Copper Modulates the Degradation of Copper Chaperone for Cu,Zn Superoxide Dismutase by the 26 S Proteosome

Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the utilization of copper when it is scarce. Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the utilization of copper when it is scarce. Copper is an essential trace metal and a co-factor for a number of metalloenzymes that function to reduce molecular oxygen. Because of its reactive nature, free copper can participate in reactions that adversely modify proteins, lipids, and nucleic acids, which can have detrimental effects on the viability of the cell. Therefore, elaborate mechanisms have evolved to control the uptake and distribution of copper so that in the cell free copper is virtually nonexistent (1Rae T.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-808Crossref PubMed Scopus (1327) Google Scholar). Copper chaperones are a family of proteins that scavenge for copper in the cell and facilitate incorporation of the metal into specific proteins through direct interaction with their target (2Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 3Lamb A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Biochemistry. 2000; 39: 14720-14727Crossref PubMed Scopus (88) Google Scholar, 4Lamb A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 751-755Crossref PubMed Scopus (244) Google Scholar). By directly inserting copper into their target, copper chaperones prevent free copper ions from engaging in deleterious reactions in the cell. The copper chaperone HAH1/ATOX1 delivers copper to the P-type intracellular membrane-bound copper transporters ATP7A and ATP7B (5Larin D. Mekios C. Das K. Ross B. Yang A.S. Gilliam T.C. J. Biol. Chem. 1999; 274: 28497-28504Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 6Hamza I. Schaefer M. Klomp L.W. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13363-13368Crossref PubMed Scopus (239) Google Scholar). Mutations in ATP7A and ATP7B have been associated with diseases of copper metabolism, namely Menkes syndrome (7Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1204) Google Scholar) and Wilson's disease (8Bull P.C. Thomas G.R. Rommens J.M. Forbes J.R. Cox D.W. Nat. Genet. 1993; 5: 327-337Crossref PubMed Scopus (1676) Google Scholar), respectively. Copper chaperones that deliver copper to the mitochondria, cytochrome c oxidase, and the nucleus have also been described (9Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 10Schulze M. Rodel G. Mol. Gen. Genet. 1989; 216: 37-43Crossref PubMed Scopus (69) Google Scholar, 11Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 12Reddy M.C. Majumdar S. Harris E.D. Biochem. J. 2000; 350: 855-863Crossref PubMed Scopus (14) Google Scholar, 13Amaravadi R. Glerum D.M. Tzagoloff A. Hum. Genet. 1997; 99: 329-333Crossref PubMed Scopus (134) Google Scholar). Cu,Zn superoxide dismutase (SOD1) 1The abbreviations used are: SOD1, Cu,Zn superoxide dismutase; CCS, copper chaperone for SOD1; Me2SO, dimethyl sulfoxide; MG132, carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal; calpain inhibitor II, N-acetylleucinyl-leucinyl-methional-H; FBS, fetal bovine serum; RT, room temperature; ctr1, copper transporter 1; 2,3,2-tetraamine, N,N′-bis-(2-aminoethyl)-1,3-propanediamine.1The abbreviations used are: SOD1, Cu,Zn superoxide dismutase; CCS, copper chaperone for SOD1; Me2SO, dimethyl sulfoxide; MG132, carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal; calpain inhibitor II, N-acetylleucinyl-leucinyl-methional-H; FBS, fetal bovine serum; RT, room temperature; ctr1, copper transporter 1; 2,3,2-tetraamine, N,N′-bis-(2-aminoethyl)-1,3-propanediamine. is an abundant homodimeric enzyme that contains one copper and one zinc atom per subunit. SOD1 functions as an antioxidant, eliminating toxic superoxide anion radicals. Recently, dominantly inherited mutations in SOD1 have been linked to the fatal motor neuron disorder, familial amyotrophic lateral sclerosis (14Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. Rahmani Z. Krizus A. McKenna-Yasek D. Cayabyab A. Gaston S.M. Berger R. Tanzi R.E. Halperin J.J. Herzfeldt B. Van den Bergh R. Hung W.Y. Bird T. Deng G. Mulder D.W. Smyth C. Laing N.G. Soriano E. PericakVance M.A. Haines J. Rouleou G.A. Gusella J.S. Horvitz H.R. Brown Jr., R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5402) Google Scholar). It has been proposed that SOD1 mutants exert their toxic effects through some uncharacterized gain-of-function (15Wong P.C. Pardo C.A. Borchelt D.R. Lee M.K. Copeland N.G. Jenkins N.A. Sisodia S.S. Cleveland D.W. Price D.L. Neuron. 