperazinediethanesulfonic acid. proteins for the high affinity transport of Cu(I) across the plasma membrane (30Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 269: 25660-25667Abstract Full Text PDF PubMed Google Scholar, 31Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Abstract Full Text PDF PubMed Scopus (563) Google Scholar, 32Kampfenkel K. Kushnir S. Babiychuk E. Inze D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 33Zhou B. Gitschier J. Proc. Natl. Acad. Sci. 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Intracellular copper is routed to copper-dependent enzymes in the cytosol such as Cu,Zn-superoxide dismutase, in mitochondria (cytochrome oxidase), and in the secretory pathway (for example, the Fet3 or ceruloplasmin ferroxidase in yeast and mammals, respectively) through the action of specific copper chaperone proteins (38Field L.S. Luk E. Culotta V.C. J. Bioenerg. Biomembr. 2002; 34: 373-379Crossref PubMed Scopus (99) Google Scholar, 39Rosenzweig A.C. Chem. Biol. 2002; 9: 673-677Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 40Finney L.A. O'Halloran T.V. Science. 2003; 300: 931-936Crossref PubMed Scopus (919) Google Scholar, 41Luk E. Jensen L.T. Culotta V.C. J. Biol. Inorg. Chem. 2003; 8: 803-809Crossref PubMed Scopus (113) Google Scholar). Recent experiments have demonstrated that S. cerevisiae cells lacking the high affinity plasma membrane transporters are capable of routing copper to both Fet3 and Cu,Zn-superoxide dismutase (42Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Crossref PubMed Scopus (93) Google Scholar). This activity was shown to be dependent at least in part on the Fet4 plasma membrane broad-specificity copper, iron, zinc transporter, and the Ctr2 protein (42Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Crossref PubMed Scopus (93) Google Scholar). Ctr2 was first identified based on its strong homology to Copt1, a plant copper transporter that is a member of the Ctr1 family, and suggested to be a low affinity copper importer (32Kampfenkel K. Kushnir S. Babiychuk E. Inze D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Two reports have localized an epitope-tagged form of Ctr2 and a Ctr2-GFP fusion protein to the vacuolar membrane and punctate spots, or the endoplasmic reticulum, respectively (42Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Crossref PubMed Scopus (93) Google Scholar, 43Huh 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 (3270) Google Scholar). However, the subcellular location of Ctr2 has not been firmly established, nor has its role in copper homeostasis been clearly defined. Here we demonstrate that S. cerevisiae Ctr2 is localized to the vacuole membrane where it functions to mobilize vacuolar copper stores to cytosolic copper chaperones. We show direct evidence that Ctr2 function affects vacuolar copper levels and that a CTR2 mutant that suppresses the requirement for the high affinity plasma membrane Cu(I) transporters is mislocalized to the plasma membrane and facilitates the kinetics of copper uptake into cells. Taken together, these studies identify a mechanism whereby cells mobilize intracellular copper stores to render this metal available for cellular signaling and a number of copper-dependent enzymes whose activity is essential for normal growth and development. Yeast Strains and Plasmids—The ctr1Δctr3Δctr2Δ strain was constructed by integrating the HIS3 gene at the CTR2 locus in the MPY17 strain (44Peña M.M. Koch K.A. Thiele D.J. Mol. Cell. Biol. 1998; 18: 2514-2523Crossref PubMed Scopus (154) Google Scholar). Wild-type CTR2 and CTR2 mutant alleles were amplified from genomic DNA by PCR. Site-directed mutagenesis was performed by the overlap extension method (45Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6812) Google Scholar). For the CTR2-GFP fusion, a NotI restriction enzyme site was introduced in-frame just upstream of the stop codon and the GFP open reading frame was inserted. Yeast strains were transformed with plasmids using standard techniques and grown in minimal selective media at 30 °C with agitation. Functional Complementation of ctr2 Mutants—ctr1Δctr3Δctr2Δ cells were transformed with wild-type, mutant CTR2, or empty plasmids and grown to exponential phase in selective media with agitation. 10-Fold serial dilutions were spotted on selective media, ethanol (2%) and glycerol (3%) media (YPEG), and YPEG containing 10, 15, 20, 25, and 100 μm copper containing 1.5% agar and incubated for 3–7 days at 30 °C. Fluorescence Microscopy—The ctr1Δctr3Δctr2Δ strain was transformed with p416GPD-CTR2-GFP and grown in selective media to exponential phase at 30 °C. FM4-64 (Molecular Probes) was added to a final concentration of 40 μm and cells were incubated at 30 °C for 15 min. Cells were then collected and resuspended in fresh media for further incubation at 30 °C without agitation for 30–60 min. Cells were visualized with a Zeiss Axioskop upright, wide field fluorescence microscope equipped with a filter wheel. Indirect immunofluorescence was performed as previously described (46Pringle J.R. Adams A.E. Drubin D.G. Haarer B.K. Methods Enzymol. 