Ctr 6 , a Vacuolar Membrane Copper Transporter in Schizosaccharomyces pombe *

Aerobic organisms possess efficient systems for the transport of copper. This involves transporters that mediate the passage of copper across biological membranes to reach essential intracellular copper-requiring enzymes. In this report, we identify a new copper transporter in Schizosaccharomyces pombe, encoded by the ctr6 gene. The transcription of ctr6 is induced under copper-limiting conditions. This regulation is mediated by the cis-acting promoter element CuSE (copper-signaling element) through the copper-sensing transcription factor Cuf1. An S. pombe strain bearing a disrupted ctr6 allele displays a strong reduction of copper,zinc superoxide dismutase activity. When the ctr6 gene is overexpressed from the thiamine-inducible nmt1 promoter, the cells are unable to grow on medium containing exogenous copper. Surprisingly, this copper-sensitive growth phenotype is not due to an increase of copper uptake at the cell surface. Instead, copper delivery across the plasma membrane is reduced. Consistently, this results in repressing ctr4 gene expression. By using a functional ctr6 epitope-tagged allele expressed under the control of its own promoter, we localize the Ctr6 protein on the membrane of vacuoles. Furthermore, we demonstrate that Ctr6 is an integral membrane protein that can trimerize. Moreover, we show that Ctr6 harbors a putative copper-binding MetX-His-Cys-X-Met-X-Met motif in the amino terminus, which is essential for its function. Our findings suggest that under conditions in which copper is scarce, Ctr6 is required as a means to mobilize stored copper from the vacuole to the cytosol.

Acquisition of copper is crucial for aerobic life, because this element is an essential component of enzymes of primary metabolism (1,2). Despite this vital role, too much copper in the cell can be detrimental, because in the presence of oxygen, copper can catalyze the production of cell-damaging hydroxyl radicals (3,4). To balance the need for copper and its poten-tially harmful effects, living organisms have developed various specialized pathways of copper transport and distribution (5)(6)(7)(8).
The use of bakers' yeast Saccharomyces cerevisiae as a model organism has provided fundamental information of copper homeostasis in eukaryotic cells (6, 8 -10). For high affinity copper transport into S. cerevisiae cells, Cu 2ϩ is reduced to Cu ϩ by the Fre1 and Fre2 cell surface reductases (11)(12)(13)(14)(15). Following reduction, copper ions are specifically transported across the plasma membrane by two distinct transporters, Ctr1 1 (16 -18) and Ctr3 (19,20). Ctr1 is characterized by the presence of eight copies of the consensus sequence Met-X 2 -Met-X-Met in its amino-terminal extracellular domain (16,17), whereas Ctr3 is rich in Cys residues with 11 cysteines found throughout the protein but lacks the Met-clustered motif. Although the eight Met-X 2 -Met-X-Met motifs found in Ctr1 play an important role in copper uptake when cells are grown under copper starvation conditions, the last methionine (amino acid residue 127) of the Met-X 2 -Met-X-Met motif 8 is essential for Ctr1 function (16). Likewise, a Met-X 3 -Met motif (residues 256 -260) within the second transmembrane domain of Ctr1 was also identified as essential for copper transport (16). Despite the fact that the S. cerevisiae Ctr3 protein exhibits a limited overall sequence homology to Ctr1, it has been demonstrated that Ctr3 bears a similar Met-X 3 -Met motif (residues 185-189) within its second transmembrane domain (16). This enables Ctr3 to transport copper across the plasma membrane in conjunction with other critical residues such as Cys-16 within the amino-terminal portion, Cys-48 and Cys-51 within the first transmembrane domain, and Cys-199 found into the third transmembrane domain of the protein (16,19). Once inside the cell, free copper ions are virtually undetectable (21). In fact, copper ions are transiently associated with small copper-binding proteins, denoted copper chaperones, that possess the ability to distribute copper to specific intracellular destinations (22). To date, three distinct copper chaperones Atx1 (23,24), CCS (also termed Lys7) (25), and Cox17 (26 -29) have been identified and found to deliver copper to the secretory compartment (into the Fet3 multicopper oxidase (30) via the intracellular copper transporter Ccc2 (31)), cytosolic copper,zinc-SOD1, and mitochondria (into the cytochrome c oxidase presumably with the aid of Sco1 (32)(33)(34) and Cox11 (35,36) proteins), respectively. Consistent with their function in discrete pathways of intracellular copper distribution, mutations in any one of the copper chaperone genes gives rise only to specific defects in its respective pathway (22). In addition to the high affinity copper transporter and copper chaperones, a gene denoted CTR2 (37) has been characterized that encodes a putative copper transporter located predominantly in the vacuolar membrane (38). Although Ctr2 may mobilize intracellular copper stores, its precise mechanism of action has not been ascertained.
The early molecular mechanisms of copper acquisition in Schizosaccharomyces pombe differ from those of S. cerevisiae. Two proteins, Ctr4 and Ctr5, form a two-component copper transporting complex at the cell surface (39,40). This association between Ctr4 and Ctr5 appears to be critical for protein maturation and secretion of the heteroprotein complex to the plasma membrane (39). Within this complex, the exact function of each protein is currently unclear. Ctr4 is a 289-amino acid protein with five repeats of the consensus sequence Met-X 2 -Met-X-Met in its amino terminus, which is predicted by topological analysis to reside extracellularly (40). The carboxylterminal residues 111-248 of the S. pombe Ctr4 exhibit strong homology to the S. cerevisiae Ctr3 copper transporter, especially with respect to several residues within the three predicted transmembrane domains (6,40). Ctr5 is a 173-amino acid protein that is structurally related to Ctr4. An elegant study has demonstrated that Ctr5 is an integral membrane protein, which is required for properly localizing Ctr4 to the plasma membrane in S. pombe cells (39). Once copper ions are transported by the Ctr4-Ctr5 complex into the cells, they are presumably taken by putative copper chaperones, which are yet uncharacterized at the molecular level (41). A hallmark of the ctr4 ϩ and ctr5 ϩ genes is the fact that they are transcriptionally regulated according to copper need (40,42). The expression of ctr4 ϩ and ctr5 ϩ is activated under copper starvation conditions, whereas repression of these genes occurs under copper-replete conditions. This regulation is mediated by the cis-acting promoter element, denoted CuSE (Cu-signaling element) with the consensus sequence 5Ј-D(T/A)DDHGCTGD-3Ј (D ϭ A, G, or T; H ϭ A, C, or T) (42). The transcription factor responsible for regulating the copper-dependent expression of genes encoding components involved in copper transport through the CuSEs is Cuf1 (40,42).