1995; 14: 1105-1116Abstract Full Text PDF PubMed Scopus (1234) Google Scholar, 16Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y.W. Deng H.X. Chen W. Zhai P. Sufit R.L. Siddique T. Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3393) Google Scholar). In vivo, insertion of copper into SOD1 is dependent on the copper chaperone for Cu,Zn superoxide dismutase (CCS). In mammals, CCS is a homodimer of ∼33-kDa subunits and is localized mainly to the cytoplasm (2Casareno R.L. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 17Rothstein J.D. Dykes-Hoberg M. Corson L.B. Becker M. Cleveland D.W. Price D.L. Culotta V.C. Wong P.C. J. Neurochem. 1999; 72: 422-429Crossref PubMed Scopus (105) Google Scholar). Mice genetically engineered to lack CCS show a reduction in SOD1 activity due to the absence of the copper co-factor required for enzymatic activity (18Wong P.C. Waggoner D. Subramaniam J.R. Tessarollo L. Bartnikas T.B. Culotta V.C. Price D.L. Rothstein J. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2886-2891Crossref PubMed Scopus (251) Google Scholar). When copper is abundant, CCS is expressed at low levels, and CCS has been estimated to be up to 30-fold less abundant than SOD1 in some tissues (17Rothstein J.D. Dykes-Hoberg M. Corson L.B. Becker M. Cleveland D.W. Price D.L. Culotta V.C. Wong P.C. J. Neurochem. 1999; 72: 422-429Crossref PubMed Scopus (105) Google Scholar). CCS is composed of three distinct domains. The N-terminal sequence contains an HAH1/ATOX1 copper-binding domain, MXCXXC (M, methionine; X, any amino acid; C, cysteine), and this region has been reported to be required for insertion of copper into SOD1 under conditions where copper is limiting (19Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The central domain is highly homologous to SOD1 and mediates interaction with SOD1. The C-terminal domain is a short sequence that is highly conserved between CCS molecules of different species and contains a CXC motif that can bind copper. Mutations within this motif abrogate copper transfer from CCS to SOD1 (19Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Despite considerable advances in our understanding of the mechanism by which copper chaperones transfer copper to their target (20O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A.C. Chem. 2001; PubMed Scopus Google Scholar), is how the activity of these proteins is We have recently reported a dose-dependent increase in CCS protein in and of rats fed copper-deficient diets J. M. J. PubMed Scopus (69) Google Scholar) suggesting that copper may control CCS expression. In this we have in the mechanism CCS expression and show that copper CCS protein level by modulating the of CCS degradation by the 26 S and proteosome inhibitors MG132 and lactacystin from and respectively. of the lysosomal degradation and and the cysteine protease inhibitor calpain inhibitor from was blocked with SOD1 and with and of the CCS used to CCS by is described (17Rothstein J.D. Dykes-Hoberg M. Corson L.B. Becker M. Cleveland D.W. Price D.L. Culotta V.C. Wong P.C. J. Neurochem. 1999; 72: 422-429Crossref PubMed Scopus (105) Google Scholar). CCS was used to CCS in The specific copper chelator 2,3,2-tetraamine was used to copper deficiency in cultured cell and to medium from and human cell lines cultured in with fetal bovine and at in in at and to and and diets have been described in J. M. J. PubMed Scopus (69) Google Scholar). rats to one of and in free to and one of diets but in copper in and copper by and copper-deficient diets and of respectively. of the rats by under and in and at was from of was in a under conditions and to a a was to the a The was by and the CCS from the with with CCS was by The and was used to the by the as a of of and of CCS mRNA was as per and in and protein by the PubMed Scopus Google Scholar). and cells with and from the in was and was to the cells. was and protein of the was under and conditions and blocked in with at room for