Paul A. Cobine

The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the CuB and CuA sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins. The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the CuB and CuA sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins. Cytochrome c oxidase (CcO) 1The abbreviations used are: CcO, cytochrome c oxidase; IM, inner membrane; IMS, intermembrane space; Ni-NTA, nickel-nitrilotriacetic acid; Sod1, superoxide dismutase. is the terminal enzyme of the respiratory chain within the mitochondrial inner membrane. Mammalian CcO consists of 13 polypeptide subunits, 3 of which (Cox1-Cox3) are encoded by the mitochondrial genome with the remaining 10 subunits encoded by the nuclear genome (1Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (523) Google Scholar, 2Poyton R.O. McEwen J.E. Annu. Rev. Biochem. 1996; 65: 563-607Crossref PubMed Scopus (434) Google Scholar). In addition to the subunits, over 30 distinct proteins are important for the assembly of CcO (3Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). A number of these accessory proteins are important in the processing and translation of COX1-COX3 mRNA transcripts, in chaperoning the assembly process, and in the synthesis or delivery of cofactors. The cofactors in CcO include two copper sites (CuA and CuB), two heme A moieties, and a magnesium and zinc ion (4Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Hakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1295) Google Scholar). The assembly of the two copper sites will be reviewed because that is the focus of the present study. Cox2 requires two copper ions in the binuclear CuA site, and Cox1 requires one copper ion in the CuB site that exists within a Cu-heme A binuclear center. Because both Cox1 and Cox2 are synthesized inside the mitochondria, the three Cu atoms must be imported from the cytoplasm. Four proteins (Cox11, Cox17, Cox19, and Sco1) have been implicated in copper ion delivery and insertion into CcO (reviewed in Ref. 5Carr H.S. Winge D.R. Acc. Chem. Res. 2003; 36: 309-316Crossref PubMed Scopus (198) Google Scholar). The first protein implicated in copper ion delivery to newly synthesized CcO was Cox17 (6Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Yeast harboring a mutant cox17–1 were shown to be deficient in respiratory growth and CcO activity, but these phenotypes were reversed by the addition of 0.4% copper salts to the growth medium (6Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Among a collection of yeast respiratory-deficient mutants defective in cytochrome c oxidase activity, only cox17–1 cells were suppressed by exogenous copper, suggesting a role for Cox17 in copper ion metallation of CcO. Sco1 was first implicated in the copper delivery pathway to CcO by the observation that the respiratory-deficient phenotype of a cox17–1 strain was suppressed by overexpression of SCO1 (7Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Sco1 is an integral inner membrane (IM) protein that is tethered to the IM by a single transmembrane helix and contains a globular domain, projecting into the intermembrane space (IMS) (8Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 9Buchwald P. Krummeck G. Rodel G. Mol. Gen. Genet. 1991; 229: 413-420Crossref PubMed Scopus (72) Google Scholar). Null sco1 cells, like cox17Δ cells, are respiratory-deficient and have diminished CcO activity. Unlike cox17Δ cells, these phenotypes are not reversed in yeast by the addition of exogenous copper. The role of Cox11 in CuB site formation was suggested by the observation that CcO isolated from Rhodobacter sphaeroides cox11Δ cells lacked CuB and Mg(II) but contained the other cofactors (10Hiser L. Di Valentin M. Hamer A.G. Hosler J.P. J. Biol. Chem. 2000; 275: 619-623Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Yeast lacking Cox11 are devoid of CcO activity (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Cox11 is also an intrinsic IM protein tethered by a transmembrane helix. A fourth protein, Cox19, is implicated in copper metallation of CcO by virtue of its limited sequence similarity with Cox17 (12Nobrega M.P. Bandeira S.C.B. Beers J. Tzagoloff A. J. Biol. Chem. 2002; 277: 40206-40211Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Yeast cells lacking Cox19 are respiratory-deficient, but this phenotype is not reversed by the addition of exogenous copper salts. The roles of Cox17, Sco1, and Cox11 in copper metallation of CcO were substantiated by the observation that each protein is a Cu(I)-binding protein and that mutations that abrogate in vivo function attenuate Cu(I) (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, T. Winge D.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, George G.N. G. Winge D.R. PubMed Scopus Google Scholar, T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Cox17 is a protein that within the and also within the cytoplasm (8Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The of Cox17 suggested that a copper ion to the we that Cox17 is tethered to the IM by a heterologous transmembrane Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The pathway of copper ion delivery to CcO not been Sco1 was to the transfer of Cu(I) ions from Cox17 to the CuA site in Cox2 J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The observation that Sco1 and Cox2 this A. M. Rodel G. 2000; Scholar). Cox17 not been shown to with of these proteins, its role a copper donor to Sco1 Cox11 Cox19, like Cox17, is a protein within the IMS, also have a copper transfer is of which in the CcO assembly is by of the in the Cox17 are in a that is important for Cu(I) T. Winge D.