Proc. Natl. Acad. Sci. USAVol. 92, pp. 2632-2636, March 1995Biochemistry

The Menkes/Wilson disease gene homologue in yeast providescopper to a ceruloplasmin-like oxidase required for iron uptakeDANIEL S. YUAN*, ROBERT STEARMAN*, ANDREW DANcIs*, TERESA DUNNt, TROY BEELERt,AND RicHARD D. KLAUSNER*t*Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; andtDepartment of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD 20814

Contributed by Richard D. Klausner, December 21, 1994

ABSTRACT The CCC2 gene of the yeast Saccharomycescerevisiae is homologous to the human genes -defective inWilson disease and Menkes disease. A biochemical hallmarkof these diseases is a deficiency of copper in ceruloplasminand other copper proteins found in extracytosolic compart-ments. Here we demonstrate that disruption ofthe yeast CCC2gene results in defects in respiration and iron uptake. Thesedefects could be reversed by supplementing cells with copper,suggesting that CCC2 mutant cells were copper deficient.However, cytosolic copper levels and copper uptake werenormal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolicdomain of the FET3-encoded protein, a ceruloplasmin homo-logue previously shown to be necessary for high-affinity ironuptake in yeast. Copper restored oxidase activity both in vitroand in vivo, paralleling the ability of copper to restore respi-ration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from thecytosol into an extracytosolic compartment, supporting theproposal that intracellular copper transport is impaired inWilson disease and Menkes disease.

Copper is a trace element required by most organisms. Inmammals, copper is indispensable as a cofactor in a number ofproteins, including cytochrome-c oxidase, superoxide dis-mutase, dopamine 3-hydroxylase, peptide a-amidating en-zyme, and lysyl oxidase (1). These copper-dependent enzymesare crucial to several processes of oxidative metabolism,including respiration, free-radical detoxification, neurotrans-mitter synthesis, and the maturation of connective tissue.Another important copper protein is ceruloplasmin, an abun-dant serum glycoprotein that contains most of the copper inthe circulation (1). Ceruloplasmin has a ferroxidase activitythat appears to be required for the delivery of iron into thecirculation (2), but the biochemical basis of this requirementis not understood. The loading of ceruloplasmin with copperis defective in Wilson disease (3, 4). Recently, the genesresponsible for Wilson disease and a closely related disorder,Menkes disease, were cloned and identified as putative cop-per-transporting P-type ATPases (5-10).The yeast Saccharomyces cerevisiae is an excellent model

organism for studying many fundamental processes of eukary-otic cells, due in large part to the clarity that yeast geneticsbrings to the analysis of protein function (11). Because of thefundamental importance of oxidative metabolism to eukary-otic cells, it is reasonable to expect that mechanisms of coppermetabolism may be well conserved between human cells andyeast. A plasma membrane protein required for high-affinitycopper uptake has been identified in yeast, where it is encodedby the CTR1 gene (12). CTRI mutants have several phenotypesattributable to copper deficiency, including loss of high-

affinity iron uptake (12, 13). Growth of cells in copper-supplemented medium reverses these phenotypes. Reversal ofthe iron uptake defect by copper, however, requires the FET3gene (12, 14). FET3 encodes a predicted transmembraneprotein with significant homology to copper-dependent oxi-dases such as ceruloplasmin, ascorbate oxidase, and laccase(14). These observations suggest that Fet3p is a copper proteinthat receives its copper through a mechanism involving Ctrlp.Because of the homology of Fet3p with ceruloplasmin, it isperhaps not surprising that yeast also contain a homologue ofthe human Wilson and Menkes disease gene products. Indeed,the predicted product of the yeast CCC2 gene possesses boththe motifs common to P-type ATPases and two of the copperinteraction motifs found in its mammalian counterparts (15).

In this paper we characterize copper-dependent phenotypesin cells lacking a functional CCC2 gene. We show that theCCC2 gene product plays a specific role in the copper-dependent activation of a ceruloplasmin-like oxidase activityof Fet3p, an activity that we demonstrate resides outside of thecytosol. Thus, CCC2 encodes a protein that delivers cytosoliccopper to the lumen of an internal organelle, analogous to theproposed mechanisms implicated in Wilson disease and Men-kes disease (16, 17).