with the for at at a in with and with an for at at a in and proteins by with and to was and at within the of the cells in and 2,3,2-tetraamine for with medium and and with for was and the cells with the medium in cell for and with with of and 2,3,2-tetraamine or at and in protease inhibitor of protein was for at with protein and for with CCS for and proteins to and the to CCS by a have reported recently J. M. J. PubMed Scopus (69) Google Scholar) that rats fed low copper diets show a dose-dependent of CCS protein in and that the reduction in SOD1 activity and in associated with copper suggest that copper may control CCS expression. In this we to the mechanism by which CCS expression is regulated and the of copper in this a C-terminal region of human CCS was used to CCS expression by in from rats fed a or copper-deficient for By this a that at in was which is in with the molecular of CCS and reported data this (17Rothstein J.D. Dykes-Hoberg M. Corson L.B. Becker M. Cleveland D.W. Price D.L. Culotta V.C. Wong P.C. J. Neurochem. 1999; 72: 422-429Crossref PubMed Scopus (105) Google Scholar). of CCS protein a increase in CCS expression in copper-deficient rats with rats fed a copper are with our data a different to CCS J. M. J. PubMed Scopus (69) Google Scholar). In to CCS, SOD1 protein in of copper-deficient rats The increase in CCS protein in copper-deficient rats could increased of the CCS or increased mRNA these was with from the used to CCS protein expression. a a of in of CCS and to the S that CCS mRNA to in and copper-deficient rats increased CCS and mRNA as the mechanism increased CCS expression. of the was by the of the S and S CCS protein any in that CCS expression is by a Copper CCS in Rodent and copper is in CCS we CCS protein in to copper deficiency in cultured cell copper we used the specific copper chelator 2,3,2-tetraamine that has been to copper deficiency in cell lines J. 1997; PubMed Scopus Google Scholar, J. 1999; PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar). our we that copper-deficient rats the increase in CCS expression in Therefore, we to the cell to CCS expression in to 2,3,2-tetraamine cells in medium with or medium or 2,3,2-tetraamine for of cells with medium up to 2,3,2-tetraamine no effects on cell or and M. R. cells and CCS expression was by CCS protein was increased to with 2,3,2-tetraamine and up to in cells with 2,3,2-tetraamine with cells cultured in medium with 2,3,2-tetraamine CCS expression in human we the with a human cell In these CCS protein a dose-dependent increase with cells cultured in the medium CCS expression was increased with 2,3,2-tetraamine with cells cultured in medium with expression was by 2,3,2-tetraamine and protein that CCS expression was in cells cultured with medium with low of with the cells in medium that the medium used in our is in copper to an that with medium with copper CCS protein of CCS for Copper increased CCS in cells with 2,3,2-tetraamine was a of copper deficiency and not deficiency of we to the metal of CCS by of CCS in cells by with of or directly to the cells and the cells for CCS level was increased in cells cultured in the presence of 2,3,2-tetraamine with cells in medium of to the cells CCS expression to with cells cultured with medium of of and not reduce CCS and with in cells with 2,3,2-tetraamine In addition of but not or a in CCS protein in cells with and M. R. Together, these that of CCS in and cells by 2,3,2-tetraamine is specific for copper and addition of copper to cells a in CCS protein Copper a in CCS in the of in CCS protein level by we a of CCS in cells with Copper was directly to the and the cells for CCS expression at and addition of copper a cells cultured in medium or medium with in the CCS protein level in cells with copper was than in cells in medium of to cells a rapid in CCS protein level and CCS protein could be as as addition of copper and By in the copper-rich CCS protein level to Copper CCS by the 26 S rapid in CCS protein cells from copper-deficient to copper-rich medium that copper increase the of The 26 S proteosome is a protease that expression of short proteins B. J.D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, T. P.J. Yang J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. B. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of CCS was dependent on degradation by the 26 S we the proteosome inhibitor MG132 could the in CCS protein in to copper. cells with 2,3,2-tetraamine in the presence of MG132 or for to addition of to the cells. CCS protein level was at and addition of copper with the reduction in CCS protein in to copper and CCS protein level to by which is with that for cells In of cells with MG132 blocked the in CCS expression. than of CCS protein in the cells in copper-rich medium The reduction in of CCS in the presence of MG132 of the 26 S proteosome in CCS expression. of the proteosome in CCS we the of the highly specific proteosome inhibitor lactacystin G. S. Science. 1995; PubMed Scopus Google Scholar) to CCS in a cells with lactacystin to CCS and In CCS protein in cells with to by of in copper-rich medium In cells cultured in medium with and with lactacystin or with to protein of with CCS expression was at and CCS protein to by and by for cells In lactacystin blocked CCS degradation and a increase in CCS expression at and The increase in CCS expression for cells a in protein protein from degradation of proteins by Because the of CCS was in a of protein a in protein in the absence of CCS degradation be by an increase in CCS The in CCS with lactacystin protein that the proteosome directly or regulates CCS expression by CCS protein degradation participate in CCS we the inhibitors of the lysosomal degradation and and the cysteine protease inhibitor calpain inhibitor CCS degradation in to copper cells with 2,3,2-tetraamine with calpain inhibitor II, or for to CCS expression was at and addition of copper. with calpain inhibitor II, or no on the of CCS with cells that and increased lysosomal in our cells under our we of the and in cells with the inhibitors to that of cells. in cells for with or was with cells increased lysosomal these show that copper CCS mainly by the 26 S Copper CCS the of copper on the degradation of CCS, cells cultured in medium with copper or 2,3,2-tetraamine with to protein of with cells for CCS expression at and in medium a in CCS as to by and CCS expression in cells cultured in the presence of 2,3,2-tetraamine in CCS protein level protein and the of copper on the of CCS, we a cells cultured in the presence of 2,3,2-tetraamine for for with and cysteine and for and or and with medium 2,3,2-tetraamine or respectively. The of CCS in cells in copper-rich or copper-deficient was to be and Together, these data that the degradation of CCS is rapid when copper is abundant than when it is scarce. The of of copper is by the associated with to copper as Menkes syndrome and Wilson's disease A. A. S. J. PubMed Scopus Google Scholar, H. 1999; PubMed Scopus Google Scholar). In addition, mutations in the human that for a copper chaperone required for cytochrome c have been linked recently to a fatal by and M. I. J. G. Hum. Mol. Genet. 2000; PubMed Scopus Google Scholar, K. I. S. W. J. Glerum D.M. G. E. R. P. S. E. M. S. Nat. Genet. 1999; PubMed Scopus Google Scholar). Copper is dependent on the interaction of transporters that copper membrane-bound transporters that regulate copper and copper chaperones that deliver copper to specific within the cell. The P-type transporters ATP7A and ATP7B have been to from the to the cell and under of copper where function to the cell of copper M. Gitlin J.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. J. 1996; PubMed Scopus Google Scholar, J.R. Cox D.W. Hum. Mol. Genet. 2000; PubMed Scopus Google Scholar, I. M. K. D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). expression and of the human copper uptake ctr1, has that cells to of copper reduce expression of and its suggesting a reduction in copper uptake K. Lee J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (244) Google Scholar). the of copper transporters have our understanding of the mechanisms in copper in the to date, is how the distribution of copper to and by copper chaperones is by copper J. M. J. PubMed Scopus (69) Google Scholar) has that copper deficiency in rats by low copper diets to CCS protein in suggesting that copper may control CCS was at the mechanism CCS expression and the role that copper in this We have that increased CCS protein by copper deficiency is not by an increase