Trafficking of a Secretory Granule Membrane Protein Is Sensitive to Copper

We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl2. Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis. We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl2. Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis. Copper plays an essential role in cellular respiration, free-radical defense, connective tissue biosynthesis, iron metabolism, peptide amidation, and the synthesis of catecholamines (1Madsen E. Gitlin J.D. Curr. Opin. Gastroenterol. 2007; 23: 187-192Crossref PubMed Scopus (61) Google Scholar). An adequate supply of copper must be distributed to copper-requiring enzymes in various subcellular compartments without allowing free cuprous ions to react with hydrogen peroxide and generate hydroxyl radical (2Linder M.C. Hazegh-Azam M. Am. J. Clin. Nutr. 1996; 63 (S): 797S-811PubMed Google Scholar). Cuproproteins can be divided into two groups, those that are involved in the uptake and delivery of copper and those that require copper for catalytic activity. Plasma membrane copper transporters like Ctr1 and copper-transporting P type ATPases like ATP7A and ATP7B, along with various copper chaperones, fall into the first group (3Camakaris J. Voskoboinik I. Mercer J.F. Biochem. Biophys. Res. Commun. 1999; 261: 225-232Crossref PubMed Scopus (204) Google Scholar, 4Harris E.D. Annu. Rev. Nutr. 2000; 20: 291-310Crossref PubMed Scopus (222) Google Scholar). Enzymes like tyrosinase, cytochrome c oxidase, Cu/Zn superoxide dismutase (SOD), 2The abbreviations used are: SOD, superoxide dismutase; BCS, bathocuproine disulfonic acid; PHM, peptidylglycine α-hydroxylating monooxygenase; PAL, peptidyl-α-hydroxyglycine α-amidating monooxygenase; TGN, trans-Golgi network; CSFM, complete serum-free medium; JP, joining peptide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; PAM, peptidylglycine α-amidating monooxygenase; PALm, membrane PAL. and peptidylglycine α-amidating monooxygenase (PAM) fall into the second group. Many proteins playing important roles in copper homoeostasis have been identified. Copper enters the cell via the high affinity uptake protein, CTR1 (5Zhou B. Gitschier J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7481-7486Crossref PubMed Scopus (474) Google Scholar). Specific copper chaperones are involved in the distribution of copper to cytosolic SOD, mitochondrial cytochrome c oxidase and the secretory pathway (6Culotta V.C. Klomp L.W. Strain J. Casareno R.L. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 7Amaravadi R. Glerum D.M. Tzagoloff A. Hum. Genet. 1997; 99: 329-333Crossref PubMed Scopus (135) Google Scholar, 8Klomp L.W. Lin S.J. Yuan D.S. Klausner R.D. Culotta V.C. Gitlin J.D. J. Biol. Chem. 1997; 272: 9221-9226Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar); sequestration of excess copper by metallothioneins prevents cellular damage. ATP7A and ATP7B deliver copper to the lumen of the secretory pathway, a transport step that is essential both for metallation of secretory pathway enzymes and for removal of excess copper from the cell (9Voskoboinik I. Camakaris J. Mercer J.F. Adv. Protein Chem. 2002; 60: 123-150Crossref PubMed Scopus (35) Google Scholar). As might be expected, expression and localization of the proteins essential for copper uptake and delivery are sensitive to copper levels (3Camakaris J. Voskoboinik I. Mercer J.F. Biochem. Biophys. Res. Commun. 1999; 261: 225-232Crossref PubMed Scopus (204) Google Scholar, 10Puig S. Thiele D.J. Curr. Opin. Chem. Biol. 2002; 6: 171-180Crossref PubMed Scopus (582) Google Scholar, 11Rees E.M. Thiele D.J. Curr. Opin. Microbiol. 2004; 7: 175-184Crossref PubMed Scopus (64) Google Scholar). The stability of several cuproenzymes is sensitive to copper levels. Both ceruloplasmin and hephaestin are normally loaded with copper in or immediately after they pass through the trans-Golgi network (12Hellman N.E. Kono S. Manchini G.M. Hoogeboom A.J. De Jong G.J. Gitlin J.D. J. Biol. Chem. 2002; 277: 46632-46638Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 13Nittis T. Gitlin J.D. J. Biol. Chem. 2004; 279: 25696-25702Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Ceruloplasmin synthesized in the absence of copper is secreted normally, but is rapidly degraded in plasma (14Sato M. Gitlin J.D. J. Biol. Chem. 1991; 266: 5128-5134Abstract Full Text PDF PubMed Google Scholar). When synthesized in the absence of copper, hephaestin, an integral membrane protein is