MATERIALS AND METHODSYeast Strains. Strain 1 is YPH252 (MA Ta, ura3-52 lys2-801

ade2-101 trpl-Al his3-A200 1u2-A1) (18) and is congenic withstrains 2 through 6. Strain 2: the CTR1 gene was replaced withthe LEU2 gene by using the plasmid AMTR-LEU (13). Strain3: the CCC2 gene was disrupted at the BspEI site by the URA3gene by using the plasmid E5-URA3.4. Strain 4: theFET3 genewas replaced with the TRP1 gene by transforming a diploidhomozygous parent of strain 1, YPH274 (18), with a DNAfragment generated by PCR to contain 50 bp of FET3 se-quence at each end flanking the TRP1 gene (19). Sporulatedcells were dissected and tested for tryptophan prototrophy.The cells chosen were MATa. Strain 5: the CCC2 gene wasdisrupted (as above) in strain 4. Genotypes for strains 2through 5 were confirmed by PCR using primers derived fromthe inserted marker gene and flanking genomic sequences.Strain 6: a rho0 mitochondrial genotype in strain 1 was createdas described (20). Strain 7 (MATa, his3-200 leu2 trpl-101ura3-52 ade5), also called 2908, was a gift from R. Wickner(National Institutes of Health). Strain 8 was derived fromstrain 7 by deletion of CCC2 as described (15). Strain 9 is theprotease-deficient strain BJ2168 (MATa, prcl-407 prbl-1122pep4-3 leu2 trpl ura3) (21) transformed with the episomalURA3-based plasmid YEpFet3ET18 (Fet3p-HA). This plas-mid contains the FET3 gene with sequences specifying thehemagglutinin (HA) epitope (22) added to the 3' end of theFET3 coding region by overlapping PCR; it was a gift from D.

Abbreviations: HA, hemagglutinin; Endo H, endoglycosidase H.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Eide (University of Minnesota) and J. Kaplan (University ofUtah).

Identification of Candidate Genes Encoding P-TypeATPases. Degenerate oligonucleotides [GCGGAYAARACIG-GNAC (5' primer) and CCGTCRTTIATNCCRTC (3' primer)],derived from conserved motifs in P-type ATPases [DKTGT andDGIND, respectively (23)], were used in PCRs to amplifysequences from genomic DNA isolated from S. cerevisiaestrain F113 (24). Cloned DNA was analyzed by Southernblotting and sequenced. Data base searches for homologousproteins were performed by using the Swiss-Prot protein database (version 29.0) and the FASTA program (Genetics Com-puter Group, version 7) (25, 26).Growth and Iron Uptake Assays. Cells were washed in water

and placed at a dilution of 1000 cells per drop on agar mediumcontaining 2% glucose (YPD) or 3% ethanol (YE) (27), aloneor with 1 mM ferric ammonium sulfate or 0.5 mM cupricsulfate as indicated and grown at 30°C for 3 days beforephotography. High-affinity iron uptake was determined asdescribed previously (12).

13-Galactosidase Assays. For studies of copper-dependentCTRl-lacZ activity, cells were grown in microtiter plates at30°C for 14 hr in a synthetic defined medium (YNB, Bio 101)lacking iron and copper, supplemented with 50 mM hemiso-dium salt of 2-morpholinoethanesulfonic acid at pH 6.1, 10 ,Mferric ammonium sulfate, copper in the indicated concentra-tion, and media supplements to maintain selection for theCTRJ-lacZ plasmid. 13-Galactosidase was assayed in perme-abilized cells as described (13, 28).

