THE JOURNAL OF BIOWGICAL Val. No. 41, Issue of ... Saccharomyces cereuisiae Copper Transport Protein (Ctrlp) BIOCHEMICAL CHARACTERIZATION, REGULATION BY COPPER, AND PHYSIOLOGIC ROLE

THE JOURNAL OF BIOWGICAL CHEMISTRY Val. 269, No. 41, Issue of October 14, pp. 25660-25667, 1994 Printed in W.S.A.

The Saccharomyces cereuisiae Copper Transport Protein (Ctrlp) BIOCHEMICAL CHARACTERIZATION, REGULATION BY COPPER, AND PHYSIOLOGIC ROLE IN COPPER UPTAKE*

(Received for publication, July 12, 1994)

Andrew Dancis, David Haile, Daniel S. Yuan, and Richard D. Klausnert From the Cell Biology and Metabolism Branch, NICHHD, National Institutes of Health, Bethesda, Maryland 20892

The CTRl gene of Saccharomyces cereuisiae encodes a protein required for high affinity copper uptake. The protein is expressed on the plasma membrane, is heavily glycosylated with 0-linkages, and exists as an oligomer in vivo. The transcript abundance is strongly regulated by copper availability, being induced by copper deprivation and repressed by copper excess. Regulation occurs at very low, nontoxic levels of available copper and is independent of ACEl, the trans-inducer of yeast metallothionein. Expression of Ctrlp is limiting for copper uptake, since overexpression from a 2p high copy number plasmid increases copper uptake. Mutations in CTRl result in altered cellular responses to extracellular copper, demonstrating a physiologic role for CTRl in the delivery of copper to the cytosol. A copper-dependent reporter gene construct, CUPl-lacZ, is not expressed in CTRl mutants to the same level as in wild-type strains, and Cu,Zn superoxide dismutase activity is deficient in these mutants. The growth arrest that occurs in CTRl mutants grown aerobically in copper-deficient media is attributable to the defect in Cu,Zn superoxide dismutase activity.

Copper is an essential nutrient required as a cofactor in a variety of proteins (Linder, 1991). Despite its importance, rela- tively little is understood about the molecular details of how organisms acquire this trace metal from the environment. We have recently identified a gene called CTRl that encodes a protein required for high affinity copper uptake in Saccharo- myces cerevisiae (Dancis et al., 1994) (Table I). The CTRl gene encodes a multispanning plasma membrane protein of 406 amino acids. An unusual methionine- and serine-rich amino- terminal domain of Ctrlp contains multiple repeats of a motif found in prokaryotic proteins involved in the handling of copper. The identification of CTRl revealed an unexpected connection between the uptake of copper and the ability of cells to accumulate ferrous iron. In fact, CTRl was initially identified by a genetic selection for mutants defective in iron uptake. A strict requirement for copper uptake was noted in order to permit high affinity ferrous iron uptake (Dancis et al., 1994; Askwith et al., 1994) through a specific, though unidentified, ferrous transporter (Eide et al., 1992). The FET3 gene product most likely explains this copper requirement. Mutations in FET3, like mutations in CTRl , abolish high affinity ferrous iron uptake. FET3 encodes a transmembrane protein with a domain containing homology to copper-containing mixed func-

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tion oxidases, such as ascorbate oxidase, laccase, and cerulo- plasmin (Askwith et al., 1994). CTRl mutants appear to be defective in the uptake of both copper and iron, while FET3 mutants are defective only in iron uptake. The deficiency of ferrous iron uptake in the CTRl mutants but not the FET3 mutants is corrected by growth in the presence of high concentrations of copper. Thus, in our current view, copper transport, either through the high affinity system requiring expression of CTRl or through a separate low affinity system, supplies Fet3p with copper, which in turn is required for ferrous iron uptake. A further connection between the uptake of these two metals in S. cerevisiae is provided by the FREl gene (Dancis et al., 1990, 1992). FREl encodes a plasma membrane reductase that is required for the reduction of iron(II1) to iron(II), which can then be transported into the cell. The expression of FREl is nega- tively regulated by both iron and copper (Lesuisse and Labbe, 1994), thus providing an additional point in the iron uptake pathway that can be influenced by copper.

In addition to this functional connection between the uptake of copper and of iron, these two metals present similar prob- lems for all cells. Both are essential nutrients and yet are highly toxic. The toxicity of these metals may relate to their ability to participate in reactions that generate free radicals from molecular oxygen (Halliwell and Gutteridge, 1988). Other mechanisms of toxicity are also likely, including direct membrane damage by copper (Oshumi et al., 1988) and misincorpo- ration of copper into metalloproteins (Zhou and Thiele, 1993). In order to maintain the cellular concentrations of these metals at levels adequate for their physiologic targets while minimiz- ing the toxicity of metal excess, regulated homeostatic controls have evolved. These controls may act at two levels: metal uptake and metal detoxificatiodsequestration (O’Halloran, 1993). The process of copper detoxification in eukaryotes has been characterized. This process is mediated by metallothionein, a cysteine-rich cytoplasmic protein that sequesters intracellular copper, thereby lowering free copper to less toxic levels (Hamer, 1986). In S. cereuisiae, expression of the CUPl gene, encoding metallothionein, is induced when the growth medium contains high concentrations of copper. This regulation occurs via the action of a copper-binding transcription factor, ACEl , that in- teracts with specific cis-acting elements in the promoter of the CUPl gene (Thiele, 1988).