rapidly degraded by the proteasome pathway (13Nittis T. Gitlin J.D. J. Biol. Chem. 2004; 279: 25696-25702Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). SOD3, tyrosinase, and lysyl oxidase also receive copper in the secretory pathway (15Qin Z. Itoh S. Jeney V. Ushio-Fukai M. Fukai T. FASEB J. 2005; 20: 334-336Crossref PubMed Scopus (81) Google Scholar, 16Wang N. Herbert D.N. Pigment Cell Res. 2006; 19: 3-18Crossref PubMed Scopus (184) Google Scholar, 17Li S. Kagan H.M. Matrix Biol. 1997; 16: 387-398Crossref Scopus (28) Google Scholar). ATP7A binds directly to SOD3, presumably facilitating the incorporation of copper (15Qin Z. Itoh S. Jeney V. Ushio-Fukai M. Fukai T. FASEB J. 2005; 20: 334-336Crossref PubMed Scopus (81) Google Scholar). Tyrosinase requires the incorporation of two copper ions into the apoenzyme during synthesis (16Wang N. Herbert D.N. Pigment Cell Res. 2006; 19: 3-18Crossref PubMed Scopus (184) Google Scholar). Two cuproenzymes, PAM and dopamine β-monooxygenase, are localized to the secretory granules that store bioactive peptides (18Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: PubMed Scopus Google Scholar, J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). is the of copper on PAM the of peptide a essential for the of Mains R.E. Cell Sci. 2000; PubMed Scopus Google Scholar). The is by two monooxygenase and PHM, an two essential copper ions that and during Mains R.E. Cell Sci. 2000; PubMed Scopus Google Scholar). Both copper are with and must be loaded with copper to have Mains R.E. Cell Sci. 2000; PubMed Scopus Google Scholar). PAM in both integral membrane and soluble a to the of copper availability on membrane and soluble forms of the through the secretory pathway, are through the of several peptide enzymes and PAM Curr. Opin. Chem. Biol. PubMed Scopus Google Scholar, M. Res. 1999; PubMed Scopus Google Scholar). PAM and enzymes are with to the secretory pathway Curr. Opin. Cell Biol. PubMed Scopus (61) Google Scholar, Biochem. J. PubMed Scopus Google Scholar). PAM is in the and secretory granules S.L. Mains R.E. J. Cell Sci. 1997; PubMed Google Scholar). The peptides produced are in secretory granules, to in response to soluble PAM is secreted along with the bioactive peptides and membrane PAM on the plasma membrane and is by S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). The essential for the of membrane PAM into secretory granules and for endocytic trafficking are cytosolic domain and and The of PAM on the plasma membrane and be into granules or Mains R.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). We used tumor cell lines expressing integral membrane and soluble forms of PAM to the of copper availability on PAM and the trafficking of soluble by copper cellular copper the return of internalized membrane PAM to secretory granules. copper levels the of internalized PAM. The cytosolic domain of PAM is essential to to to copper availability. Cell proteins are to and lines expressing (18Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: PubMed Scopus Google or J. Biol. Chem. Full Text PDF PubMed Google or S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google were in with bovine expressing to Mains R.E. 1997; PubMed Scopus Google were in with and bovine were loaded with copper by for in or CuCl2. were depleted of copper by incubation in bathocuproine disulfonic acid and the copper or copper loading cell were from were from PAM α-hydroxylating monooxygenase were using Mains R.E. J. Cell Biol. 1999; PubMed Scopus Google Scholar). Mains R.E. 1997; PubMed Scopus Google Scholar). by were to with in in for using J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). using Mains R.E. J. Cell Biol. 1999; PubMed Scopus Google Scholar). using a to the luminal domain S.L. Mains R.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). using horseradish and When were with a using The effect of copper on of and using to cell using Mains R.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Mains R.