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Cu(I) ions to Cox17 within a that a of and George G.N. G. Winge D.R. PubMed Scopus Google Scholar). A in Cox17 the The cox17–1 a C57Y that in vivo function Cu(I) or with Cox17 mutants a role in mitochondrial with Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The two IM proteins Sco1 and Cox11 are to be the direct copper donors to Cox2 and Sco1 and Cox11 have domains that into the Sco1 a sequence that with a the Cu(I) site T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the Sco1 that these two within an E. L. S. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that the single Cu(I) ion to Sco1 is and for a transfer Cox11 Cu(I) by three (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). of of these Cu(I) and CcO activity. The that Sco1 and Cox11 copper ion insertion into Cox2 and respectively, is with copper metallation of other yeast proteins. insertion into in yeast requires the function of the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a copper ion copper ions from the J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, R.A. A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R.A. P. J.E. Science. 1997; PubMed Scopus Google Scholar). both and copper transfer occurs in of the protein and its specific Thus, the is that copper transfer from Sco1 to Cox2 and Cox11 to Cox1 is copper ion metallation of Sco1 and Cox11 is to in two the of the copper to Sco1 and Cox11 and the of copper to the mitochondrial In this report we have the first and demonstrate for the first that Cox17 is the specific copper donor to both Sco1 and Cox11. Yeast strain and cox17Δ (6Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (408) Google were used for Yeast were on or in in medium lacking for or lacking for were with or were a to to a sequence a was from T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google by with and sites to the and mutant SCO1 the with the was from with and terminal The SCO1 were into the and for of cells L. PubMed Scopus Google Scholar). The is A of and lacking the first of the was from the (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google and into a Cox11 mutant with a of and was a from an mutant for into The of and was Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). expression with the occurs in medium lacking whereas expression occurs in medium the The contains the of the mitochondrial sequence and transmembrane of to Cox17. was by insertion of the C57Y of Cox17 in R. M. Res. PubMed Scopus Google Scholar). The of in this were by Cox17 was purified by George G.N. G. Winge D.R. PubMed Scopus Google Scholar). E. with on a were used for the Cox17 on an protein The C57Y mutant of Cox17 was purified from cells harboring the Sco1 and mutant Sco1 were purified from harboring SCO1 or mutant by T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The Sco1 purified were lacking the that the mitochondrial sequence and transmembrane was used for the of the Sco1 proteins. was isolated from E. in the of copper to the growth these Sco1 is with of copper. E. strain harboring a lacking the first was used for the of a was used (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). was isolated from in the of copper to the growth Yeast with or were in medium to an of was to expression of the Sco1 or Cox11. were for of by of a and from The overexpression of the of Sco1 or Cox11 not mitochondrial in vitro CuCox17 and mutants were purified from E. and in or purified from E. was in and into transfer The transfer was by of the Cox17 into the transfer by the addition of the were a and and were and were by by or by on a in was by the addition of to the protein by a with was by and the was The was in of transfer in of was by with an was by copper of the protein was a or a was by in in on a was on a was on a with from the mitochondrial or was on a and to were in and to with and with a to was from Cox11 and were (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of CuCox17 was used to in vitro copper transfer to proteins. The of the copper of Cox17 from Cu(I) ions from George G.N. G. Winge D.R. PubMed Scopus Google Scholar). The were expressed domains of Sco1 and Cox11 lacking transmembrane shown that the of each protein was competent for specific Cu(I) and that the Cu(I) of each protein was (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The addition of or to a of CuCox17 to an in with of the CuCox17 or copper ion transfer The was within a studies suggested that the of not from the addition of the to a of CuCox17 not in in the addition of bovine serum albumin or carbonic not attenuate not The of CuCox17 by the addition of or was of Cu(I) ion transfer to this transfer was by the of Sco1 or Cox11 from the and of copper. The of Sco1 and Cox11 contained only copper to with with CuCox17 and by both Sco1 and Cox11 were with copper Cox17 was not on the and not with Sco1 or Cox11. The contained only Sco1 or Cox11 Thus, the copper in the was with Sco1 or Cox11. copper in the was with Cox17. The of the transfer was in two carbonic was used a of Cu(I) by carbonic in a enzyme that be by exogenous Winge D.R. Biochem. PubMed Scopus Google Scholar). a of CuCox17 with the enzyme was by the addition of to the suggesting that Cu(I) ion transfer not not the C57Y mutant Cox17 encoded by the was used a of transfer that C57Y Cox17 Cu(I) exists in and to the was T. Winge D.