Regulation of copper uptake could also provide a level of homeostatic control for cellular copper levels. By analogy with iron uptake systems operating in bacteria (Nielands et al., 1987) and complex eukaryotes (Klausner et al., 19931, if the Ctrlp represents the rate-limiting step in the delivery of copper to the cytosol, we would expect it to be regulated by copper availability. Our initial study of the CTRl gene demonstrated that it was required for high affinity copper accumulation and indirectly for copper-dependent ferrous iron accumulation (Dancis et al., 1994). Thus, CTRl probably encodes a eukaryotic copper uptake transport protein, which to date has not

25660

The S. cerevisiae Copper Dansport Protein (Ctrlp) 25661 TABLE I

Genes to which reference is made in this paper

Gene Encodes Required for Ref.

CTRl Copper transporter Copper, ferrous transport Dancis et al. (1994) FET3 Multifunction oxidase Ferrous transport Askwith et al. (1994) FREl External reductase Ferric iron uptake Dancis et al. (1990) ACEl Transcription factor Transactivation of CUP1 Thiele (1988)

SOD1 Cu.Zn suDeroxide dismutase Superoxide detoxification Gralla and Kosman (1992) CUP1 Metallothionein Copper detoxification Hamer (1986)

-

been identified in other species. In this paper we extend our understanding of this unique protein by: 1) beginning its biochemical characterization, 2) demonstrating the homeostatic regulation of the CTRl transcript by copper via a mechanism independent of ACEl, and 3) providing evidence that the delivery of copper to the cytosol is, in fact, mediated by the expression of CTRl.

MATERIALS AND METHODS

Yeast Strains The strains used in this paper are listed in Table I1 with their geno-

types and source. The following strains were generous gifts from other laboratories. Strain RSYl2 (sec53-6) was provided by Randy Schek- man. Strains Dm22 and DTY26 were provided by Dean Hamer. Strains DEY1397-6A and DY150 were provided by Jerry Kaplan and David Eide.

Growth Conditions For growth of yeast under nonselective conditions, yeast extract-

peptone-dextrose medium (YPD) was used. When moderate copper or iron starvation was required, a modified defined medium was made from components of SD medium (Sherman et al., 19891, omitting iron and copper and adding MES buffer (50 mM, pH 6.2). To achieve more severe copper starvation, the copper chelator bathocuproinedisulfonate (BCS,I Fluka) was added to the above medium. For growth of yeast on a solid matrix, agarose was used rather than agar. To achieve anaerobic growth conditions, yeast strains were incubated in the presence of 0.1% Tween 80 and 30 pg/ml ergosterol in a chamber rendered anaerobic using a BBL GasPak (Becton Dickinson). Copper starvation for induction of CTRl expression was accomplished by inoculation into SD modified as noted above at a density of OD,,, = 0.2 with 10 p~ BCS followed by growth for 4-6 h. For induction of invertase expression under the control of the pH05 promoter, cells were grown in phosphate-depleted medium as described (Kramer et al., 1984).

Plasmids and DNA Constructions InterruptionlDeZetion of CTRI-The construct FTRUN was used as

described (Dancis et al., 1994) when selection for uracil prototrophy was possible. Alternatively, the gamma strategy (Sikorski and Hieter, 1989) was used to insert a LEU2-containing vector at the CTRl locus. CTRl genomic fragments between HindIII and XhoI and between EagI and HindIII were cloned into the polylinker of pRS405 (Stratagene, La Jolla, CA). The plasmid was linearized at the unique HindIII site and transformants were selected for leucine prototrophy. In each case the genomic locus was examined by Southern blot to verify the correct integration.

Epitope Tagging of CTRI-An AfZII-PstI genomic fragment of CTRl was cloned into the SmaI and PstI sites of a high copy number vector (YEp352) which was modified by destruction of the EcoRI site, and this construct was termed 2pCTR1. The construct was further modified by insertion of sequences encoding a myc epitope at a unique EcoRI site creating the plasmid 352-myc as has been described (Dancis et al., 1994). Similarly, complementary oligonucleotides encoding the 9-amino acid HA epitope from the influenza hemagglutinin protein (Kolodziej and Young, 1991) were inserted at the EcoRI site of 2pCTR1, creating 352-HA. The insert from 352-HA was transferred to a low copy number LEU2-containing vector by removing the 2.8-kilobase insert of 352-HA with PstI and Sac1 and inserting this fragment into the corresponding sites of pRS415. This construct was termed 702-HA.

P-Galactosidase Reporters-The plasmid YEp24 (Schneider and Guarente, 1991) was modified by insertion into the BamHI site of se-

TEMED, N,N,N',N'-tetramethylethylenediamine; PAGE, polyacrylam- The abbreviations used are: BCS, bathocuproinedisulfonate;

ide gel electrophoresis.

quences encoding the bacterial gene for p-galactosidase (ZacZ) beginning at codon 8 and flanked by a BamHI site at the 5' end and a BglII site at the 3' end. This construct was called YEGal4. The 5'-flanking region of CTRl between nucleotides -893 and +3 was amplified by polymerase chain reaction (Innis and Gelfand, 1990) from the genomic clone pdl (Dancis et al., 1994) and inserted into the SalI-BamHI sites 5' of the lac2 sequences of YEGal4, creating plasmid CTR-lac2 (Table 111). This generated a fusion of the CTRl promoter region and ATG start codon with codon 8 of the bacterial P-galactosidase gene. The sequence of the fusion was determined by dideoxynucleotide sequencing to be: attcaaATGGATCCCGTC, with the CTRl flanking sequences represented by lower case letters, the ATG start codon represented by italics, and the lac2 coding represented by upper case letters. The fusion of the CYCl upstream activating sequences with the P-galactosidase gene has been described (Guarente and F'tashne, 1981).

Invertase Expression Construct-pPHO-INV consists of a fusion of the pH05 promoter and invertase coding region on a high copy number plasmid obtained from Juan Bonifacino.