In this study, we identify a novel gene, termed ctr6 ϩ , which is regulated at the transcriptional level in a copper-and Cuf1dependent manner. A deletion of the ctr6 ϩ gene (ctr6⌬) results in a significant reduction of copper,zinc-SOD1 activity. Cells overexpressing ctr6 ϩ are unable to grow on medium containing exogenous copper. Surprisingly, this copper toxicity phenotype resulting from ctr6 ϩ overexpression is not due to an increase of copper uptake. Instead, the cell surface copper transport activity is reduced, and consistently the steady-state levels of the ctr4 ϩ mRNA are diminished. By using a ctr6 ϩ -HA 4 epitopetagged allele, which retains wild type function, we have localized Ctr6-HA 4 to the vacuolar membrane when cells are grown under conditions of low copper availability. Interestingly, we show that the amino terminus of Ctr6 harbors a Met-X-His-Cys-X-Met-X-Met sequence, which is essential for its intracellular copper transport activity. Furthermore, we demonstrate that Ctr6 is an integral membrane protein, which can assemble into a homotrimer complex. Taken together these results suggest that under copper scarcity, Ctr6 may serve to mobilize intravacuolar stores of copper in fission yeast.
Analysis of ctr6 ϩ Gene Expression-For Northern blot analysis, the ctr6 ϩ gene was isolated by PCR using primers that corresponded to the start and stop codons of the ORF from an S. pombe cDNA library (ATCC number 87284, deposited by S. Elledge) (kind gift of Dr. Dennis J. Thiele, University of Michigan, Ann Arbor, MI). This PCR product was purified and 32 P-labeled as described previously (48). Hybridization was carried out according to the Schleicher & Schuell protocol. The S. pombe act1 ϩ probe (40) was used as an internal control. For RNase protection analyses (49), three plasmids for making antisense RNA probes were utilized. The plasmids pKSlacZ and pSKact1 ϩ used were described previously (40,50). The plasmid pSKctr6 ϩ was constructed by inserting a 173-bp BamHI-EcoRI fragment of the ctr6 ϩ cDNA into the same sites of pBluescript II SK. The antisense RNA hybridizes to the first 173 ribonucleotides of the ctr6 ϩ transcript. To assess the ability of the CuSEs (42) to regulate the ctr6 ϩ gene expression, the plasmid pSP1ctr6 ϩ -546lacZ containing the ctr6 ϩ promoter region up to Ϫ546 from the start codon of the ctr6 ϩ gene in addition to the Escherichia coli lacZ gene was created. The plasmid was constructed via three-piece ligation by simultaneously introducing the EcoRI-StuI fragment of YEp357R (51) and the BamHI-EcoRI fragment from the ctr6 ϩ promoter containing 546-bp of the 5Ј-noncoding region and the first 11 codons of the ctr6 ϩ gene into the BamHI-SmaI cut pSP1 vector (52). Furthermore, the plasmid pSKctr6 ϩ -546 containing nucleotides from position Ϫ546 to position ϩ33 with respect to the start codon of the ctr6 ϩ ORF was created to introduce mutations in the CuSEs (positions Ϫ210 to Ϫ201; positions Ϫ196 to Ϫ187) by site-directed mutagenesis. Precisely, the oligonucleotide 5Ј-Ϫ226 TATACCATTAGTGTACGGGGTATGAG-AGTGGGTCGATGAATATATCGTTACTTGC Ϫ172 -3Ј (letters that are underlined represent multiple point mutations in the CuSEs) was used in conjunction with pSKctr6 ϩ -546 and the Chameleon mutagenesis kit (Stratagene, La Jolla, CA). The DNA sequence of the mutant promoter was verified by dideoxy sequencing and then used to replace the equivalent wild type ctr6 ϩ promoter in pSP1ctr6 ϩ -546lacZ.
Disruption of the S. pombe ctr6 ϩ Gene-A functional ura4 ϩ cassette was isolated from pUR18 (53) by PCR. The primers were designed to create SmaI and BglII sites to the beginning and the end of the ura4 ϩ genetic marker, respectively. After digestion at these sites, the ura4 ϩ fragment was inserted to replace the ctr6 ϩ ORF, leaving 710 and 438 bp each side of the ctr6 ϩ locus for homologous recombination, creating pctr6⌬::ura4 ϩ . The gene disruption fragment (5Ј-ctr6-ura4 ϩ -ctr6-3Ј) was generated by restriction endonuclease digestion using unique flanking sites (BamHI and Asp718) and then transformed into the S. pombe FY435 strain by electroporation (54). The allele status of the disrupted locus was verified using Southern blotting and diagnostic PCR as described previously (55). The ctr6⌬ ctr4⌬ double mutant disruption strain was created as follows. The ctr6 ϩ gene inactivation was conducted as described above, except that the 710-bp BamHI-BglII fragment of the 5Ј region of ctr6 ϩ and 438-bp BamHI-KpnI fragment of the 3Ј-flanking fragment of ctr6 ϩ were inserted to the 5Ј and 3Ј ends of hisG-ura4 ϩ -hisG in plasmid pDM291 (57). Once the hisG-ura4 ϩ -hisG cassette recycled, the ctr4 ϩ locus was disrupted as described previously (40).
SOD Enzymatic Activity and Sod1 ϩ mRNA Analysis-The S. pombe isogenic strains FY435 (wild type), DBY31 (ctr6⌬), and DBY11 (ctr6⌬ ctr4⌬) were grown in yeast extract plus supplements (YES) medium. Copper treatment of yeast strains was conducted as described previously (40). Once treated, cultures were divided in half and harvested by centrifugation. One-half of cells from each culture were washed and disrupted with the lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA) in the presence of protease inhibitors. Aliquots of equal concentrations of protein extracts were analyzed for assay of SOD

Fission Yeast Vacuolar Copper Transporter Ctr6
activity by standard in-gel assay with nitro blue tetrazolium staining (40). Spectrophotometric determination of SOD activity was also performed from these protein extracts by measuring the inhibition of the reduction rate of cytochrome c by SOD, which competes for reactive oxygen species produced from the xanthine-xanthine oxidase system (56). The other half of cells of each culture was stored at Ϫ80°C until total RNA was extracted as described previously (50). For analysis of sod1 ϩ gene expression by Northern blot, a 545-bp genomic DNA fragment from S. pombe FY435 was isolated by PCR using primers that corresponded to the start and stop codons of the sod1 ϩ gene. The PCR product purification, 32 P-labeling, and hybridization were performed as described above. 64 Cu Uptake Measurements-S. pombe cells were grown to midlogarithmic phase prior to uptake experiments. At A 600nm of ϳ1.0, cells were harvested and washed twice with citrate buffer (50 mM sodium citrate, pH 6.5, 5% glucose) as described previously (17). Radioactive copper (250 Ci/g of 64 Cu in the form of 64 CuCl 2 in 0.1 M HCl) was produced at the 64 Cu production facility at the Sherbrooke PET Center. 64 CuCl 2 was added to 2 ml of cells to a final concentration of 2 M, and cultures were incubated for 10 min either at 30 or 0°C. Uptake of 64 Cu was terminated by adding ice-cold EDTA (10 mM in PBS). Samples were collected by suction through nitrocellulose membrane filters (0.45 m) loaded onto a 1225 Sampling Manifold (Millipore, Bedford, MA). After filtration, the cells were washed with 25 ml of ice-cold PBS, pH 7.4, air-dried, and then counted using a ␥-counter (Canberra-Packard Cobra II). Counts obtained at 0°C were subtracted from the values at 30°C to give net uptake values. Furthermore, the values were normalized to culture density as described previously (39).