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The addition of not attenuate the of the C57Y Cox17 In the of both and C57Y Cox17 was by the addition of of Sco1 with C57Y Cox17 not copper, whereas of Cox11 a with C57Y Cox17 copper ion transfer that the C57Y a specific for Sco1. The of the copper transfer was a CuCox17 and the of the to that only a of the Cu is to Sco1 by the CuCox17 A and the addition of Cu(I) in Cox17. of the Cu(I) is of the of Sco1 The copper transfer from Cox17 from an within Cox17 and not Sco1. the Cox17 the of Sco1 that be the metallation of Sco1, the of the that copper transfer to Sco1 observation is corroborated by of Sco1, the a transfer with Sco1 The copper was In a series of Sco1 and the CuCox17 we a for copper transfer from Cox17 Cox17 10 Cox17, transfer to Sco1 is the in Cox17 from to George G.N. G. Winge D.R. PubMed Scopus Google Scholar). Cox17 be in the copper transfer Cox17 in the and of was by and the of Cox17 was by of the In the of Sco1, Cox17 was in and was shown George G.N. G. Winge D.R. PubMed Scopus Google with Cox17 were of Cox17 copper CuCox17 exists in with and George G.N. G. Winge D.R. PubMed Scopus Google Scholar). Cox17 are by CuCox17 into and The was with and A to these were from of CuCox17 in the or of of the was with to of CuCox17 to that only a of the Cu was also to Cox11 of the Cu(I) was to Cox11. of Sco1 and Cox11 not in of Cu(I) not The of Cu(I) to Cox11 to be of Cox11 with CuCox17 copper transfer to Sco1 Cox17 Cu to Cox11 to in this a copper was by of Cox11 the in vitro studies suggested that Cox17 is a specific copper donor to both Sco1 and Cox11. these we expressed the domains of each protein in a the yeast cytoplasm. the domains of SCO1 and was in a yeast expression the expression of each The domains the mitochondrial sequence and transmembrane A was to each to for of each was to the medium with cells harboring the for to and of of by single of Sco1. of the protein by Sco1 the of the copper by and protein by that Sco1 contained The copper in Sco1 was to the cells were in the of suggested that copper metallation of Sco1 in the yeast cytoplasm was but be by exogenous of copper. is to the copper metallation of in cells lacking the R.A. Science. PubMed Scopus Google Scholar). Cox17 is a specific for Sco1, cells harboring the were with a the of the of in of Cox17 in the with a in the (8Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, T. Winge D.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Sco1 was isolated from in to an of by the addition of for to Sco1 from these cells of Cox17 contained of copper of the expression by the in in a copper in the Sco1 of not that the copper was with the Cu(I) site and not with the the was a Sco1 mutant with both in the by of the mutant and in the of Sco1 with of copper Cox17 not with Sco1 in the single suggesting that the two proteins not The metallation of Sco1 from direct copper transfer from Cox17. that Cox17 contains copper, we expressed a Cox17 the on a of expression by the to medium for in of Cox17 in the cytoplasm shown by of the protein by copper and in Thus, a of the Cox17 are in cells in growth in vitro copper transfer studies with no copper transfer from the C57Y mutant Cox17. this mutant was of copper transfer in the yeast both and C57Y were in by in Sco1 purified from a copper a to cells not Cox17 The expression of C57Y Cox17 was the Cox17 used in the of the Cox17 by cells in medium not copper metallation of Sco1, but the Cox17 protein were with the of the C57Y mutant protein not Thus, the C57Y Cox17 is an copper donor in both the in vitro and in vivo In the of Cox17 the copper of the Sco1 is because of of Cox17. not the metallation of Sco1 in cox17Δ cells because in the limited copper from Cox17, we used cox17Δ cells a is and within the mitochondrial IM Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and were in the by in Sco1 from these cells a copper of suggesting that copper ion metallation of Sco1 is dependent on the Cox17 protein growth The was in the and of In the of co-expression of Cox17, Cox11 was from yeast The of the Cox11 was for of the the were purified by on a Cox11 was with of copper from cells in medium copper of Cox11 from cells in medium with in of copper The protein from the in a with a that Cox11 purified from E. a (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). of and in cells copper in copper metallation of Cox11 The copper from in the of Cox17 to in the of Cox17. is that is expressed in E. cells in medium (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The copper ions in the Cox11 purified from E. are (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google the of Cox11 from the yeast with only of copper a exists with a single copper ion the a mutant Cox11 was in which and were by no copper was in the protein Thus, copper by Cox11 is dependent on this with this we that Cox11 purified from E. a Cu(I) to Cox11 and not the (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). the