Biochemical Procedures Cell Lysates-Cells were washed once with lysis buffer (25 mM Tris-

C1, pH 7.5, 150 mM NaC1,l mM EDTA, 0.5 mM aminoethylbenzylsulfonyl fluoride, 10 pg/ml leupeptin, 1 pg/ml pepstatin) and disrupted by vor- texing in the presence of glass beads. Triton X-100 was added to a final concentration of 1%, and cells were incubated for 10 min prior to centrifugation at 15,000 x g for 15 min. The protein content of the lysate was determined by assay with bicinchoninic acid (Pierce Chemical Co.).

Immunoprecipitations-Cell lysates with freshly added protease in- hibitors were incubated for 1 h on ice with specific antibody. The antibodies used were the monoclonals 9E10 directed against the myc epitope (Evan et al., 1985), 12CA5 directed against the HA epitope (Wilson et al., 19841, or a polyclonal antibody directed against yeast invertase obtained from Juan Bonifacino. Protein A-Sepharose was added, and the lysate was tumbled for 1 h at 4 "C. The beads were washed with lysis buffer with 1% Triton X-100 prior to boiling for 10 min in Laemmli buffer and P-mercaptoethanol.

Immunoblotting-Cell lysates were boiled under reducing conditions in Laemmli buffer prior to separation on a 6 2 0 % gradient SDS-polyacrylamide gel and electrophoretic transfer to nitrocellulose. The blots were incubated with 9E10 supernatant a t 1:30 dilution or 12CA5 asci- tes at 1:lOOO dilution and visualized using an alkaline phosphatase conjugate substrate kit (Bio-Rad).

Metabolic Labeling-Cultures were incubated in modified SD lacking iron or copper for 20 min with 150 pCi/ml Tran3%-label (1000 Ci/ mmol; ICN Radiochemicals). Cells were lysed in the standard manner; an amount of lysate containing 1.6 million cpm precipitable by 10% trichloroacetic acid was immunoprecipitated with 12CA5 prior to separation by SDS-polyacrylamide electrophoresis and visualization by fluorography.

Copper Uptake Radionuclide copper uptake was performed as described previously

(Dancis et al., 1994). The radionuclide 64Cu (1.9-2.2 mCi/mg of copper, DuPont NEN) was used at 10 p~ concentration in citrate-containing assay buffer (5% glucose, 50 mM sodium citrate, pH 6.5). The yeast cells were grown to early logarithmic growth phase prior to assay, and uptake was measured as temperature-dependent uptake, i.e. as uptake at 30 "C subtracted by uptake at 0 "C.

Assays

as described (Miller, 1972). P-Galactosidase-Activity was assayed using buffers and substrate

Superoxide Dismutase-Lysates were prepared and a quantity equiv- alent to 20 pg of protein was separated on a nondenaturing polyacrylamide gel. The gel was developed as has been described (Beauchamp and Fridovich, 1971) by incubating first with 2 mg/ml nitro blue tetrazolium in 50 mM potassium phosphate buffer, pH 7.0, followed by incu-

25662 The S. cerevisiae Copper Dunsport Protein (Ctrlp) TABLE I1

Yeast strains used in this study Strain name Source Genotype

CM3262 61 - MATa trpl-63 leu2-3, 112gcn4-IO1 his3-609 ura3-52 FREl-HIS3::URAJ 64 FTRUNBl 66 (2pCTRl) This study MATa trpl-63 leu2-3, 112 gcn4-101 his3-609 ura3-52 FREI-HIS3:LEU2 65 This study DEY1397-6A

MATa trpl-63 leu2-3, 112 gcn4-101 his3-609 ura3-52 FREl-HIS3::URA3 Afrel:ZEU2

DY150 DTY22 DTY22Actrl This study MATa his6 ura3-52 YIPCL::LEU2 ACEl Actr1::URAJ DTY26 - MATa his6 ura3-52 LEU2::YIpCL Aace1::URAS RSY12 - I MATa ura3-52 leu2-3, 112 sec53-6

Dancis et al. (1994).

D - MATa inol-13 leu2-3, 112 gcn4-101 his3-609 ura3-52

- MATa inol-13 leu2-3, 112 gcn4-101 his3-609 ura3-52 Actr1::URAJ

- MATa ura3-52 leu2-3, 112 trpl-1 his3-11, 15 ade2-1 canl-100 fet3:flIS3 - MATa ura3-52 leu2-3, 112 trpl-1 his3-11, 15 ade2-1 cad-100 - MATa his6 ura3-52 YIPCL:AEU2 ACEl

This study MATa trpl-63 leu2-3, 112gcn4-101 his3-609 ura3-52 FREl-HIS3::URA3 Actrl:ZEU2

h

h

Askwith et al. (1994). Yang et al. (1991). Kepes and Schekman (1988).

TABLE 111 Copper regulation of CTR-lac2 fusion

Transformants of strain 66 were grown in modified SD without uracil and without copper (-Cu) or with 10 p~ added copper (+Cu) for 18 h prior to preparation of cell lysates and measurement of P-galactosidase. Values are normalized to protein concentration and incubation time (Miller, 1972) and are means of triplicate determination with standard deviations shown in parentheses.

Plasmid /3-Galactosidase activity

-cu +cu -Fold change

units CTR-lac2 10,320 (179) 424 (48) 24 pLG699-Z 772 (24) 801 (18) 0.96

bation with 63 p~ riboflavin and 300 mM TEMED in 36 mM potassium phosphate buffer and exposing the gel to light. Superoxide dismutase activity was visualized as areas lacking the color change indicative of tetrazolium reduction. The Cu,Zn form of superoxide dismutase activity was distinguished by its sensitivity to cyanide at pH 9 (Chang et al., 1991). The negative image was converted to a positive image by placing the gel on film and flashing using a camera flash.