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar); were levels of were to of using a to the cytosolic domain of by were with in for with in for and with in for (18Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: PubMed Scopus Google Scholar). were with in for or with PBS, were in the or for in the were with using were using a and were with a to an using of and were in acid with and with were divided into two to and were were in with lacking were for protein using the acid were used for used and the in the of is Mains R.E. J. Cell Biol. 1999; PubMed Scopus Google Scholar). used and the does not with or but does peptides Mains R.E. J. Biol. Chem. 261: Full Text PDF PubMed Google Scholar). and is for the of A. Mains R.E. J. Biol. Chem. Full Text PDF PubMed Google were used for of endocytic were in complete serum-free a of for and and immediately a of or in for a of using second PAM protein using a to the cytosolic domain of PAM and second were and using As S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google PAM is internalized in a of endocytic trafficking using to and by to protein on a using of into a were with for and immediately a of or for or in complete serum free were were to and to of the were and into by incubation for by levels were by of to the a The after by and using a were in and for and immediately or in for PAM using to and second lines into were to a incubation in lacking were in or for and immediately or for in were in to cell PAM using to or were by and synthesized proteins were by of the with were in in the of the were in with for of the were with in the to react for The by the with with were immediately or for into with and and were with for were with and proteins were into by for by for of into were by in for were with to ATP7A Mains R.E. PubMed Scopus Google or PAM cytosolic domain S.L. Mains R.E. J. Cell Sci. 1997; PubMed Google for were using protein were with proteins into were by and to of ATP7A and by to Copper the of PAM and of incubation of with is used to the availability of copper (12Hellman N.E. Kono S. Manchini G.M. Hoogeboom A.J. De Jong G.J. Gitlin J.D. J. Biol. Chem. 2002; 277: 46632-46638Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 13Nittis T. Gitlin J.D. J. Biol. Chem. 2004; 279: 25696-25702Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, J. 1996; PubMed Scopus Google Scholar). The of to copper availability in by peptide amidation, a that is the of copper to PAM Mains R.E. PubMed Scopus Google Scholar). normally two and Mains R.E. J. Biol. Chem. 261: Full Text PDF PubMed Google Scholar). of were levels of were The used to to the with copper, or to the for copper levels are an effect on protein synthesis in copper levels the synthesis and secretion of PAM, expressing were depleted of copper by incubation with BCS, in or to levels of synthesized proteins were by in of copper and immediately or for on of protein with the copper of the not protein synthesis not PAM from cell and and synthesized PAM by the the synthesized PAM protein after the incubation through the enters secretory granules and to cleavage S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). cleavage soluble and membrane can be or and of granules with the plasma membrane S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). of and to effect of on the synthesized PAM first endoproteolytic cleavage the stability of many cuproenzymes is copper is not (13Nittis T. Gitlin J.D. J. Biol. Chem. 2004; 279: 25696-25702Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16Wang N. Herbert D.N. Pigment Cell Res. 2006; 19: 3-18Crossref PubMed Scopus (184) Google Scholar, Mains R.E. 117: PubMed Scopus Google the of PAM proteins after the the of PAM after the to it is that lack of copper excess copper the of PAM proteins for or of the synthesized and the plasma membrane and during the in cytosolic domain endocytosed PAM secretory granules, it is to endoproteolytic cleavage to PALm, or We cleavage of PAM to were by copper levels. the of PAM to can be or the of the PAM protein is soluble in the The of secretion of lack of copper enzyme Mains R.E. Annu. Rev. 1992; PubMed Scopus Google PAM is not copper levels are levels of copper PAM and The effect of on secretion is not to of is for levels of effect on secretion Copper the by the of secretory granules membrane PAM and soluble along with peptide to the of on the plasma membrane and to the secretion of the effect of copper levels on the endocytic trafficking of PAM, and were in to for The and were immediately or The of the cell and not using a uptake by type the Copper loading not uptake of the internalized from the during the first depleted of copper internalized internalized for a The effect after the the of the PAM protein in is localized to endocytic compartments in the (18Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: PubMed Scopus Google Scholar). for PAM not by copper levels the effect of copper on the endocytic trafficking of PAM, loaded with copper or depleted of copper were to PAM and in and As for the copper loading in the of internalized copper in the of internalized and uptake of not affected by copper Copper levels effect on the of internalized the of the copper Copper the of PAM to to endocytic trafficking of membrane PAM is by in cytosolic domain Mains R.