1991; 194: 565-602Crossref PubMed Scopus (600) Google Scholar). Polyclonal antibody against Ctr2 was generated by Bethyl Laboratories, Inc. from a peptide sequence (CVHKRQLSQRVLLPNRSLTK) in the intracellular loop region of Ctr2 and affinity purified. Vacuole Isolation and ICP-MS—Vacuoles were isolated from spheroplasted yeast cells by previously described methods (47Bankaitis V.A. Johnson L.M. Emr S.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9075-9079Crossref PubMed Scopus (278) Google Scholar). Briefly, spheroplasts were resuspended in buffer containing PIPES and 15% Ficoll. DEAE-dextran was added to permeabilize the plasma membrane. Lysates were then loaded into Beckman SW41 ultraclear tubes and overlaid with the same buffer containing 8 and 4% Ficoll, and then tubes were filled completely with buffer. Gradients were centrifuged at 30,000 rpm for 1.5 h at 4 °C. Samples were then collected from each of the three interfaces of the gradients (0/4, 4/8, and 8/15%) and total protein concentrations were determined using the BCA method. 50 μg of total protein was used for ICP-MS analysis. For ICP-MS analysis, cells were incubated with 20 μm copper for 1 h prior to harvesting the cells. Immunoblotting—Whole cell extracts made by the alkali extraction method (48Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (179) Google Scholar) or samples from the vacuole isolation were separated by 10 or 15% SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-GFP (Chemicon), anti-Pma1 (Santa Cruz), anti-alkaline phosphatase, anti-3-phosphoglycerate kinase, or anti-carboxypeptidase Y (all from Molecular Probes). Identification of CTR2 Mutants—A PCR-based random mutagenesis strategy was used to generate CTR2 mutant alleles (49Lin-Goerke J.L. Robbins D.J. Burczak J.D. BioTechniques. 1997; 23: 409-412Crossref PubMed Scopus (92) Google Scholar). The CTR2 mutant alleles were cloned into the constitutive GPD promoter-driven yeast expression vector. Approximately 1 × 104 expression plasmids containing CTR2 alleles were generated. This CTR2 expression library was transformed into ctr1Δctr3Δ cells, and the yeast cells were selected on YPEG media. Plasmids containing Ctr2 mutant alleles were isolated from yeast cells growing on YPEG media and sequenced. Copper Accumulation and 64Cu Uptake—Whole cells were used to measure copper accumulation and 64Cu uptake. Radioactive 64Cu was purchased from the Mallinckrodt Institute of Radiology at Washington University, St. Louis, MO. Copper accumulation was measured by ICP-MS, and 64Cu was quantified in a Packard Cobra II γ-counter as described before (37Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Ctr2 Contributes Copper to Mitochondrial Respiration—All characterized members of the Ctr1 family of high affinity plasma membrane copper transporters possess specific conserved structural features (50Puig S. Thiele D.J. Curr. Opin. Chem. Biol. 2002; 6: 171-180Crossref PubMed Scopus (575) Google Scholar), which include three putative transmembrane domains, the second of which contains a Met-X3-Met motif required for function (Fig. 1A). Additionally, many Ctr1 family members identified to date possess a methionine-rich amino terminus and a cysteine/histidine-rich carboxyl terminus. Along with these structural features, all Ctr1 family members possess a methionine residue, shown to be essential for function, ∼20 amino acids upstream of the first transmembrane domain (37Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Interestingly, Ctr2 has two methionine residues separated by one amino acid in this position. Whereas S. cerevisiae Ctr2 shares only ∼23% amino acid identity overall with Ctr1, the majority of sequence conservation occurs within the hydrophobic transmembrane domains and the ∼20 amino acid residues from the conserved methionine to the first transmembrane domain. Fig. 1A summarizes the structural features that are conserved in S. cerevisiae Ctr1, Ctr2, and potential Ctr2 homologs from Schizosaccharomyces pombe, mouse, and humans. Work by Culotta and colleagues demonstrated that ctr2Δ mutants, in the context of a ctr1Δctr3Δ double mutant background, exhibited decreased activity of both Cu,Zn-superoxide dismutase and the Fet3 multicopper ferroxidase that functions in high affinity iron uptake (42Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Crossref PubMed Scopus (93) Google Scholar). To further explore the physiological role of Ctr2 in copper homeostasis we ascertained whether ctr1Δctr3Δctr2Δ strains displayed a more pronounced defect in delivery of copper to mitochondrial cytochrome oxidase than the