RNA Blot Hybridization Total RNA was isolated from cells growing in logarithmic phase. 10

pgAane was separated on a formaldehyde gel, blotted to nylon mem- branes, and hybridized to a 32P-labeled DNA fragment, following standard protocols (Sambrook et al., 1989). The probe for evaluation of the CTRl transcript was derived from the XhoI-EcoRI in the genomic sequence. The actin probe was derived from pUCACT, a plasmid into which coding sequences of the yeast ACT1 gene were subcloned.

RESULTS Biochemical Characterization of Ctrlp-A CTRl genomic

fragment was engineered to add an epitope tag derived from either c-myc (Evan et al . , 1985) or the influenza hemagglutinin ( H A ) protein (Wilson et al . , 1984) to the carboxyl-terminal domain of the protein. The tagged Ctrlp complemented the mutant phenotype of a CTRl deletion strain. The tagged protein was localized to the plasma membrane by immunofluorescence and immunoelectron microscopy (Dancis et al., 19941, consistent with its putative role in mediating copper acquisition from the medium. We then used the epitope-tagged Ctrlp to inves- tigate some of the biochemical properties of the protein. On immunoblots of cell lysates, we noted two specific bands, migrating on SDS-PAGE with an apparent molecular mass between 100 and 105 kDa (Fig. lA). These were significantly larger than the molecular mass of 46 kDa predicted from the amino acid sequence. There was no change in this gel mobility as a function of sample reduction or temperature treatment prior to loading of the gel, suggesting that the aberrant migra- tion was not due to incomplete reduction or denaturation. To assess the possibility that carbohydrate addition might be re-

97 -

46-

A B C 1 2 3 4 5 6 7

II " . " FIG. 1. Glycosylation of Ctr lp . A , immunoblotting of the epitope-

tagged Ctrlp. Cell lysates from strain CM3262 transformed with plasmid 352-myc (lane I ) or 352-HA (lane 2 ) and induced for Ctrlp expression were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and visualized with a mouse monoclonal anti-HA antibody and a goat anti-mouse alkaline phosphatase conju-

RSYl2 (sec53-6) transformed with the epitope-tagged CTRl on plasmid gate. B, glycosylation of Ctrlp. The temperature-sensitive mutant

352-HA and induced for Ctrlp expression was subjected to metabolic labeling at the permissive temperature of 24 "C (lane 3 ) or at the restrictive temperature of 37 "C (lane 4 ) prior to bead lysis of the cells, immunoprecipitation from the lysate with anti-HA antibody, separation

N-linked glycosylation of invertase. Strain CM3262 transformed with on SDS-polyacrylamide gel, and analysis by fluorography. C, evidence of

plasmid pPHO-INV was induced by phosphate starvation and subjected to metabolic labeling in the absence (lanes 5 and 7) or presence (lane 6 ) of tunicamycin. Cell lysates were prepared, and one of the samples (lane 7 ) was treated with Endo H prior to immunoprecipitation with anti- invertase antibody, polyacrylamide gel separation, and fluorography.

sponsible for this decreased mobility, we expressed the epitope- tagged protein in the temperature-sensitive sec53 mutant (Kepes and Schekman, 1988). At nonpermissive temperatures, this mutant does not produce the lipid-linked precursors of either N- or 0-linked oligosaccharides. The epitope-tagged protein expressed in sec53 mutant cells was metabolically labeled at permissive or restrictive temperatures. When lysates from the permissively labeled cells were immunoprecipitated with antibody directed against the HAepitope and analyzed by SDS- PAGE, a major band migrating at approximately 100 kDa was found (Fig. lB). On shorter exposures this signal was seen to be composed of a doublet similar to the one seen on the immuno- blot. At the restrictive temperature, a band of approximately 49 kDa was seen, indicating that protein labeled at the permissive temperature was glycosylated (Fig. lB). Incubation with tunicamycin during the labeling, or digestion with Endo H, did not affect the electrophoretic mobility of Ctrlp (not shown), indicating that the added oligosaccharides are most likely in the form of 0-linked mannose residues (Orlean et al . , 1991). An

The S. cerevisiae Copper Dansport Protein (Ctrlp) 25663

1 2 3 4

Ctrlp - CTRl pHA f + -

CTRl p-myc tttti - FIG. 2. Oligomerization of C t r l p . Strain CM3262 was transformed

with both HA-tagged Ctrlp (702-HA) and myc-tagged Ctrlp (352-myc) (lanes 1 and 3 ) or individually with the HA-tagged construct (lane 2 ) or the myc-tagged construct (lane 4 ) . The transformants were grown in order to induce Ctrlp expression, and lysates were immunoprecipitated with anti-HA (lanes 1 and 2 ) or anti-myc (lanes 3 and 4) antibody. The immunoprecipitates were separated on a 4-20'70 acrylamide gel, blotted to nitrocellulose, and visualized with the anti-myc antibody (lanes 1 and 2 ) or anti-HA antibody (lanes 3 and 4). The doublet derived from the Ctrlp protein is indicated by an arrow; an arrow identified by Ig indi- cates the position of the immunoglobulin heavy chain from the immunoprecipitating antibodies.

invertase control was markedly altered by either treatment (Fig. 1C). The high proportion of serine and threonine residues (>40%) in the first third of this membrane protein is, in fact, typical of proteins bearing 0-linked sugars (Kuranda and Robbins, 1991). The marked SDS-PAGE mobility shift pro- duced by the see53 mutation suggests the possibility of a large number of such linkages.