Ctr6 Epitope Tagging-The plasmid pSKctr6 ϩ -StuI-BspEI carries a 15-bp StuI-BspEI linker inserted in-frame within the ctr6 ϩ gene at position ϩ208 relative to the first nucleotide of the initiator codon. The linker was introduced by the overlap extension method as described by Ho et al. (58). The insertion generated 5 extra amino acids after the arginine residue at position 70 (Arg 70 -Pro-Asp-Tyr-Thr-Ser) within a predicted hydrophilic loop that is located immediately after the first transmembrane domain of Ctr6. We used the restriction sites StuI and BspEI created within ctr6 ϩ to swap the linker region with four copies of the Haemophilus influenzae HA epitope (59). To generate the four copies of the HA epitope, a short DNA region of pCTR3-C-HA 3 /315 (20) harboring three copies of the HA tag was isolated by PCR using primers that contained StuI and BspEI restriction sites. The fragment was digested and cloned into pSKctr6 ϩ -StuI-BspEI vector. This plasmid was digested with StuI, and a fourth copy of the HA epitope was subcloned into the StuI site. The ctr6 ϩ -HA 4 fusion allele was verified by sequencing, and the HA 4 epitope-tagged Ctr6 protein was judged to be fully functional because of its ability to mobilize intracellular copper stores. To create the ctr6-M1 allele, the primers CTR6MUT1-A (5Ј-CGGAAT-TCATGAATCACGGCGGTAATTCTACGGCGCGAGCCTGTTCAATG-AAGATG-3Ј) and CTR6TAA (5Ј-CGCGGATCCGTTAATGGCATAATC-CTACAGTTTGAACAG-3Ј) were made corresponding to the beginning and the end of the ctr6 ϩ gene with mutations (underlined) in the sequence that generated the Met-9Ala, His-11Ala substitutions at the amino-terminal region of Ctr6. For the ctr6-M2 and ctr6-M3 alleles, a similar approach was used, except that the substitutions C12A, M14A, M16A, M9A, H11A, C12A, M14A, and M16A were created, respectively, as specified in Fig. 8.
Protein Extraction and Immunoblotting-For Ctr6-HA 4 detection, ϳ5 A 600 units of ctr6⌬ mutant strain harboring the indicated expression plasmid were spheroplasted as described by Pasion and Forsburg (60), except that the cell wall was digested with 0.8 mg of Zymolyase 20T (Seikagaku, Tokyo, Japan) and 80 units of Glusulase (PerkinElmer Life Sciences) per ml of Spheroplasting buffer (50 mM Tris-HCl, pH 7.4, 1 M sorbitol, 1 mM dithiothreitol, 1 mM 2-mercaptoethanol). Spheroplasts were resuspended in 0.6 ml of HEGN 100 buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 10% glycerol, 100 mM NaCl) supplemented with 1 mM phenylmethylsulfonyl fluoride, 8 g/ml aprotinin, 4 g/ml pepstatin, and 2 g/ml leupeptin (Sigma). Spheroplasts were lysed by three rounds of freezing in a nitrogen liquid bath and rapid thawing at 30°C. Lysates were centrifuged at 100,000 ϫ g for 30 min at 4°C. The pellet fraction was resuspended in 0.6 ml of buffer A (1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, and the above-mentioned protease inhibitors in PBS, pH 7.4) and incubated on ice for 30 min. Solubilized Ctr6-HA 4 was enriched by immunoprecipitation using 2 g of monoclonal antibodies against HA (F-7) (Santa Cruz Biotechnology, Santa Cruz, CA) bound to the protein A-Sepharose CL-4B (Amersham Biosciences). After incubation at 4°C on a rotating wheel for 2 h, immunoprecipitated complexes were washed three times with buffer B containing 1 mM EDTA and 0.5% Triton X-100 in PBS (pH 7.4) and three times with PBS (pH 7.4). Once washed, the attached complexes were resuspended in 4ϫ SDS loading buffer containing 4.0 M urea and then dissociated from the beads by heating them to 85°C. Samples were analyzed by immunoblotting with anti-HA (F-7), horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) and developed with enhanced chemiluminescent detection reagents. For protein expression analysis of Ctr4-FLAG 2 , GST-Ptc4, and PCNA, the following antisera were used for immunodetection: monoclonal anti-FLAG antibody M2 (Sigma); polyclonal anti-GST antibody Z-5 (Santa Cruz Biotechnology, Santa Cruz, CA); and monoclonal anti-PCNA antibody PC10 (Sigma).
Fractionation and EGS Cross-linking-Cells were spheroplasted and lysed as described above. Membrane fractions were isolated by centrifugation at 100,000 ϫ g. The supernatant that contained soluble proteins were precipitated in 10% trichloroacetic acid and washed with acetone before separation on SDS-PAGE. Membrane fractions (pellets) were untreated or treated with 0.2 M Na 2 CO 3 (pH 11) or 1% Triton X-100 for 30 min on ice and re-fractionated at 100,000 ϫ g for 2 h as described previously (39). The Ctr4-FLAG 2 fusion protein (39) and PCNA (61) were used as control. For in vitro cross-linking experiments, Triton X-100-treated fractions were incubated with increasing concentrations of EGS (Pierce) as described previously (19). The cross-linked complexes were immunoprecipitated as described above and analyzed by SDS-PAGE under denaturing conditions and immunoblotting but employing, this time, a polyclonal goat anti-HA (Y-11) to counterblot the immunoprecipitated material.