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar, Mains R.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Johnson R.C. N. B. Mains R.E. Biol. PubMed Scopus (28) Google Scholar). uptake is an of the of membrane PAM. a of the effect of copper on the endocytic trafficking of PAM. depleted of copper by with BCS, loaded with copper or were to for and immediately or for and The proteins in cell and were using and PAM proteins were using to PALm, PHM, and of soluble and PAM and are in secretory granules by cleavage of (18Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: PubMed Scopus Google the cleavage that does not in secretory granules. a of the PAM and in the cell is on the plasma membrane and to cell of the and of the is during a the of to is levels of copper the of the that is during a The of to is not affected by copper levels. in the of in a delivery of to the plasma membrane or removal of from the plasma cell and from the were and the that it on the plasma membrane can be into or into The endoproteolytic cleavage to in secretory granules. to the of PAM after the in in The of in the after the is not by copper availability. PAM and from that on the plasma membrane can in the cell or be first the of from it is that is in of is not sensitive to copper levels. the endoproteolytic cleavage that in secretory granules, the effect of copper levels on secretion of data from the secreted by to copper in a in secretion of excess copper decreases of Copper in an in the cleavage of synthesized and in an in the secretion of from synthesized with copper both that been internalized from the cell and into secretory granules and into that is for basal An in copper levels decreases the stability of on the plasma is into in secretory granules and is The and of to Copper effect of copper on the trafficking of be through to or be an through trafficking in the cytosolic domain of for of copper to the of PHM, expressing soluble were depleted of copper by incubation with BCS, in or to for were in for and immediately or for with of Cell and were by of were by the of from to the of after both and of by copper levels. The protein in the a that is by the in cell J. Biol. Chem. Full Text PDF PubMed Google to of a synthesized secreted during the is the the of cleavage is affected by copper levels The protein in the does not the a to transit of through the secretory pathway Mains R.E. Cell Sci. 2000; PubMed Scopus Google Scholar, S.L. Mains R.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Unlike not secretory granules that store bioactive synthesized in the with of the in the after a The secretion is by copper availability Copper of copper to the of PHM, lacking a were expressing or with or two the were with and by of synthesized not by the of the for secretion the of protein in the after the We from that soluble the secretory pathway in the copper is to it or The of PAM a in to trafficking of membrane PAM is by of and in cytosolic domain Mains R.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). protein truncated immediately after the to in secretory granules and does not on the plasma membrane S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar); is expressing were depleted of copper with BCS, in or to with in of of copper levels. does not in secretory granules, a of the synthesized protein is a and the a of proteins that not in the cell S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). The of to PAM, and is not by copper availability and The secretion of during the is not by copper availability The effect of copper on trafficking requires the of cytosolic ATP7A and are both localized to the (18Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: PubMed Scopus Google Scholar, Mains R.E. PubMed Scopus Google Scholar). ATP7A and to the proteins were with a to ATP7A or a to the cytosolic domain of PAM, the but not many be by the that copper-sensitive machinery the trafficking of and copper availability by with the catalytic of PAM and PHM, the synthesized are a of the synthesized PAM and synthesized be copper levels were or is from that of secretory pathway a