Oligomeric Nature of Ctrlp-The primary sequence of CTRl, while predicting a transmembrane protein capable of travers- ing the membrane three times (Dancis et al., 1994), does not otherwise resemble any known protein and, in particular, does not resemble any known transmembrane transporter. A wide array of membrane transport proteins have now been characterized (Serrano, 1991). While these transporters fall into dif- ferent classes, they generally include between 6 and 12, or sometimes more, transmembrane domains. This led us to won- der whether Ctrpl existed as an oligomer. Oligomerization of Ctrlp was first assessed by expressing the gene from two dif- ferent plasmids. One plasmid encoded the protein modified by the addition of the HA epitope, and the other plasmid carried Ctrlp with a myc epitope. Lysate from a strain transformed with both plasmids was analyzed by immunoprecipitation using an antibody directed against HA, resolution of the immu- noprecipitate by SDS-PAGE, transfer to nitrocellulose, and visualization by immunoblotting with an antibody directed against the myc epitope (Fig. 2, lane 1 ). The converse experi- ment was also performed by immunoprecipitating with the antibody against myc and immunoblotting with an antibody against HA (Fig. 2, lane 3). In both cases, the specific doublet between 100 and 105 kDa was observed, signifying that molecules carrying the myc tag assembled with molecules carrying the HA tag, thereby allowing co-precipitation. Co-transforma- tion with the myc- and HA-containing plasmids was required in order to observe Ctrlp self-assembly (Fig. 2, lanes 2 and 4) . In addition, sucrose density gradient centrifugation demonstrated that the Ctrlp complex migrates with an apparent size consistent with a dimer (not shown). These experiments demonstrate that the protein exists in vivo as at least a homodimer.

Control of CTRl Expression by Copper-If CTRl mediates the uptake of copper, its activity might be homeostatically controlled to respond to copper deprivation and copper overload. CTRl transcript levels did exhibit regulation in response to available copper. Furthermore, the direction of this regulation was as would be predicted for such a homeostatic system, in that copper deprivation induced CTRl expression and copper

A ACEl ace1 "

1 2 3 4 5 6

I' 'I- CTR1

ACTl

CTRl ctrl FET3 fet3

- + - + - + - + +10pMCu 1 2 3 4 5 6 7 8

CTR 1

ACTl

independence ofACEl expression. Strain DTY22 ( A C E l ) was grown in FIG. 3. Copper-dependent regulation of the CTRl transcript.A,

YPD in the presence of a 100 p~ concentration of the copper chelator bathocuproinedisulfonate (lane 1 ), with no addition (lane 2 1 or with 10 PM added copper sulfate (lane 3 ) . Strain Dm26 (Aacel::URA3) was grown under the same conditions (lanes 4, 5, and 6). Total RNA was extracted, and 10 pg was separated on a formaldehyde gel, blotted, and probed with a coding fragment from the CTRl gene or from the ACTl gene. Separate gels were run for analysis with each of the probes. B, induction by copper deprivation, not by iron deprivation. Strains were grown in YF'D medium without (lanes 1 , 3 , 5 , and 7) or with 10 PM added copper sulfate (lanes 2,4,6, and 8 ) prior to isolation of RNA. The RNA was analyzed as in A . The strains analyzed were related strains 61

DEY1397-6A (fet3xHIS3). ( C T R l ) and M10 (ctrl-IO), and related strains DY150 VET31 and

. '""1 80

I DTY22 f /

r I

04 .01 .I 1 10 100 1000

COPPER ADDED (pM)

FIG. 4. CUP1-lac2 regulation. Strain DTY22 containing an inte- grated CUPl-lac2 fusion was modified by intenuptionldeletion of CTRZ to make DTY22Actrl. The strains were grown for 4 h in YPD with varying amounts of added copper, and lysates were prepared for assay of P-galactosidase. Values were normalized to the lysate protein concentration and the time of incubation.

excess repressed CTRl expression (Fig. 3A). That this regulation was most likely exerted at the transcriptional level was demonstrated by utilizing a fusion of 893 bases of the 5'-flanking region of the CTRl gene with the coding region of P-galactosidase. This CTR-lac2 construct conferred copper-dependent regulation on the P-galactosidase reporter as contrasted with the CYCl-lac2 fusion pLG699Z (Guarente and Ptashne, 1981) which was unaffected by manipulation of medium copper concentrations (Table 111).

The ACEl protein, which mediates the regulated expression of CUP1 (Thiele, 1988, is a well-characterized mediator of copper-dependent gene transcription. We therefore examined whether the homeostatic regulation of CTRl depends on ACEl. To this end, mRNA was isolated from a congenic pair of strains, one of which carried an inactivated copy of ACEl (Yang et al.,

25664 The S. cerevisiae Copper Dansport Protein (Ctrlp) FIG. 5. Phenotypes result ing h m

various CTRI Eenotypes. Culture plates were poured containing modified SD medium with 39 p~ hathocupminedi- sulfonate Clef/ side) , no copper addition (middlcp), or 870 p~ copper sulfate IrIght side I . A patch of a dilute inoculum of each of a panel of strains was spread onto the plates and allowed to grow for 48 h prior to photographing. The strains included the parental strain 61 (CTRI ), strain 64 (Actr l ) , strain 65 (Afrel) , and strain 66 (2pCTRl ).

1991). This mutation had no apparent effect on CTRI transcript levels or their regulation by copper availability (Fig. 3A ). Thus, a distinct regulator of copper-dependent gene expression must exist.

The initial characterization of mutants of CTRI revealed defects in both copper uptake and ferrous iron uptake. We therefore wondered whether cells deprived of iron might also induce CTRl expression. In order to achieve iron deprivation without manipulation of the media conditions, we elected to study a FET.? mutant. Such a mutant possesses a selective defect in high afinity ferrous iron uptake (Askwith et al., 1994) but no defect in copper uptake (not shown). When a FET3 mutant strain, DEY1397-6AiA, was grown in the presence of 10 pv added copper, CTRI expression was repressed (Fig. 3B, lane 8). The degree of repression of the CTRI transcript by copper in the FET3 mutant was complete, as was observed for the matched parental strain DYl.50 (compare lane 6 and lane 8 of Fig. 3B ). Thus, repression of CTRl expression is mediated pri- marily by copper rather than by iron.