Cell Homogenization and Fractionation. Exponentiallygrowing cells were washed and homogenized in ice-cold buffer[150 mM NaCl/25 mM Tris-HCl, pH 7.4/1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (ICN)/10 ,M pepstatinA/30 ,uM leupeptin, and additives as indicated] by mixing withglass beads. After removal of large particles, homogenateswere centrifuged (16,000 x g, 4°C) for 30 min. Pellets werewashed in buffer, resuspended in buffer containing 1% TritonX-100, and centrifuged for 30 min to yield a clarified extract.Extracts were stored at -20°C.Western Blotting. Samples were heated in Laemmli sample

buffer containing dithiothreitol and analyzed for the HAepitope by Western blotting using rabbit antiserum HA1l(Berkeley Antibody, Richmond, CA) at a dilution of 1:5000.Bound antibody was detected as enhanced chemiluminescence(Amersham).Oxidase Assays. Samples (30 gg of protein), mixed with

Laemmli sample buffer lacking dithiothreitol, were separated

GROWTH ON AGAR MEDIA

Strain Mutations YE YE/Fe YE/Cu YPD

without prior heating by SDS/PAGE. The gel was thenequilibrated with 50 vol of 0.05% Triton X-100 in 10% (wt/vol)glycerol, soaked for an equal time in 5 vol of 3 mM p-phenylenediamine dihydrochloride (Sigma) (29)/100 mM so-dium acetate, pH 5.7/1 mM NaN3, and air-dried in the dark at23°C for 24 hr before photography.

RESULTSCloning of Genes Encoding S. cerevisiae P-Type ATPases.

We used PCR to identify genes encoding P-type ATPases in S.cerevisiae, using S. cerevisiae genomic DNA as template anddegenerate oligonucleotide primers derived from two con-served amino acid motifs common to P-type ATPases (23).This approach yielded the three previously identified P-typeATPase genes in S. cerevisiae, PMR1, PMA1, and ENA1/PMR2(30), as well as a fourth gene, whose predicted productcontained two of the GMTCXXC amino acid motifs (31)found in the Wilson and Menkes disease gene products. Thisfourth gene proved to be identical to CCC2, a gene recentlyidentified as a suppressor of calcium sensitivity in CSGImutants (15, 32). Comparison of the entire CCC2-predictedamino acid sequence with a protein data base revealed that theCCC2 gene product shares significant similarity with theWilson disease and Menkes disease gene products (31%identity over 604 residues and 29% over 814 residues, respec-tively).

Disruption of the CCC2 Gene-Metal-Correctable Pheno-types. To examine the role of the CCC2 gene, a CCC2disruption mutant was compared with two congenic mutantstrains for the ability to grow on an ethanol-based medium(YE), a test of respiratory competence (27). These strains haddisruptions in either CTR1, essential for high-affinity copperuptake (12), or FET3, essential for high-affinity iron uptake(12, 14). As shown in Fig. 1, each of these mutations resultedin impaired growth on ethanol medium. The growth deficiencyfor each could be corrected by a particular pattern of metalsupplementation. The CTR1 mutant could be rescued by highconcentrations of copper, as expected, but not by iron, pre-sumably because copper is required for respiration as' acofactor in cytochrome-c oxidase (1) in addition to iron.Conversely, the FET3 mutant could only be rescued by highconcentrations of iron. Surprisingly, the CCC2 mutant couldbe rescued by high concentrations of either copper or iron.High-affinity ferrous transport activity was undetectable in allthree mutant strains. In all of these cases, the ability of addedmetal to correct metal deficiency is presumably due to the

FERROUS IRON UPTAKE(pmol/l O 6 cells/h)

YPD YPD/Cu

5.0 3.11 (Wild-type)

2 Actrl

3 ccc2

4 Afet3

5 ccc2 Afet3

6 rho'

-0.3 10.6

-0.1 9.6

0.0 0.0

-0.2 0.0

8.6 4.4

FIG. 1. Metal-dependent phe-notypes of a CCC2 disruption mu-tant. The effect of added iron orcopper on growth in ethanol-basedmedium was determined for theindicated yeast strains. High-affinity ferrous iron uptake wasdetermined for the same strainsgrown in YPD medium alone orwith 50 ,uM added CUSO4. Theresults are the means of triplicatedeterminations; standard devia-tions were <20% of the means.