C57Y Cox17 was an copper donor to Sco1 in vitro or in C57Y Cox17 was Cox17 a copper donor to Cox11 in the yeast Cox11 purified from contained of copper, that C57Y Cox17 is an copper donor to Cox11 but not Sco1. The copper transfer by the C57Y mutant to Cox11, but not Sco1, that the transfer to only one The of the mutant copper transfer is with Cox17 with each in distinct Cox17 not with Sco1 or Cox11 in the a of Cox17 with Sco1 or Cox11 the purified Sco1 and Cox11 were by for of of Cox17. Cox17 was not in purified Sco1 or Cox11 Cox17 only with these In an to a Cox17 and Sco1, we and a mutant SCO1 a of the two Cu(I) T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was of Cox17 and the Sco1 mutant lacking the to Cu(I) not Cox17 was to function a copper donor to Sco1 an in the copper metallation of the CuA in Cox2 assembly of cytochrome c oxidase (7Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, Glerum D.M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). was on the of the respiratory of a cox17Δ strain by overexpression of SCO1 (7Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (281) Google and on the that Sco1 is a protein T. George G.N. Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In the present studies we this and the of Cox17 to show for the first that is also a copper donor to Cox11. Sco1, Cox11 is a protein, suggesting that copper metallation of Sco1 and Cox11 is an in the transfer of copper to the CuA site in Cox2 and CuB site in (11Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). were used to the of the copper transfer to both Sco1 and Cox11. in vitro studies with purified proteins copper transfer from CuCox17 to Sco1 or Cox11. of this copper transfer was by the of transfer to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, the C57Y mutant of Cox17 to transfer copper to Sco1 but was competent for copper transfer to Cox11. the in vitro transfer studies were corroborated by a yeast cytoplasm Soluble domains of Sco1 and Cox11 lacking the mitochondrial targeting sequence and transmembrane domains were expressed in the yeast cytoplasm in the and of Cox17 of Sco1 or Cox11 that copper metallation of protein was dependent on growth of cells in medium exogenous copper ions or co-expression of Cox17. The metallation of Sco1 or Cox11 in yeast in copper was in Sco1 from yeast encoded Cox17. copper metallation of Sco1 from the of Cox17 within the cytoplasm (8Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). metallation of Sco1 was purified from cells Cox17 tethered to the mitochondrial inner membrane. in the yeast cytoplasm was by a of copper transfer to Sco1 by the C57Y Cox17 copper transfer by the mutant to Cox11 in with the in vitro Yeast harboring C57Y Cox17 are respiratory-deficient and cytochrome c oxidase activity (6Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). The mutant protein fails to to a within the mitochondrial in the or of a heterologous targeting sequence T. Winge D.R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The C57Y Cox17 protein Cu(I) and the protein George G.N. G. Winge D.R. PubMed Scopus Google Scholar, Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the of the protein not from defective copper ion The present show that C57Y Cox17 is defective only in copper transfer to Sco1. The of the mutant protein to transfer copper to Cox11 that Cox17 have for Sco1 and Cox11. that the an helix Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the protein be an important for Sco1, but not Cox11. were to Cox17 and Sco1 or Cox11 with in vitro or yeast cytoplasm by that its with Sco1 and Cox11 be A exists with the other two copper of the and and proteins not been The observation that copper transfer not Cox17 of Cu(I) in the in vitro was with Sco1 or Cox11, copper with Cox17. Because Cox17 contains a George G.N. G. Winge D.R. PubMed Scopus Google the that only a of the Cu(I) ions is for The is that the copper ions within the are not have to by to the number of transfer studies the of metallation of CcO. Cox17 be within the by transfer of copper from the copper Winge D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). CuCox17 Cu(I) ions to both Sco1 and Cox11 for to Cox2 and The metallation of Cox2 is to of the Cox2 into the by Mol. Biol. 2002; Scopus (91) Google Scholar). transfer Sco1 and Cox2 shown A. M. Rodel G. 2000; Scholar). is this both copper ions in the CuA site are from are to this yeast cytoplasm a to copper transfer from Sco1 to Cox2 and to the function of from the of to the for synthesis the of

We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu(+)-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu(+) in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome c oxidase 1 and 2 proteins (SCO1 and SCO2), each of which is required for assembly and metalation of functionally active cytochrome c oxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a global copper deficiency at the whole cell level, total copper and exchangeable mitochondrial Cu(+) pools in SCO1 and SCO2 patient fibroblasts are largely unaltered relative to wild-type controls. Our findings reveal that the cell maintains copper homeostasis in mitochondria even in situations of copper deficiency and mitochondrial metallochaperone malfunction, illustrating the importance of regulating copper stores in this energy-producing organelle.