parental CTR2 strain, as measured by growth on the non-fermentable carbon sources glycerol and ethanol. As shown in Fig. 1B, a ctr1Δctr3Δ strain cannot grow on ethanolglycerol media (YPEG), as it possesses a defective mitochondrial respiratory chain because of the inability of cytochrome c oxidase to obtain its copper cofactor. The addition of exogenous copper to the growth medium at concentrations of 15 to 20 μm or higher rescues this phenotype, indicating the mitochondrial defect is because of a lack of copper and not general mitochondrial dysfunction. Deletion of CTR2 in the ctr1Δctr3Δ background resulted in a more severe respiratory deficiency, with ctr1Δctr3Δctr2Δ cells unable to grow even when supplemented to 20 μm copper, but retaining growth in the presence of 100 μm copper (Fig. 1B). Furthermore, overexpression of the wild type CTR2 gene or a CTR2-GFP fusion allele restored growth on YPEG containing 10 μm copper. Taken together, these results suggest that inactivation of CTR2 causes an exacerbated copper deficiency and that increased levels of Ctr2, or a Ctr2-GFP fusion protein, are beneficial for cell growth under conditions of copper deprivation. Ctr2 Is a Vacuolar Membrane Protein—Despite the primary structural and topological similarities between Ctr2 and the Ctr1 family and their involvement in copper-dependent physiological processes, Ctr2 cannot complement the growth defect associated with ctr1Δctr3Δ mutants (Fig. 1B) (32Kampfenkel K. Kushnir S. Babiychuk E. Inze D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 42Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Crossref PubMed Scopus (93) Google Scholar). Although Ctr2 was initially proposed to function as a low affinity copper importer (32Kampfenkel K. Kushnir S. Babiychuk E. Inze D. Van Montagu M. J. Biol. Chem. 1995; 270: 28479-28486Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), a partially functional epitope-tagged CTR2 allele was localized to intracellular membranes that were postulated to be largely vacuolar (42Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Crossref PubMed Scopus (93) Google Scholar). Moreover, a genome-wide GFP fusion analysis placed Ctr2-GFP in the endoplasmic reticulum (43Huh 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 (3270) Google Scholar). To establish Ctr2 subcellular location, the functional Ctr2-GFP fusion protein was localized both by fluorescence microscopy and by vacuolar purification on discontinuous Ficoll density gradients. As shown in Fig. 2A, ctr2Δ cells bearing the CTR2-GFP expression plasmid displayed co-localization of Ctr2-GFP with the perimeter of the vacuole, as ascertained by DIC imaging and by overlapping fluorescence with the red fluorescent lipophilic dye FM4-64, which is endocytosed to the vacuole membrane. We also observed vacuolar localization of Ctr2-GFP for a chromosomal CTR2-GFP fusion allele (data not shown), and this localization did not change in the presence or absence of copper or the plasma membrane copper transporters CTR1 and CTR3 (data not shown). Based on the microscopic localization of Ctr2-GFP, we independently confirmed the vacuolar localization of Ctr2-GFP by vacuole purification and immunoblotting experiments. The dynamic nature of yeast vacuoles allows them to be isolated intact from whole yeast cells; centrifugation of permeabilized yeast spheroplasts on a discontinuous density gradient causes the vacuoles to expand and float to the top, whereas the other organelles and cellular debris fractionate elsewhere in the gradient. A sample from each interface of the density gradient was collected and separated by SDS-PAGE, followed by immunoblotting to detect Ctr2-GFP. As shown in Fig. 2B, Ctr2-GFP co-fractionates with the integral vacuolar membrane protein alkaline phosphatase as well as carboxypeptidase Y, a protein contained in the lumen of the vacuole. The GFP fusion does not affect localization of Ctr2, as a non-tagged plasmid-born copy of Ctr2 shows an identical fractionation pattern (Fig. 6A). 3-Phosphoglycerate kinase is a cytosolic protein that does not appear in the same fraction as Ctr2-GFP, alkaline phosphatase, and carboxypeptidase Y, indicating the vacuoles have indeed been purified away from other cellular components. Marker proteins in the mitochondria (Cox2) and trans-Golgi apparatus (Kex2) also do not appear in the fraction with the vacuoles (data not shown). Whereas there is slight contamination of the vacuole fraction with the plasma membrane ATPase Pma1, there is no enrichment of the plasma membrane in the vacuole samples over the other fraction where Pma1 is observed (Fig. 6A). Given the vast abundance of Pma1 in the plasma membrane, the actual plasma membrane contamination present in the vacuole samples is likely very low. 2A. Chang, personal communication. Together with the results of the fluorescence microscopy, these