Effrcts of Interruption /Deletion or Overexpression of the CTRl Gene on Copper Uptake, Toxicity, and Deprivation- Expression of Ctrlp correlated with measurements of cell-associated radioactive copper. Interruptioddeletion strains mani- fested a deficiency of copper accumulation (Dancis et al., 1994). On the other hand, the expression of Ct r lp from a high copy number plasmid 2pCTR1 resulted in an increased rate of radioactive copper accumulation (13 pmoVIOfi cell.s/h compared with 7 pmol/lOfi ce1ls.h). Such measurements are likely to reflect the combined effects of copper binding to the cell wall and uptake to the cytosol (Lin et al., 1993). In order to obtain more direct evidence that the radioactive copper accumulation represents true delivery to the interior of the cell, we asked whether an intracellular biosensor of copper would report effects of altered expression of CTRI. A fusion of CUPI to the bacterial lacZ reporter (Yang et al., 1991) was used to evaluate the copper-dependent induction of CUPI in a wild-type and CTRI-deleted strain. Addition of graded concentrations of copper to the growth medium resulted in a marked increase in 0-galactosidase activity derived from the CUP1-lacZ fusion inserted into the wild-type strain ( D W Z Z ) , while the congenic strain carrying a deleted CTRI allele (DTY22Actrl) exhibited no such increment (Fig. 4). Since CUP1 induction is mediated through the ability of the ACE1 gene product to respond to intracellular copper levels, the implication of these result.. is that CTRl expression is affecting intracellular copper levels.

Interestingly, CUPI expression was not induced in the CTRI deletion strain, even a t high medium copper concentrations. These concentrations (100 p11) exceed theK,,, of the high afinity uptake system (1-4 PM) dependent upon Ctrlp (Dancis et al., 1994). One prediction of these data is that it would be dificult to deliver toxic levels of copper to cells carrying a CTRI deletion. This could manifest itself as resistance to the growth inhibitory effects of high concentrations of the metal in the growth medium. Copper toxicity was therefore evaluated by examining the strain 64 (Actr l ) , the wild-type 61 (parent), and

"

33uM BCS 0 370yM Cu

*O 1

0 ,001 .01 .1 1 10 100 1000

COPPER ADDED (pM)

FIG. 6. Copper requirement for growth result ing from Actrl mutation. Strain 61 (CTRI ) or related strain FTRUSBl ( A c t r l ) were diluted to a density of OD, 0.001 in modified SI) medium without (circles) or with the addition of 10 p~ femc chloride (squares). Copper sulfate was added at the indicated concentrations. The cell numhw of the cultures was determined after 14 h of growth.

the overexpressing strain (2pCTRl) for the effects of copper- mediated growth inhibition and killing. The Actrl strain was most resistant to toxicity (see right-hand plate labeled 370 pu in Fig. 5). The parental strain 61 was somewhat less resistant, and the overexpressing strain, 2pCTR1, was the most sensitive to copper toxicity (not shown). Thus, the CTRI-dependent copper uptake system mediates the eficient delivery of copper to the cells, even a t high external copper concentrations.

The requirement for copper as a nutrient suggested that the loss of CTRI function could make the cells particularly suscep- tible to copper deprivation and allow us to readily evaluate the manifestations of copper deficiency. In contrast to wild-type cells, a congenic Actrl strain did not grow in defined medium without the addition of copper (Fig. 6). Although copper uptake via Ctrlp is required for high afinity iron uptake (Dancis et al . , 1994), iron addition was unable to rescue the CTRl deletion strain from its growth arrest (Fig. 6). Restriction of growth of a Actrl strain on low copper solid medium was also seen (see central plate labeled 0, for no added copper, Fig. 5). This obser- vation was extended by comparing the growth of the wild-type strain, 61 (parent), with a related strain carrying CTRI on a high copy number plasmid (2pCTRl). When these strains were spread as a dilute inoculum on plates containing 33 pu bathocuproinedisulfonate (BCS) to lower free copper concentrations beyond the levels that can be achieved by omitting copper addition to the medium, strain 61 (parent) exhibited markedly retarded growth. In contrast, the cells overexpressing Ctrlp from a high copy number plasmid (2pCTRl) were resistant to this level of copper limitation (see left plate labeled 33 pw BCS in Fig. 5). Addition of 20 p~ copper to a control plate containing 33 pu BCS restored growth to the test strains, enabling them to form colonies of similar size (not shown). These results together suggest that CTRI gene expression is a major deter-

The S. cerevisiae Copper Dansport Protein (Ctrlp) 25665

A

61 (CTR1)

64(Actrl)

Oxygen

Copper

B Gl(CTR1)

64(Actrl)

+ + - - - + - +

Methionine - + - +

Lysine - - + + FIG. 7. Characteristics of arrested growth of Actrl strain sub-

jected to copper deprivation. A, oxygen dependence of growth arrest resulting from copper deprivation in a Artrl mutant. Strains 61 (CTRI) or 64 (Actrl ) were plated on defined medium plates with minimal aux- otrophic markers, Tween 80, ergosterol, and bathocuproinedisulfonate (10 p~). For copper addition, 10 p~ copper sulfate was added, and for anaerobic growth an anaerobic chamber was used with a gas pack (BBL). Plates were incubated 48 h prior to photographing. B, amino acid additions bypass copper requirement for growth of a Actrl mutant. Defined medium plates prepared as in A were incubated aerobically with added methionine (20 mglliter) and/or lysine (30 mg/liter). Amino acid stocks were pretreated with Chelex-100 resin to ensure that they were free of contaminating metals. Strains and plating were as in A.

minant of both copper-dependent growth a t low external copper concentrations and copper-inhibited growth at high external copper concentrations.