Indirect Immunofluorescence Microscopy-For localization of Ctr6-HA 4 , ctr6⌬ mutant cells were transformed with the plasmid pctr6 ϩ -HA 4 , which expresses from its own promoter a functional HA 4 epitopetagged Ctr6 protein. Transformed cells were grown to early log phase. After no treatment or incubation in the presence of either CuSO 4 (100 M) or BCS (100 M) for 9 h as described previously (47), the cells were fixed by adding formaldehyde (methanol-free) (Polysciences, Warrington, PA) to 3.7%. Fixed cells were harvested and washed with 0.1 M potassium phosphate, pH 6.5, containing 1.2 M sorbitol. Cells were spheroplasted as the above-described procedure and adsorbed to polylysine-coated multiwell slides. After a 30-min block with TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% bovine serum albumin), cells were incubated with anti-HA antibody (F-7) and anti-GST antibody (Z-5) diluted 1:200 in TBS. After a 2-h reaction, cells were washed with TBS and incubated for 1 h with the appropriate secondary antibodies as follows: goat anti-mouse Alexa-Red conjugate or goat anti-rabbit Alexa-Green conjugate (Molecular Probes, Eugene, OR) both diluted 1:500 in TBS. After cells were washed, mounting solution containing 4,6-diamino-2-phenylindole (DAPI) was added to each well. The cells were observed with an Olympus BX60 epifluorescent microscope (Olympus America, Melville, NY). To localize GST-Ptc4, S. pombe cells were co-transformed with pctr6 ϩ -HA 4 and pDS473aGST-ptc4 ϩ . To generate this latter plasmid, the ptc4 ϩ gene was isolated by PCR from S. pombe FY435 genomic DNA using primers that corresponded to the start and stop regions of the ptc4 ϩ ORF (62). The PCR product obtained was digested with BamHI and SmaI and cloned into the corresponding sites of pDS473a vector (63).

Ctr6 Is a Putative Member of the Ctr Transporter Family-
Protein data base searches from the S. pombe Genome Project (64) revealed an ORF (SPBC23G7.16) encoding a putative copper transporter related to the Ctr transporter family (1). This was supported by the following observations. First, the amino terminus of this putative transporter harbors a Met-X-His-Cys-X-Met motif (residues 9 -14) that contains only one difference, which is a cysteine (fourth position) instead of a methionine residue to be identical to the Met-X 2 -Met-X-Met motifs identified in the Ctr transporter family as potential copper ion-binding motifs (1). Second, like the Ctr4 (40) and Ctr5 (39) proteins that form a two-component copper transporting complex at the cell surface of S. pombe, the SPBC23G7.16-encoded protein contains three transmembrane regions according to TOP-PRED II analysis (65). Third, the overall sequence homology with the S. pombe Ctr4 (32% identity in 140-amino acid overlap) and Ctr5 (27% identity in 138-amino acid overlap) proteins was noteworthy, especially within the putative transmembrane spanning domains.

Fission Yeast Vacuolar Copper Transporter Ctr6
Thus, we termed the locus encoding this novel and uncharacterized polypeptide, ctr6 ϩ (Fig. 1). Among the known Ctr family members from yeast and mammals, Ctr6 displays the highest sequence identity with the S. cerevisiae Ctr2 protein (37), exhibiting 37% identity in 126-amino acid overlap. Although the Ctr2 protein from bakers' yeast has been localized in the vacuolar membrane and may participate in the mobilization of intracellular pools of copper ions (38), its precise function in copper homeostasis has not yet been ascertained. Taken together, these observations suggest that ctr6 ϩ encodes a new member of the Ctr transporter family, and for that reason the gene was isolated for further analysis.
Copper-specific Transcriptional Repression of the ctr6 ϩ Gene-Based on these structural features of Ctr6, we ascertained whether the ctr6 ϩ gene was transcriptionally regulated by copper availability via the Cuf1 copper-sensing transcription factor. As shown in Fig. 2A, the S. pombe ctr6 ϩ mRNA expression in a wild type strain was repressed (ϳ6-fold) when cells were exposed to 100 M CuSO 4 and derepressed (ϳ3-fold) as compared with the basal levels when cells were grown in the presence of the copper chelator BCS. Furthermore, using isogenic strains harboring a wild type cuf1 ϩ gene and an insertionally inactivated cuf1 allele, we found that the copper-dependent regulation of ctr6 ϩ mRNA required the copper sensor Cuf1 (Fig. 2B). Indeed, in the absence of Cuf1, although a low level of ctr6 ϩ mRNA was still observed, its expression was clearly unregulated by cellular copper status. Interestingly, within the ctr6 ϩ promoter region up to Ϫ546 from the start codon of the ctr6 ϩ ORF, two copies of a repeated sequence, 5Ј-D(T/A)DDHGCTGD-3Ј (D ϭ A, G or T; H ϭ A, C, or T), termed CuSE (42), were found at positions Ϫ210 to Ϫ201 and Ϫ196 to Ϫ187. It is important to note that Cuf1 factor directly interacts with CuSEs to mediate transcriptional copper regulation of the ctr4 ϩ and ctr5 ϩ genes, which encode high affinity copper transport proteins in fission yeast. To ascertain if the CuSEs play a role in ctr6 ϩ regulation by copper, we fused 546-bp of the 5Ј-noncoding region and the first 11 codons of ctr6 ϩ in-frame with the E. coli lacZ gene. ctr6 ϩ -lacZ expression from the reporter plasmid was down-regulated in the presence of copper (ϳ7-fold) and up-regulated in the presence of BCS (ϳ3-fold) (Fig. 3). When we inserted multiple point mutations that mimic changes known to abolish binding of Cuf1 to CuSEs in both elements within the ctr6 ϩ promoter, a low and constitutive basal level of expression was observed (Fig. 3). In fact, there was a complete lack of either down-or up-regulation of the ctr6 ϩ -lacZ fusion. Taken together, these data show that

Fission Yeast Vacuolar Copper Transporter Ctr6
ctr6 ϩ is regulated at the transcriptional level through CuSEs in a copper-and Cuf1-dependent manner.