biochemical data firmly support a vacuolar localization for Ctr2. Ctr2 Mobilizes Vacuolar Copper Stores—Genetic evidence, subcellular localization studies, and the homology of Ctr2 to the Ctr1 family of plasma membrane copper transporters are consistent with the hypothesis that Ctr2 functions in copper homeostasis by transporting copper across the vacuolar membrane. To date, however, little information is available on the mobilization of copper from the lumen of internal organelles to the cytosolic copper chaperones. If Ctr2 functions in the export of vacuolar copper stores to the cytosol, we reasoned that vacuolar copper levels should vary with changes in the levels of expression or functional state of Ctr2 protein. To test this hypothesis we quantified vacuolar-associated copper by inductively coupled plasma mass spectrometry (ICP-MS) in an otherwise wild-type ctr2Δ strain transformed with either an empty vector or CTR2 under the control of the constitutive GPD promoter. One hour prior to harvesting cells for the vacuole purification, cultures were supplemented with 20 μm copper, and vacuoles were isolated as described under "Experimental Procedures." The data in Fig. 3, which represent three independent experiments, demonstrate that cells lacking CTR2 possess vacuolar copper levels that are ∼4-fold higher than cells constitutively expressing CTR2. This same trend was also observed when cells were supplemented with 100 μm copper for 30 min prior to harvesting, and in the ctr1Δctr3Δctr2Δ strain supplemented with 20 μm copper prior to harvest (data not shown). Taken together, these results demonstrate that loss of CTR2 results in vacuolar copper accumulation, and suggest that Ctr2 functions during copper deficiency by mobilizing vacuolar copper stores from the vacuolar lumen to the cytosol. Methionine Residues and Multimerization Are Critical for Ctr2 Function—Much is known about the mechanisms by which copper transporting P-type ATPases pump copper into the lumen of the secretory compartment in both yeast and mammals (51Lin 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 (352) Google Scholar, 52Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 53Lutsenko S. Petris M.J. J. Membr. Biol. 2003; 191: 1-12Crossref PubMed Scopus (173) Google Scholar). In contrast, little is known about the mechanisms by which cells mobilize stores of intracellular copper. Given the significant primary structural and topological similarity to Ctr1, and the data pointing to a role for Ctr2 in vacuolar copper mobilization, we carried out structure-function studies to ascertain whether Ctr2 and Ctr1 might utilize a similar mechanism to move copper across distinct subcellular compartments. Both Ctr1 and Ctr2 possess conserved methionine residues in the amino terminus as well as a Met-X3-Met motif within transmembrane domain two, which are essential for the function of Ctr1 in high affinity copper import from the plasma membrane (Figs. 1A and 4A). To determine whether these conserved methionine residues are required for Ctr2 function, mutant alleles of CTR2 were tested for complementation of the respiratory-deficient phenotype of ctr2Δ cells in the context of ctr1Δctr3Δ. The Ctr2 amino terminus contains two methionine residues, Met-59 and Met-61, that are 22 and 20 residues upstream of the first transmembrane domain, respectively. CTR2 mutant alleles with either single alanine mutations (M59A or M61A) or a double mutation (M59A,M61A) were transformed into the ctr1Δctr3Δctr2Δ triple mutant strain and grown on YPEG media. As seen in Fig. 4B, both single mutant alleles (M59A and M61A) still complemented the growth-defect phenotype. However, substitution of both methionine residues with alanine (M59A,M61A) resulted in a non-functional Ctr2 protein that could not rescue the respiratory deficiency (Fig. 4B). Biochemical fractionation data indicated that this non-functional Ctr2 protein retained proper expression and localization (see Fig. 6A). Analogous to Ctr1 (37Puig S. Lee J. Lau M. Thiele D.J. J. Biol. Chem. 2002; 277: 26021-26030Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), changing both methionine residue substitutions in Ctr2 to cysteine or histidine residues (M59C,M61C and M59H,M61H), other potential copper ligands, restored growth with the addition of 20 μm copper (data not shown), suggesting these methionine residues are not only critical for function, but may potentially coordinate copper ions at some point during the copper mobilization process. Mutations were also generated, singly (M148L or M152L) and in combination (M148L,M152L), in the Met-X3-Met motif in the second transmembrane domain of Ctr2. Each of the mutant alleles, when expressed in the ctr1Δctr3Δctr2Δ strain and seeded on a non-fermentable carbon source supplemented with 20 μm copper, re
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