The FREl gene encodes an externally directed reductase important for ferric iron uptake (Dancis et al., 1990). This reductase activity is also capable of reducing copper (Lesuisse and Labbe, 1994), thus raising the question of whether it might play a role in copper uptake. However, a Afrel strain, 65, was indistinguishable from the wild-type strain in assays of copper- dependent growth or toxicity, suggesting that the role of this gene in copper uptake is minimal (Fig. 5).

Superoxide Dismutase and the Phenotypes of Copper Depri- vation-The mechanism by which copper deprivation leads to arrested growth of Actrl strains was investigated further. The deficiency of high affinity ferrous iron uptake exhibited by these strains does not account for the observed growth defect, as demonstrated by two observations: the addition of iron to the medium did not restore growth in a Actrl strain (Fig. 6), and a congenic FET3 mutant strain with a selective defect in ferrous uptake grew well under these conditions (not shown). Several of the growth characteristics of the mutants of CTRl resembled mutants of SODl, the gene encoding Cu,Zn superoxide dismutase. Similar to what has been described for SODl mutants (Gralla and Kosman, 1992), the growth arrest in CTRl mutants occurred only during aerobic growth (Fig. 7A). Under

-cu +cu 61 64A 61 64A 1 2 3 4

Y Ur*'

FIG. 8. Activity of superoxide dismutase. Strains 61 (CTRl) and 64 (Actrl) were grown in YPD medium or YPD with 50 VM added copper sulfate for 4 h. Cell lysates were prepared, and 20 pg of protein per sample was separated on a nondenaturing gel. Superoxide dismutase activity was visualized using the nitro blue tetrazolium reduction inhibition assay. A negative image is shown.

aerobic conditions, the copper requirement for growth could be by passed by addition of methionine and lysine to the minimal medium lacking copper (Fig. 7 B ) . A similar dual requirement for methionine and lysine during aerobic growth has been described for the Asodl mutants (Galla and Kosman, 1992). For the Actrl strains grown under air, as for the Asodl strains (Liu et al., 1992), intermediates of lysine biosynthesis distal to the transaminase step (i.e. a-aminoadipic acid) could substitute for the lysine requirement while intermediates proximal to this step (i.e. a-ketoadipic acid) could not substitute (not shown). Also as described for Asodl strains (Gralla and Valentine, 1991), paraquat sensitivity in air was observed for the Actrl strains. In this assay performed in rich (YPD) medium, the concentration required for 50% growth inhibition was 15 times decreased in the Actrl compared with the wild type (0.15 versus 2.5 mM) as contrasted with 100-fold decrease reported for the Asodl strain (0.01 M versus 2.5 mM) (Gralla and Valentine, 1991). This difference may derive from either minimal residual superoxide dismutase activity in the Actrl strains under the growth conditions of the assay or the protective effects of copper deficiency on paraquat toxicity.

To more directly assess the effect of the Actrl mutation on superoxide dismutase, we assayed the enzyme by means of an activity gel. The activity was found to be absent in the Actrl strain (64) grown in rich (YPD) medium (Fig. 8), although a small amount of residual activity below the sensitivity threshold for the assay cannot be ruled out. The activity was restored by supplying high concentrations (50 PM) of copper sulfate to the mutant for 6 h (Fig. 8). This activity probably represents copper-zinc superoxide dismutase since it was cyanide-inhibited (Chang et al. 1991) (not shown) and copper-dependent (Greco et al., 1990). Thus, a Actrl strain resembles a Asodl strain with respect to its growth phenotypes and lacks measured Cu,Zn superoxide dismutase activity, suggesting that the phenotypes may derive from deficiency of this enzyme activity.

DISCUSSION

The studies presented here characterize the CTRl gene product, first identified as required for the high affinity uptake of both copper and iron in S. cerevisiae (Dancis et al., 1994). Not only was CTRl expression required for high affinity copper uptake but overexpression increased copper uptake in a wild- type strain, suggesting that CTRl expression was the limiting factor in copper uptake. Experiments presented in this paper were designed to demonstrate that Ctrlp mediates the delivery of copper to the cytosol. The results support the conclusion that the measurements of radioactive copper accumulation which

25666 The S. cerevisiae Copper Dunsport Protein (Ctrlp)

we previously observed to be dependent on Ctrlp (Dancis et al., 1994) are likely to reflect internalization, as distinguished from association with the cell exterior (Lin et al., 1993). This is supported by the shift in the concentration dependence for CUP1-lacZ expression attributable to CTRl expression. The Actrl strain grown in copper-containing media did not induce CUPl-lacZ, probably because sufficient copper did not reach Acelp in the nucleus. In addition, several phenotypes were associated with either deletion of CTRl or overexpression of the protein from a high copy number plasmid. Yeast strains with the former genotype exhibited an increased growth requirement for copper and resistance to copper toxicity. In the latter genotype, the strains exhibited the converse phenotypes of decreased growth requirement for copper and enhanced sensitivity to copper toxicity.

A copper transporter would be predicted to interact with copper and mediate its translocatiorl from the exterior of the cell across the plasma membrane to the cytoplasm. To our knowledge, this has not been demonstrated for any copper transport protein in a reconstituted system. We proposed that the multiple MWZM motifs present in the Ctrlp amino acid sequence that also appear in proteins of bacterial copper metabolism might mediate the essential interaction with copper. The heavy glycosylation of Ctrlp, most probably entirely on 0-linked residues, may also be important for interaction with copper. Alternatively, the sugar linkages might be required for the protein to assume a functional conformation. The glycosylation of Ctrlp, therefore, may have to be considered in design- ing an experimental approach to demonstrate the interaction of this protein with copper, since in vitro translation systems or bacterial expression systems would be unlikely to reconsti- tute the authentic glycosylation pattern. A feature of many transporters is the presence of numerous transmembrane domains; permeases for various nutrients have between 6 and 12 predicted transmembrane domains (Serrano, 1991). The number of transmembrane domains in the Ctrlp is most likely 3, judging from the lengths of the predicted hydrophobic amino acid stretches (Dancis et al., 1994). Data from experiments utilizing the co-expression of myc- and HA-tagged Ctrlp suggest that multimerization occurs. The total number of transmembrane domains in a multimer might then equal those in other permeases.