Effects of Deletion and Overexpression of ctr6 ϩ on Cell Growth and Regulation of Cell Surface Copper Transporter-To understand the role of Ctr6, we inactivated the ctr6 ϩ locus by deletion and replacement with the S. pombe ura4 ϩ gene. Whereas the ctr6⌬ mutant cells exhibited no obvious defect to use respiratory carbon sources (e.g. glycerol) or to grow on medium containing an iron chelator (e.g. BPS, ferrozine) (data not shown), the copper,zinc-SOD1 activity in ctr6⌬ cells was strongly diminished as compared with wild type cells (Fig. 4, A and B). As observed for Ctr6, deletion of the ctr4 ϩ gene (ctr4⌬) dramatically lowered copper,zinc-SOD1 activity, whereas the ctr6⌬ ctr4⌬ double disruptant was devoid of measurable activity (Fig. 4, A and B). In all cases, loss of endogenous SOD1 activity was repaired to ϳ40 -85% that of the wild type starting strain by the addition of exogenous copper (Fig. 4, A and B). Importantly, under low basal copper conditions (Fig. 4, C and D, untreated (Ϫ)), the sod1 ϩ transcript levels remained virtually unchanged and clearly visible in all isogenic strains used, whereas under the same conditions, inactivation of the ctr6 ϩ and ctr4 ϩ genes resulted in an ϳ6and ϳ19-fold reduction in SOD activity, respectively (Fig. 4B). These data clearly suggest a physiological and post-translational function for Ctr6 and Ctr4 in copper delivery to copper-,zinc-SOD1. Although in S. pombe the sod1 ϩ mRNA expression was found to increase ϳ2-fold with the addition of exogenous CuSO 4 to the growth medium, the effect of the disruptions (ctr6⌬, ctr4⌬, and ctr6⌬ ctr4⌬) should be mainly considered under low copper conditions, because copper transport proteins become critical for cell function only under these conditions. Furthermore, the fact that the reduction in SOD activity in ctr6⌬, ctr4⌬, and ctr6⌬ ctr4⌬ strains is largely reversed by addition of exogenous copper clearly implicates Ctr6 and Ctr4 in copper metabolism.
Interestingly, when the ctr6ϩ gene was overexpressed from the thiamine-inducible nmt1 ϩ promoter, the cells were hypersensitive to copper and unable to grow on medium containing 100 M CuSO 4 (Fig. 5A). Furthermore, this phenotype appeared to be highly copper-specific because among 10 different metal ions, CuSO 4 , AgNO 3 , HgCl 2 , CdCl 2 , FeCl 3 , NH4Fe(SO) 4 , CoCl 2 , Pb(C2H 3 O 2 ), MnCl 2 , and ZnCl 2 , tested at many concentrations, only copper and silver, a metal that is electronically similar to the reduced form of Cu 2ϩ , gave rise to that growth defect ( Fig. 5A and data not shown). To ascertain whether this copper toxicity phenotype resulting from ctr6 ϩ overexpression was due to an increase of copper uptake, we measured 64 Cu transport. Surprisingly, as shown in Fig.  5B, activation of the ctr6 ϩ gene resulted in an ϳ60 -70% reduction in the high affinity 64 Cu transport. Consistently, in the ctr6⌬ strain overexpressing the wild type ctr6 ϩ gene, the steady-state levels of the ctr4 ϩ mRNA were strongly diminished (ϳ12-fold) as compared with the levels observed in the same strain (ctr6⌬) harboring either the plasmid alone or a mutated ctr6 allele (Fig. 5C). This diminution of the ctr4 ϩ steady-state mRNA levels was particularly striking under

Fission Yeast Vacuolar Copper Transporter Ctr6
copper-limiting conditions (Fig. 5C, medium under copperlimiting conditions due to the presence of the copper chelator BCS, Fig. 5B). These data may suggest a copper redistribution within the cell, perhaps because of a release of copper from intracellular organelle(s). To confirm that the copper toxicity phenotype was linked with the overexpression of ctr6ϩ, a mutant version of the gene was created. Precisely, site-directed mutagenesis was used to convert the methionine (Met-9) and histidine (His-11) codons to that encoding alanine. Although the Ctr6-M1 mutant localized properly (Fig. 8), the copper toxicity phenotype was lost, indicating that the phenotype was specifically associated with the presence of the wild type Ctr6 protein when overproduced.
Subcellular Location of Ctr6 -To begin to ascertain the mechanism by which Ctr6 functions in copper mobilization in S. pombe, we conducted experiments to determine the Ctr6 subcellular location. Ctr6 was tagged by inserting four tandem repeats of the HA epitope within a predicted hydrophilic loop region located between the first and second transmembrane domains of the protein. A ctr6⌬ mutant strain transformed with a plasmid harboring the ctr6 ϩ -HA 4 gene gave rise to the above-mentioned phenotypes (Fig. 5) as observed for the wild type ctr6 ϩ gene, indicating that the Ctr6-HA 4 protein is functional. This strain was grown without treatment or was incubated in the presence of either CuSO 4 (100 M) or BCS (100 M). Protein extracts were prepared, and the Ctr6-HA 4 fusion protein was enriched by immunoprecipitation using equal amounts of protein extract with anti-HA F-7 antibody. Immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting. As shown in Fig. 6A, a polypeptide species of ϳ22 kDa was detected, in keeping with the expected size of Ctr6-HA 4 fusion protein (21.4 kDa) as predicted by its primary DNA sequence. Consistent with the regulation of ctr6 ϩ mRNA steady-state levels, the Ctr6-HA 4 protein levels were dramatically reduced in cells grown in the presence of 100 M CuSO 4 (Fig. 6A).
The primary sequence of Ctr6 predicted that this protein was integrated into a cellular membrane. To investigate this, ctr6 ϩ -HA 4 and ctr4 ϩ -FLAG 2 (39) fusion genes were co-transformed and expressed in a ctr6⌬ ctr4⌬ double mutant disruption strain. Whole cell extracts, prepared from cells grown under conditions of low copper availability, were subjected to ultracentrifugation at 100,000 ϫ g to collect membranes. The supernatant that contains soluble and detached peripheral membrane proteins was precipitated, washed with acetone, resuspended, and left untreated before analysis by Western blotting. The pellet fractions were resuspended and left untreated, or were adjusted to 0.2 M Na 2 CO 3 or 1% Triton X-100, and then re-fractionated at 100,000 ϫ g. As shown in Fig. 6B, in the absence of treatment, or in the presence of Na 2 CO 3 , which dissociates peripheral but not integral membrane proteins from the membrane, Ctr6-HA 4 and Ctr4-FLAG 2 proteins were not detected into the supernatant fractions but only in the pellet fractions. Conversely, the PCNA protein, which is soluble, was only found into the supernatant fraction. In the presence of Triton X-100, a nonionic detergent that solubilizes membranes, both Ctr6-HA 4 and Ctr4-FLAG 2 proteins were detected in the pellet and supernatant fractions, implying that Ctr6-HA 4 is an integral membrane protein as shown previously for Ctr4-FLAG 2 (39) and reproduced here as a control.