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ability of the high levels of added metal to gain access to thecell through mechanisms other than specific high-affinityuptake pathways. In the CTR1 or CCC2 mutants, ferroustransport could be recovered by growth in the presence of 50,uM added copper (Fig. 1). Copper could not rescue ferroustransport in either the FET3 mutant or in CTR1 or CCC2mutants with deletions in FET3, consistent with Fet3p func-tioning downstream of both Ctrlp and Ccc2p.The CCC2 mutant thus resembled the CTR1 mutant in that

its defects in ethanol-based growth and iron uptake werecorrectable by copper (Fig. 1). The CTR1 mutant is deficientin copper uptake from the environment to the cytosol (12, 13).We therefore evaluated the cytosolic copper pool of the CCC2mutant by using a copper-responsive reporter gene construct,CTR1-lacZ (13). As shown in Fig. 2, nanomolar concentra-tions of copper added to the growth medium equally repressedlacZ expression in both a CCC2 mutant and its wild-typeparent, indicating that cytosolic copper levels were not signif-icantly decreased. By comparison, the repression of lacZ fromthe CTR1-lacZ plasmid in a CTRI mutant required 300-foldhigher concentrations of copper. Radioactive copper uptakeand cytosolic Cu,Zn-dependent superoxide dismutase activitywere also normal in the CCC2 mutant grown in YPD medium(D.S.Y. and A.D., unpublished observations), in contrast withthe greatly diminished activities in CTRI mutants (12, 13).

Fet3p: An Extracytosolic Transmembrane Oxidase. CCC2mutants thus presented a paradox: the copper-correctable phe-notypes suggested copper deficiency, and yet external copperuptake and cytosolic copper levels were not low. This could beexplained if CCC2 were required for the delivery of cytosoliccopper to an intracellular membrane-bound compartment. Wetherefore asked whether Fet3p is an extracytosolic copper pro-tein. Reexamination of the open reading frame in the FET3sequence revealed a typical hydrophobic leader sequence at the

A12000

0

8000 o

i 2908 ACCC2

4000.4a

cjt BR 120000~~~~~~~%00 0~~~A

8000

BCS

1pM

0 0.001 0.01 0 10 0.001 0.01 0.1 1.0

COPPER CONCENTRATION (pM)

FIG. 2. Copper repression of a CTR1-lacZ reporter gene in CCC2and CTRJ mutants. Cells transformed with the plasmid CTR1-lacZ(13) were grown in defined medium containing the indicated concen-trations of copper and assayed for ,B-galactosidase activity. BCS,bathocuproinedisulfonate. (A) 0, Strain 7 (wild type; 2908); 0, strain8 (Accc2). (B) 0, Strain 1 (wild type; YPH252); 0, strain 2 (Actrl).

amino terminus of the predicted protein. This was followed by alarge hydrophilic domain separated from a short carboxyl-terminal hydrophilic tail by a predicted transmembrane domain.Fourteen potential N-glycosylation sites and the four proposedcopper-binding motifs (14, 33) were identified within the largehydrophilic domain. Direct biochemical proof of the protein'sorientation was sought. We studied Fet3p by Western blottingagainst a carboxyl-terminal HA epitope tag (22). The taggedprotein (Fet3p-HA) fully complemented the iron-uptake defectin a FET3 deletion mutant. All Fet3p-HA was localized to themembrane fraction of cell homogenates and migrated morerapidly on SDS/PAGE after treatment with endoglycosidase H(Endo H) (Fig. 3A), demonstrating that Fet3p is a membrane-bound glycoprotein and confirming its predicted topology.The homology between Fet3p and ceruloplasmin had sug-