How a Ctrlp oligomer may bind copper outside the cell and translocate it into the cytoplasm is not clear. The energy for translocation is unlikely to be directly derived from ATP hy- drolysis, since the Ctrlp lacks consensus motifs for ATP binding (Green and Stokes, 1993). In this respect it differs from a recently identified family of P-type copper-transporting ATPases that include copA and copB from the bacterium En- terococcus hirae (Odermatt et al., 1993) and the defective genes responsible for the human disorders of Menkes disease (Vulpe et al., 1993) and Wilson disease (Bull et al., 1993). The energy for uptake could be derived from H' symport, or alternatively a second sequestering step into an internal cellular pool or or- ganelle might drive the uptake process (Kosman, 1994).

Because of the essential but toxic nature of copper, organisms have evolved means to homeostatically regulate the concentration of available copper within cells (O'Halloran, 1993). As previously characterized, in S. cerevisiae, transcription of the CUPl gene is induced through the ACE1 transcription factor when cells are exposed to toxic concentrations of copper. The increased biosynthesis of metallothionein that ensues leads to sequestration of copper in the cytoplasm and protection against damage of cellular constituents by the excess copper. Another means of copper homeostasis, analogous to that seen for iron (Eide et al., 1992), is the regulation of cellular uptake.

In fact, several characteristics of the copper-dependent regulation of the CTRl and CUPl genes suggested that they would not be mediated by the same regulator. CTRl and CUPl regulation proceed in opposite directions, with CTRl being repressed and CUPl being induced by copper. In addition, the range of copper concentrations over which the CTRl regulation occurs (<lo VM) (Fig. 3, A and B ) is less than the toxic threshold at which CUPl transcription is induced (>lo p~ in YPD). Here we demonstrate that these two homeostatic mechanisms for copper metabolism are controlled by distinct molecular path- ways. Thus, in the presence of copper depletion, CTRl is maxi- mally induced while in the presence of moderate levels of copper, CTRl expression is repressed. This regulation, which probably controls the level of high affinity copper uptake to the cell, occurs through trans-acting factor(s) distinct from Acelp.

The effects of CTRl expression on the cell could be mediated through any of a number of copper-utilizing proteins. The effects on high affinity ferrous iron uptake and iron-dependent growth have been described in a previous paper (Dancis et al., 1994) and are likely to involve Fet3p. However, the growth arrest of Actrl strains in low copper-containing medium does not seem to be related to iron deficiency, since it is not rescued by iron addition, nor does a FET3 mutant, exhibiting deficient high affinity ferrous iron uptake to a comparable degree, show such a growth arrest. The growth inhibition of CTRl mutants deprived of copper occurs only during aerobic growth and may be an indirect effect resulting from lack of Cu,Zn superoxide dismutase activity. Enzyme assay confirms the virtual absence of this activity in Actrl strains. This may relate to lack of SOD1 gene expression or failure to provide the protein with copper. Although copper regulation of this enzyme has been described previously, the degree of cellular copper starvation character- istic of the Actrl strains has heretofore not been readily achiev- able. Growth of these strains under low copper conditions re- veals a novel phenotype very similar to the phenotype of the Asodl strains. The growth arrest is oxygen-dependent and can be rescued by lysine and methionine only when they are added together (Gralla and Kosman, 1992). The lysine requirement can be substituted by aminoadipic acid but not by ketoadipic acid, consistent with a block at the transaminase step of lysine biosynthesis (Liu et al., 1992). The apparent methionine aux- otrophy appears to result from toxicity of partially reduced sulfur intermediates generated when extracellular concentrations of methionine (or cysteine) are inadequate to provide the sulfur needs of the cell (Gralla and Kosman, 1992). Finally, there is a marked decrease in the growth-inhibitory threshold for paraquat, a redox cycling drug that enhances the levels of superoxide (Gralla and Valentine, 1991). As expected, and distinct from the Asodl strains, all of the above mutant phenotypes are corrected by copper addition to the growth medium.

Further study of CTRl should provide insight into mechanisms involved in copper uptake into eukaryotic cells, loading of copper into proteins, and copper-dependent gene regulation. If copper uptake into yeast cells is mediated through this single-protein transporter, questions arise of how the copper is chemically coordinated, how it is moved through the membrane barrier, and how and in what form it is released into the cell interior. Also, one might expect that there should be homolo- gous systems in higher eukaryotes, in view of the ubiquitous need to take up copper from the environment in a regulated fashion. The ability to deprive cells of copper to the point that loading of copper proteins such as Sodlp is impaired may provide a setting in which to study the mechanisms by which these proteins are loaded with copper, addressing questions such as whether reduction is necessary, whether accessory proteins are involved, whether there are specialized compartments where

The S. cerevisiae Copper Dunsport Protein (Ctrlp) 25667

copper loading occurs, and whether apoproteins are stable. In both Menkes disease and Wilson disease, where the defective genes are thought to encode copper-transporting ATPases, the pathophysiology can be traced, in part, to the failure to load copper-containing proteins (Chelly and Monaco, 1993). Under- standing the pathophysiology of these diseases will require more detailed understanding of cellular copper homeostasis, a process that we hope will be aided by the yeast system described in this study.

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THE JOURNAL OF BIOWGICAL Val. No. 41, Issue of ... Saccharomyces cereuisiae Copper Transport Protein (Ctrlp) BIOCHEMICAL CHARACTERIZATION, REGULATION BY COPPER, AND PHYSIOLOGIC ROLE