in CCS mRNA a mechanism for CCS and cells to the specific copper chelator 2,3,2-tetraamine a dose-dependent increase in CCS protein with our in and of rats fed diets in copper J. M. J. PubMed Scopus (69) Google Scholar). CCS by 2,3,2-tetraamine was to be specific for copper deficiency as addition of copper to these cells CCS protein to In addition, of iron or zinc to the cells no on CCS copper deficiency as the for increased CCS protein level with 2,3,2-tetraamine 2,3,2-tetraamine CCS in and the of was less in cells. this we for the in is due to species or specific of the cell In this in the of have also been reported with different cell K. Lee J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (244) Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Van Berger R. Klomp L.W. Biochem. J. PubMed Scopus (154) Google Scholar). It is that different tissues have evolved to to copper and the may on the specific copper for that cell We that cells from a copper-deficient to a copper-rich a in CCS protein in CCS protein was as as addition of copper. The rapid in CCS protein is with the rapid of copper reported for cells in J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are also in with the rapid of SOD1 activity when are from copper-deficient to copper-rich conditions P.J. C. Culotta V.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). that CCS functions by for copper and the metal to SOD1, of CCS increase the of copper transfer to SOD1. The of CCS in copper-deficient cells may for the rapid insertion of copper into when it SOD1 is there be no for CCS to increase of copper to the and CCS expression is protein and CCS degradation in cells cultured in copper-deficient or copper-rich medium that the of CCS is when copper is When cells cultured in copper-rich CCS to in protein with In cells in copper-deficient medium by the presence of 2,3,2-tetraamine a to degradation of an for CCS when cells cultured in copper-deficient as to copper-rich medium in to the rapid in CCS expression cells from copper-deficient to copper-rich CCS cells in copper-deficient medium was We a increase in CCS protein level in cells cultured in the presence of 2,3,2-tetraamine the of which could be to in copper to the Together, our are with a in which low copper increase CCS which its accumulation proteins function is dependent on their rapid in expression are by the 26 S a In our we that the proteosome inhibitor MG132 and the specific proteosome inhibitor lactacystin blocked of CCS In addition, lactacystin blocked CCS degradation when protein was with We also that calpain inhibitor II, a cysteine protease and inhibitors of the lysosomal degradation not degradation of that copper regulates CCS expression by its degradation by the 26 S a M. Rothstein J.D. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) has that CCS mutants with a that is to a in SOD1 that familial amyotrophic lateral sclerosis and could be by of cells with an inhibitor of the that copper CCS degradation by the it be to of these CCS mutants is by copper of the cell. The of proteins is by in of the When copper is abundant it is that CCS can bind copper than when copper are It is that CCS a that the protein less its of degradation by the is with that have that of copper an in CCS (19Schmidt P.J. Rae T.D. Pufahl R.A. Hamma T. Strain J. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1999; 274: 23719-23725Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). increased of CCS in copper-deficient cells may be by a interaction with or from our data we a where the proteosome regulates CCS expression by modulating the activity of a protein that is to copper of the cell and that the of In we have that copper regulates CCS expression level by modulating its of degradation by the 26 S our this is the of any of the copper chaperones in higher eukaryotes. how the expression and activity of copper chaperones and their are by copper deficiency information regarding the utilization of copper when it is our into the mechanisms in copper and suggest a novel role for CCS in prioritizing the distribution of copper when it is scarce. We for the of the and cell lines and Rothstein for the CCS We are also to the of the for of the and for the

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