To determine the cellular location of Ctr6-HA 4 , indirect immunofluorescence microscopy was carried out using anti-HA antibody. When S. pombe ctr6⌬ cells expressing the Ctr6-HA 4 fusion protein were grown under copper starvation conditions, Ctr6-HA 4 fluorescence appeared to localize in vacuole membranes (Fig. 7A). These organelles around which Ctr6-HA 4 was detected appear as indentations by Nomarski optics (Fig. 7A,  DIC). Conveniently, when ctr6 ϩ -HA 4 was induced by copper removal, the number and size of the vacuoles decreased and became bigger, respectively, as a consequence of nutrient limitation (data not shown) (62), facilitating the Ctr6-HA 4 localization. Importantly, the fluorescence was absent when ctr6⌬ mutant cells expressing the Ctr6-HA 4 fusion protein were grown under copper-replete conditions (100 M CuSO 4 ) (Fig. 7A). Furthermore, no fluorescence was observed in cells expressing the untagged ctr6 ϩ allele (data not shown). To further confirm the FIG. 4. ctr6⌬ cells exhibit low levels of SOD activity. A, an S. pombe strain bearing a disrupted ctr6⌬ allele displayed a strong reduction of copper,zinc-SOD1 activity, as demonstrated by a representative in-gel activity assay. As observed for Ctr6, deletion of the ctr4 ϩ gene (ctr4⌬) dramatically lowered copper,zinc-SOD1 activity, whereas the ctr6⌬ ctr4⌬ double disruptant was devoid of measurable activity. In all cases, loss of endogenous SOD1 activity was repaired nearly to that found in the wild type parental strain by the addition of exogenous CuSO 4 (100 M). B, SOD activity determined from isogenic wild type, ctr6⌬, ctr4⌬, or ctr6⌬ ctr4⌬ double mutant strains by using a cytochrome c/xanthine oxidase method. The values of SOD activities are the means of three replicates Ϯ S.D. C, shown is a representative RNA blot assay of sod1 ϩ and act1 ϩ (as control) mRNA steady-state levels. Total RNA was prepared from aliquots of the same cell cultures used for assay of SOD activity. D, values are the averages of triplicate determinations Ϯ S.D.

Fission Yeast Vacuolar Copper Transporter Ctr6
Ctr6-HA 4 localization, cells were co-transformed with plasmids expressing both the Ctr6-HA 4 and GST-Ptc4 fusion proteins. The use of GST-Ptc4 fusion protein, which is known to localize in the vacuolar membrane (62), served as a positive control. As shown in Fig. 7B, double immunofluorescence labeling carried out with anti-HA and anti-GST antibodies revealed that Ctr6-HA 4 and GST-Ptc4 proteins were both visualized at the vacuolar membrane. Taken together, the copper-mediated repression of Ctr6-HA 4 protein levels, the integral membrane nature of Ctr6-HA 4 , and the vacuolar membrane staining are suggestive of a mechanism whereby Ctr6 provides copper ions from the vacuole to cytosolic copper-requiring enzyme(s) when cells are grown under copper starvation conditions.
Identification of Amino-terminal Residues Necessary for Ctr6 Function-To gain insight into the mechanisms by which Ctr6 transport copper ions, we carried out a functional dissection of a potential metal-binding motif, Met-X-His-Cys-X-Met-X-Met (residues 9 -16), within the amino-terminal region of Ctr6. Although a cysteine was found (fourth position) instead of a methionine to be identical to the Met-X 2 -Met-X-Met motif identified in the Ctr transporter family as potential copper-binding motif, the chemical nature of cysteine with an external SH group may replace the methionine to coordinate copper. Recently, an elegant study (16) has demonstrated that a conserved methionine located 20 amino acid residues from the beginning of the first transmembrane domain in S. cerevisiae Ctr1 protein is essential for copper transport. Analogous to the situation described for Ctr1, the last methionine of the putative Met motif of Ctr6 was found 18 amino acid residues from the first transmembrane domain. Because of these observations, site-directed mutagenesis was used to convert codons encoding residues, which have the potential to bind copper, to codons encoding alanine (Fig. 8A). To assess the effects of these mutations on Ctr6 function, plasmids expressing the mutant proteins shown in Fig. 8A were transformed into an S. pombe ctr6⌬ strain. As controls, subcellular localization was performed to ensure that the mutant proteins were produced and properly localized (Fig. 8B). For each mutant, we measured 64 Cu uptake. As shown in Fig. 8C, all three Ctr6 mutant proteins failed to diminish high affinity 64 Cu transport as compared with the reduction observed in the same strain (ctr6⌬) expressing wild type Ctr6. Furthermore, despite the fact that these mutant alleles were overexpressed in the ctr6⌬ strain, the steady-state levels of ctr4 ϩ mRNA were still robustly induced under copper deprivation conditions as opposed to the ctr4 ϩ mRNA levels detected in the ctr6⌬ mutant strain overexpressing the wild type ctr6 ϩ allele (Fig. 8D). These results suggest that the methionine (Met-9, Met-14, and Met-16), histidine (His-11), and cysteine (Cys-12) residues, which compose the copper-binding motif, Met-X-His-Cys-X-Met-X-Met (residues 9 -16), within the amino-terminal region of Ctr6 are involved in the process of copper transport mediated by Ctr6. However, whether these Absorption of 64 CuCl 2 was carried out at 30°C, and the values were corrected with respect to culture density and temperature (i.e. uptake at 30°C subtracted by uptake at 0°C). Results are the mean of triplicate samples. WT, wild type strain FY435 (ctr6 ϩ ) was used as control. p, plasmid alone. C, RNase protection assay from aliquots of cultures grown to midlogarithmic phase for copper uptake measurements. Cells were incubated in the absence (Ϫ) or presence of CuSO 4 (1 and 100 M), or BCS (100 M). After total RNA extraction, the ctr4 ϩ steady-state mRNA levels were analyzed. Results illustrated are representative of three independent experiments.

Fission Yeast Vacuolar Copper Transporter Ctr6
residues equivalently contribute in copper transport must await a comprehensive dissection of the copper-binding motif.