gested that Fet3p might be a copper-dependent oxidase (14).When a modified in situ SDS/PAGE assay for ceruloplasmin(29) was used, a single band of oxidase activity was observed.This activity comigrated with the HA-tagged protein, detectedby Western blotting, with or without Endo H treatment(D.S.Y., unpublished observations). The oxidase activity wasabsent from a FET3 mutant and was repressed in cells grownwith added iron (Fig. 3B), consistent with the transcriptionalrepression of FET3 by iron (14). Oxidase activity was absentfrom cells grown in the presence of the copper chelatorbathocuproinedisulfonate (BCS), despite normal levels ofFet3p (Fig. 3B). Oxidase activity was also absent from theCCC2 mutant grown in YPD medium (Fig. 3C). That Fet3plevels were normal in these cells was demonstrated by theability to fully reconstitute oxidase activity in vitro by homog-enizing the cells in the presence of copper (Fig. 3C). Oxidaseactivity in the CCC2 mutant could also be fully restored in vivoby growth of the cells in medium supplemented with high levelsof copper (Fig. 3C), paralleling the ability of copper to restoreboth iron uptake and respiratory competence (Fig. 1).

DISCUSSIONPrevious data (12, 14) revealed an essential role for Fet3p inthe high-affinity uptake of external ferrous iron. We havepresented biochemical evidence that the FET3 gene indeedencodes a ceruloplasmin-like oxidase, as predicted, and thatcopper, Ctrlp, and Ccc2p are all required for both Fet3poxidase activity and high-affinity ferrous transport. Essentialto understanding this pathway is the characterization of thetopology of the Fet3 protein. The biochemical data reportedhere clearly localize the oxidase domain to the lumenal orextracellular side of the membrane.:A scheme for the coupledpathways of iron and copper uptake in S. cerevisiae is shown inFig. 4. Ferric iron is first reduced to ferrous iron outside thecell by either of two externally directed ferric reductases(Frelp and Fre2p) (34, 35). The reduced iron is then trans-ported into the cell by a ferrous transporter that has not yetbeen molecularly identified. Ferrous transport requires thecopper-dependent oxidase activity of Fet3p. In turn, Fet3preceives its copper from Ctrlp and Ccc2p acting in sequenceto transport copper first across the plasma membrane and thenout of the cytosol across one or more intracellular membranes.Ctrlp, localized to the plasma membrane, is required forhigh-affinity copper uptake into the cell. Ctrlp providescopper to several cellular targets, including Fet3p (12, 14).Ccc2p, presumably localized to the secretory pathway, isnecessary for delivering copper from the cytosol to the site atwhich Fet3p is loaded with copper. The scheme shown in Fig.4 suffices to explain why either copper or iron corrects therespiratory deficiency of the ccc2 mutant. In these cells, therespiratory deficiency results entirely from lack of sufficientiron. The iron deficiency, in turn, is due to the selective failureto provide copper to Fet3p. In contrast, CTR1 mutants are

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a q-j~~~~~C

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2 3 4 1 2 3 4llHomog.bufferA

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globally copper deficient, and therefore dependienous copper for the copper-dependent steps ofA question that remains unaddressed is the loc

the secretory pathway at which copper loadingInterestingly, we have observed that a variety ofdefective in post-Golgi sorting (36), fail to loaccopper (D.S.Y., unpublished observations). Thipost-Golgi compartment, perhaps in the endocyti(the site of Fet3p loading. Another question pertaiioxidase activity of Fet3p in yeast, or the oxidas

FIG. 4. Model of copper and iron uptake in

FIG. 3. Te FET3 protein (Fet3p) is a glycosylated membraneprotein with copper-dependent oxidase activity. (A) Western blot ofepitope-tagged Fet3p. Cells expressing epitope-tagged Fet3p (strain 9)and grown in a defined medium containing 100 gM Ferrozine and 5 jiMCuS04 were homogenized in buffer containing I mM bathocuproinedi-sulfonate and 1 mM EDTA. Proteins (18 jig) from the particulate fraction(lanes 1-3) or the soluble fraction (lane 4) were treated with 1000 unitsof Endo H (New England Biolabs) (lane 2) or with the buffer alone (lane3) at 37°C for 4 hr. Proteins were separated on a 7.5% polyacrylamide geland analyzed for the HA epitope by Western blotting. The band at 72 kDacould be due to Endo H protein, which migrated at the same position. (B)Detection ofFet3p-HA by Western blotting and oxidase assay. Lanes 1-3,strain 9 (transformed with a plasmid encoding HA-tagged Fet3p); lanes4 and 5, strains 1 and 4 (wild-type and Afet3; see Fig. 1). Strains were

grown as in A, except for lane 2 (Fe-grown), in which Ferrozine wasreplaced by 10 jiM ferric ammonium sulfate, and lane 3 (BCS-grown), inwhich CuS04 was replaced by 10 jiM bathocuproinedisulfonate. Proteins