type membrane protein, (13Nittis T. Gitlin J.D. J. Biol. Chem. 2004; 279: 25696-25702Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). the absence of copper, is secreted but rapidly degraded in plasma (14Sato M. Gitlin J.D. J. Biol. Chem. 1991; 266: 5128-5134Abstract Full Text PDF PubMed Google Scholar). of apoenzyme to be an response to copper availability. The that PAM does not type of response suggests that it a function or be involved in in a in the high levels of PAM in not with high levels of peptidylglycine Mains R.E. Cell Sci. 2000; PubMed Scopus Google Scholar). When copper is in for PAM, The of PAM an in the of Copper on binds to two in the and the Mains R.E. Biol. 1999; 6: PubMed Scopus Google Scholar). of of the catalytic of Mains R.E. 1997; PubMed Scopus Google Scholar). of the copper the data secretion of Mains R.E. 1997; PubMed Scopus Google Scholar). When produced a soluble protein, the stability and secretion of are not sensitive to copper levels. the of copper to the and of must are not to The that secretion of from membrane PAM is sensitive to copper levels our on an in the trafficking of membrane PAM. The of synthesized to secretory granules it the is not affected by copper levels. after a both the of from synthesized and the secretion of synthesized were copper levels were and secretion of soluble secretory from a of of cell R.E. J. Cell Biol. PubMed Scopus Google Scholar). a of the synthesized have on the plasma membrane and during a a domain but the cytosolic that secretory of PAM from granules and the of PAM S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). of the synthesized the plasma membrane of of the synthesized the plasma membrane in S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). of synthesized to to a of synthesized is S.L. Mains R.E. J. Cell Biol. PubMed Scopus Google Scholar). of from and secretion of the from were not affected by copper availability basal secretion were sensitive to copper to an effect on secretion from The that not an effect on secretion to that copper availability the endocytic trafficking of Copper the of our first that the endocytic trafficking of sensitive to copper and uptake not affected by copper copper levels were internalized by from copper loaded internalized PAM in the an of protein localization and were the to the effect of copper on PAM trafficking We the of PAM proteins a and after a The stability of on the of is copper levels are and copper levels are high or the of the that both after it is that copper the of from that on the cell is produced in secretory granules, have to that the of internalized into secretory granules is copper levels are of to J. Biol. Chem. Full Text PDF PubMed Google Scholar); peptide is the step in the pathway to of bioactive cleavage of is a the stability of on the of is by copper levels integral membrane PAM is to copper levels. from the and are in a of the are with a copper-sensitive trafficking step that endocytosed PAM to return to the secretory pathway or to When copper levels are PAM to secretory it can cleavage to PHM, and the in basal When copper levels are internalized PAM does not return to granules, the of PHM, and basal internalized PAM is the of synthesized PAM into secretory granules, the of secretory granules the basal of granules be The of on the of can the cleavage of in secretory granules. be to the endocytic trafficking that are sensitive to proteins the of the endocytic pathway normally and in to Mains R.E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google the copper-sensitive are to Copper the of to be a plasma membrane levels of Ctr1 are copper levels are levels of copper the of Ctr1 J. Thiele D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). levels of are copper levels are response for an in copper levels ATP7A on the plasma membrane and in Mercer J.F. Camakaris J. J. 1996; PubMed Scopus Google Scholar, Camakaris J. S. Mercer J.F. Hum. Genet. 7: PubMed Scopus Google Scholar). ATP7B, an in copper levels in of ATP7B into a M. Gitlin J.D. Am. J. 1999; Google Scholar, M. S. Mercer J.F. Biochem. J. 2007; PubMed Scopus Google Scholar). The trafficking of ATP7A and ATP7B is with to a Voskoboinik I. M. B. Camakaris J. Mercer J.F. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). the in S. J. Hum. Genet. 7: PubMed Scopus (81) Google the in the cytosolic domain R.L. S. J. Cell Sci. 1999; PubMed Google or cytosolic copper and Hum. 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