Ctr6 Assembles into an Oligomeric Complex-On the basis of hydropathy profiling, the majority of membrane transport proteins are thought to contain 6 Ϯ 3 and up to 12 Ϯ 2 transmembrane domains (1,19,67). Because three, four, or six transmembrane domains are probably insufficient to form a translocation path, these transport proteins may form oligomeric complexes (67). To examine the possibility that Ctr6 adopts an oligomeric conformation, the Ctr6-HA 4 fusion protein was expressed in a ctr6⌬ deletion strain under copper starvation conditions. Triton X-100-solubilized membrane protein fractions prepared from these cells were incubated with increasing concentrations of EGS. This cross-linker reacts predominantly with the ⑀-amine group of lysine residues, which are predicted to be accessible for such reaction in Ctr6. 2 Once cross-linked, the tagged Ctr6 molecules were immunoprecipitated with anti-HA F7 antibody, and immunoprecipitates were resolved by SDS-PAGE and then revealed by immunoblotting, but employing this time a polyclonal goat anti-HA (Y-11) to counterblot the immunoprecipitated material. In the absence of EGS, we noted that Ctr6-HA 4 migrates as an ϳ22-kDa monomeric protein (Fig. 9), which is consistent with its predicted molecular mass of 21.4 kDa. As the EGS concentration was increased, the monomeric form of Ctr6-HA 4 protein disappeared, with concomitant appearance of homodimeric (ϳ44 kDa) and homotrimeric (ϳ66 kDa) forms of Ctr6-HA 4 . Although only a very low level of Ctr6-HA 4 homodimer was detectable, the Ctr6-HA 4 homotrimer was clearly visible (Fig. 9). Taken together, these results strongly suggest that the Ctr6-HA 4 protein forms a homotrimer as part of a copper transporter unit in the vacuolar membrane in fission yeast. DISCUSSION Because of their property to promptly gain and lose electrons, copper ions are redox-active co-factors that serve as catalytic centers of numerous proteins involved in a variety of essential enzymatic processes (2,68). Despite this crucial role, copper ions, when present in excess, can have detrimental effects due to their proclivity to engage in redox reactions or by competing with other metal ions for enzyme-active sites (4,69). Thus, distinct pathways have evolved for the signaling, transport, trafficking, and sequestration of copper ions within cells to keep the delicate balance between essential and toxic levels (70).
In this study, we identified a novel S. pombe copper-responsive gene, termed ctr6 ϩ , which encodes a vacuolar membrane transporter. Like the ctr4ϩ and ctr5ϩ genes encoding the high affinity copper heteromeric transport complex at the cell surface in S. pombe (39), ctr6ϩ is activated at the transcriptional level in response to copper limitation by the Cuf1 nutritional copper-sensing transcription factor through the CuSE recognition sequence. Based on this observation that ctr6 ϩ is transcriptionally regulated by copper in the same direction as the genes encoding components of the high affinity copper uptake machinery suggests a function for Ctr6 in copper utilization as opposed to copper detoxification. Given the fact that genetic studies have implicated the vacuole as playing a role for copper storage (38,71,72), and assuming that vacuolar copper is present in a usable form, we envision Ctr6 as an intracellular transporter to mobilize stores of copper from the organelle, thereby representing a specialized pathway by which copper could be distributed within cells (Fig. 10). The proposed model is supported by the fact that a deletion of the ctr6 ϩ gene (ctr6⌬) results in a significant reduction of copper,zinc-SOD1 activity, suggesting a role for S. pombe Ctr6 in delivering copper to cytosolic copper-dependent enzyme(s) under conditions of copper scarcity. Furthermore, when Ctr6 was overexpressed from the thiamine-inducible nmt1 ϩ promoter, the cells exhibited a copper-sensitive growth phenotype, which was not attributable to an increase of copper uptake. Consistently, in response to the action of Ctr6, there was loss of Cuf1-dependent activation of the cell surface copper transporter ctr4 ϩ gene expression, which represents an additional argument indicating the increased of copper cellular levels within the cell.
Because some membrane proteins that function in the secretory pathway (e.g. endoplasmic reticulum/Golgi apparatus) or at the plasma membrane may be mis-localized to the vacuole when overexpressed (73), for subcellular localization of Ctr6, we used a functional epitope-tagged ctr6 ϩ allele that was expressed under the control of its own promoter. This latter system ensured low levels of ctr6 ϩ gene expression. Moreover, localization of Ctr6 to the vacuolar membrane was also observed using the disruption strain (ctr6⌬) in which a ctr6 ϩ -HA 4 allele was re-integrated. 2 To examine further the localization of Ctr6, we compared the labeling pattern of Ctr6-HA 4 to the GST-Ptc4 fusion protein known to function in the vacuolar membrane (62). Indirect immunofluorescence microscopy dem-  4 and grown in a similar manner to that described in A but with only the addition of BCS (100 M). Equivalent amounts of total lysate (Total) and supernatant (S) or pellet (P) fractions were loaded. Pellet fraction obtained from cell lysates was either untreated or incubated in the presence of 0.1 M Na 2 CO 3 at pH 11, or adjust to 1% Triton X-100, and centrifuged at 100,000 ϫ g for 30 min before analysis by immunoblotting. The positions of the Ctr6-HA 4 , Ctr4-FLAG 2 , and PCNA proteins are indicated with arrows.

Fission Yeast Vacuolar Copper Transporter Ctr6
onstrated that Ctr6 localizes on the membrane of vacuoles in a manner identical to that observed for GST-Ptc4. Interestingly, similar subcellular localization has been reported recently (38) for the S. cerevisiae Ctr2 protein. Analogous to the situation for Ctr2, Ctr6 was visualized surrounding the vacuole with points of concentration of the protein around the organelle. This observation further supports a possible common role for these two proteins in the mobilization and transmission of intracellular pools of copper to metalloenzymes.
Complementary to immunofluorescence localization, subcellular fractionation experiments demonstrate that Ctr6 is an integral membrane protein that is undetectable in soluble fractions, unless cell extracts were supplemented with Triton X-100, a detergent that solubilizes membrane structures (19). As demonstrated for the S. cerevisiae Ctr3 (19) and human Ctr1 (74), EGS cross-linking experiments revealed that Ctr6 can assemble as a trimer. Importantly, the homo-multimeric state of Ctr6 may be required to form a functional translocation path, which contains, in general, 6 Ϯ 3 and up to 12 Ϯ 2 transmembrane domains within transport proteins (67). Nine transmembrane domains from three Ctr6 molecules could be sufficient to form a pore by which copper can be translocated from the vacuole into the cytoplasm. The oligomeric state may also play a role in other functions of Ctr6, including its stabilization into the membrane structure, or interaction with the cytosolic domain of delivering copper proteins.