Oxidase in membrane extracts were separated on 4-20% polyacrylamide gels andOxidase assayed concurrently for the HA epitope and oxidase activity. (C)

Copper-dependent oxidase activity due to Fet3p in mutant strains. Yeaststrains 1-4 (wild type, Actrl , ccc2, and Afet3, respectively; see Fig. 1) weregrown in YPD medium alone or with 50 AiM CuS04. Cultures were thendivided and homogenized in homogenization buffer containing 1 mM

Anti-HA bathocuproinedisulfonate (buffer A)

Ant-HA jiM CuSO4 (buffer B). Proteins in membrane extracts were separated on4-20% polyacrylamide gels and assayed for oxidase activity.

ent on exog- ceruloplasmin in mammals, functions in iron transport. WeE respiration. currently know of no function for Fet3p other than its role incation within iron transport. Preliminary observations in external proteo-takes place. lysis studies suggest that mature Fet3p resides at the cellvps mutants, surface (D.S.Y., unpublished observations). The strict corre-J Fet3p with lation between Fet3p oxidase activity and ferrous transportis suggests a strongly suggests that oxidase activity is essential for the ironc pathway, as transport function. The molecular target for the oxidase is notns to how the known. Ferrous iron may be the target, with oxidation perhaps;e activity of serving to trap ferric iron on a transporter molecule. Alter-

natively, the ferrous transporter protein itself might be thetarget, with transport being driven by an oxidation-reductioncycle. Finally, Fet3p might function in the production of acofactor which is required for iron transport.The pathway described here for copper loading of Fet3p in

yeast shares many features with that proposed for ceruloplas-min in mammals. In the human disorder of intracellular coppermetabolism, Wilson disease, the affected gene encodes aprotein believed to function in the delivery of cytosolic copperto ceruloplasmin in a yet-to-be-determined organelle of liver

Fet3t ~ cells (4, 37). Menkes disease is believed to result from a defectin a homologous gene product, expressed in a variety of tissues,for delivery of copper to a number of copper proteins (4, 38).These proposed functions of the Wilson and Menkes diseasegene products are analogous to the function of the homologousCCC2 gene product in yeast. Another point of similaritybetween the yeast and mammalian systems is the role thatFet3p and ceruloplasmin, both glycosylated copper-dependent

yeast. oxidases, appear to play in the mobilization of iron across

coC,

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membranes. In copper-deficient swine, ceruloplasmin defi-ciency results in functional iron deficiency (39). These animalsaccumulate iron in enterocytes and reticuloendothelial cells.The iron deficiency presumably results from the inability tomobilize the iron from these sites. In Wilson disease, severeceruloplasmin deficiency is associated with signs of irondeficiency (2). Thus, in the mammalian case, the extracellularcopper-dependent oxidase appears to be involved in themovement of iron out of cells. Recently, Gitlin and colleagues(40) have identified patients with defects in the ceruloplasmingene. These patients have neurologic abnormalities associatedwith deposition of iron in cells in the central nervous system,again pointing to the possible role of an oxidase in ironmobilization. The intriguing analogies between these clinicalsituations and the ferrous uptake defects in the respective yeastCTR1, CCC2, and FET3 mutants suggest that the mechanismsof copper and iron transport in eukaryotes are well conservedand that abnormalities of copper metabolism should be con-sidered as possible contributing factors in clinical disorders ofiron homeostasis.

We thank D. Eide and J. Kaplan for the Fet3p-HA plasmid and D.Eide, J. Kaplan, S. Kaler, J. Gitlin, G. Storz, and T. Rouault for helpfuldiscussions.

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