Based on computer algorithm analysis, the amino-terminal 33 amino acids of Ctr6 are predicted to be inside the vacuole. Within this region of Ctr6 lies a putative copper coordination motif, Met-X-His-Cys-X-Met-X-Met (residues 9 -16), that may function in copper capture within the vacuole. This is supported by the observation that mutations in which the methionine (Met-9) and histidine (His-11) or cysteine (Cys-12) and methionines (Met-14 and Met-16), or all five of these residues, were substituted to alanine altered copper transport activity of Ctr6. The methionine and histidine residues at positions 9 and 11, respectively, when mutated (mutant M1), gave rise to a stronger alteration with respect to Ctr6 activity compared with the mutant M2 in which the cysteine and methionine residues at position 12, 14, and 16 were mutated. However, whether one residue contributes more than another one in copper transport must await a fine mapping dissection of each amino acid that could play a role in the handling of copper. Similarly to the situation for the S. cerevisiae Ctr1 and Ctr3, and human Ctr1 (16), Ctr6 contains in its second transmembrane domain a conserved Met-X 3 -Met motif (residues 111-115). Although we have not ascertained its function, this Met-X 3 -Met motif may play a critical function in copper translocation across the vacuolar membrane.
In the presence of excess iron or copper ions, the vacuole has been proposed to play an important role to detoxify the cell, preventing their accumulation in the cytosol to toxic levels. Once inside the vacuole, perhaps, these metal ions could be bound under a bio-unavailable form as Fe 3ϩ /Cu 2ϩ to polyphosphates or other molecules. Conversely, when grown under copper starvation conditions, Ctr6 would mobilize stored copper from the vacuole to replenish the cytosol according to copper need. The similarity in the potency of silver in fostering the FIG. 7. Ctr6 localizes to the vacuolar membrane. A, ctr6⌬ deletion strain expressing Ctr6-HA 4 was grown to early logarithmic phase in Edinburgh minimal medium and incubated in the absence (Ϫ) or presence of BCS (100 M) or CuSO 4 (100 M). Cells were fixed, permeabilized, and labeled with anti-HA monoclonal antibody (Ab). DAPI staining visualized DNA and Nomarski microscopy was used to determine cell morphology. The indentations seen by Nomarski (DIC) represent the vacuoles. B, the vacuolar S. pombe Ptc4 (fused to GST without loss of function) (62) was expressed and viewed as a control.

Fission Yeast Vacuolar Copper Transporter Ctr6
copper-sensitive growth phenotype because of the expression of ctr6 ϩ and the electronic similarity of Ag ϩ to Cu ϩ , but not Cu 2ϩ , suggest that the intracellular copper transporter Ctr6 may pump Cu ϩ rather than Cu 2ϩ . This would suggest a role for a vacuolar membrane metalloreductase. So far, analysis of genomic DNA sequences from the S. pombe Genome project has revealed two open reading frames (SPBC1683.09C, denoted frp1ϩ, and SPBC947.05C) related to Cu 2ϩ /Fe 3ϩ ion reductases found in S. cerevisiae. Although the frp1ϩ-encoded reductase can reduce Fe 3ϩ to Fe 2ϩ at the cell surface of fission yeast, its role in the metabolism of other metal ions (e.g. copper) is unknown. Regarding the second ORF, SPBC947.05C, its potential role in Fe 3ϩ /Cu 2ϩ reductase activity is still uncharacterized. Finally, given the extended amino acid sequence homology between Ctr6 and all Ctr family members, especially within the regions that encompass the transmembrane do- FIG. 9. Ctr6 multimerizes. Representative EGS cross-linking experiment of Triton X-100-solubilized cell lysates prepared from ctr6⌬ cells expressing Ctr6-HA 4 . After incubations with 0, 0.5, 1.0, 2.5, 3.0, and 5.0 mM EGS for 30 min at room temperature, the cross-linked complexes were immunoprecipitated, separated on 9% SDS-PAGE, and detected by immunoblotting. Monomeric (ϳ22-kDa, 1 oval), dimeric (ϳ44-kDa, 2 ovals), and trimeric (ϳ66-kDa, 3 ovals) forms of Ctr6 were detected. M, reference marker.

FIG. 8. The amino-terminal Met-X-
His-Cys-X-Met-X-Met motif is necessary for the copper transport activity of Ctr6. A, schematic representation of the Ctr6 protein tagged with four copies of the HA epitope. The primary sequence of the Met-X-His-Cys-X-Met-X-Met motif is shown below, and the putative copperbinding ligands are underlined. TM1-3, putative transmembrane domains. The amino acid numbers refer to the position relative to the first amino acid of the protein. The sequence of the mutations (M1, M2, and M3) in the Met-X-His-Cys-X-Met-X-Met motif are shown corresponding to the residues in the wild type Ctr6. B, representative cells from M1, M2, and M3 mutants of Ctr6. Cells from cultures grown in the presence of BCS (100 M) were fixed, probed for the HA epitope, and viewed by epifluorescence. DAPI staining was used to determine the location of the nucleus. Shown are matched images of anti-HA-GAM-Alexa Red fluorescence and DAPI merged images. C, ctr6⌬ cells expressing the wild type ctr6 ϩ -HA 4 gene display a distinct 64 Cu uptake rate to that observed with cells expressing ctr6-M1-HA 4 , ctr6-M2-HA 4 , and ctr6-M3-HA 4 alleles. Cells were incubated with 2 M 64 Cu in citrate buffer (pH 6.5) (17) for 10 min. Copper uptake was quantitated and normalized to culture density and temperature-dependent transport. Error bars represent the S.D. for three independent experiments. D, ctr6⌬ strain, transformed with pctr6 ϩ -HA 4 , pctr6-M1-HA 4 , pctr6-M2-HA 4 , and pctr6-M3-HA 4 , was grown under low copper conditions. Cultures were untreated or treated with CuSO 4 (100 M) or BCS (100 M) for 1 h. Total RNA was prepared from culture aliquots. ctr4 ϩ and act1 ϩ mRNAs (arrows) were detected using RNase protection assays. Results shown are representative of three independent experiments.

Fission Yeast Vacuolar Copper Transporter Ctr6
mains, it will be interesting to determine what motif of the intracellular copper transporter Ctr6 is required for sorting the molecule to the vacuolar membrane, whereas the other members of the family, except for the S. cerevisiae Ctr2, are sorted to the plasma membrane. FIG. 10. Effect of Ctr6 activity on copper transport. Ctr6 assembles as homotrimer in the vacuolar membrane to mobilize stored copper out of the organelle. Because of the activity of Ctr6 that put extra copper into the cytoplasm/nucleus, Cuf1 copper-sensing transcription factor is inactivated by the intracellular pool of labile copper, preventing futile expression of the ctr4 ϩ and ctr5 ϩ cell surface transport genes.