Cytochrome c Oxidase Subassemblies in Fibroblast Cultures from Patients Carrying Mutations in COX10, SCO1, or SURF1
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m309232200
ISSN1083-351X
AutoresSiôn L. Williams, Isabelle Valnot, Pierre Rustin, Jan‐Willem Taanman,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoCytochrome c oxidase contains two redox-active copper centers (CuA and CuB) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the CuA center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1·COX4·COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1·COX4·COX5A subassembly, suggesting that MTCO2 associates with the MTCO1·COX4·COX5A subassembly after the CuA center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1·COX4·COX5A subassembly. Cytochrome c oxidase contains two redox-active copper centers (CuA and CuB) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the CuA center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1·COX4·COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1·COX4·COX5A subassembly, suggesting that MTCO2 associates with the MTCO1·COX4·COX5A subassembly after the CuA center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1·COX4·COX5A subassembly. Cytochrome c oxidase (EC 1.9.3.1) belongs to the superfamily of terminal oxidases present in all aerobic organisms (1Calhoun M.W. Thomas J.W. Gennis R.B. Trends Biochem. Sci. 1994; 19: 325-330Abstract Full Text PDF PubMed Scopus (163) Google Scholar). In eukaryotic cells, cytochrome c oxidase is embedded as a dimer in the mitochondrial inner membrane. Electron transfer by the enzyme from cytochrome c to molecular oxygen is coupled to proton pumping across the membrane, contributing to the mitochondrial membrane potential. The redox centers involved in electron transfer are two copper centers (CuA and CuB) and two heme A moieties (a and a3) (2Michel H. Behr J. Harrenga A. Kannt A. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 329-356Crossref PubMed Scopus (378) Google Scholar). The eukaryotic enzyme contains three mitochondrial DNA (mtDNA)-encoded subunits: MTCO1, MTCO2, and MTCO3. These subunits are homologous to polypeptides found in bacterial cytochrome c oxidases (3Saraste M. Q. Rev. Biophys. 1990; 23: 331-366Crossref PubMed Scopus (341) Google Scholar). They form both the catalytic and structural core of the enzyme (4Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1914) Google Scholar). The hydrophobic interior of MTCO1 coordinates the heme a group and a binuclear center composed of heme a3 and CuB. The intermembrane space domain of MTCO2 coordinates the binuclear CuA center (4Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1914) Google Scholar). In addition to the three mtDNA-encoded subunits, mammalian cytochrome c oxidase contains 10 nuclear DNA-encoded subunits: COX4, COX5A, COX5B, COX6A, COX6B, COX6C, COX7A, COX7B, COX7C, and COX8. In humans, COX6A and COX7A exist as skeletal/cardiac muscle (COX6A2 and COX7A1) and ubiquitously expressed (COX6A1 and COX7A2) isoforms (5Taanman J.-W. J. Bioenerg. Biomembr. 1997; 29: 151-163Crossref PubMed Scopus (79) Google Scholar). Although not confirmed at the protein level, human isoforms of COX4, COX6B, and COX8 have also been identified (6Hüttemann M. Kadenbach B. Grossman L.I. Gene (Amst.). 2001; 267: 111-123Crossref PubMed Scopus (144) Google Scholar, 7Hüttemann M. Schmidt T.R. Grossman L.I. Gene (Amst.). 2003; 312: 95-102Crossref PubMed Scopus (47) Google Scholar, 8Hüttemann M. Jaradat S. Grossman L.I. Mol. Reprod. Dev. 2003; 66: 8-16Crossref PubMed Scopus (97) Google Scholar). Although significant progress has been made concerning the structure and catalysis of cytochrome c oxidase (2Michel H. Behr J. Harrenga A. Kannt A. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 329-356Crossref PubMed Scopus (378) Google Scholar), the assembly of the mammalian enzyme has remained relatively unexplored. Studies in yeast have indicated that assembly is dependent on numerous nuclearly encoded proteins in addition to the constituent subunits and prosthetic groups (9Barrientos A. Barros M.H. Valnot I. Rötig A. Rustin P. Tzagoloff A. Gene (Amst.). 2002; 286: 53-63Crossref PubMed Scopus (157) Google Scholar, 10Carr H.S. Winge D.R. Acc. Chem. Res. 2003; 36: 309-316Crossref PubMed Scopus (194) Google Scholar). Human homologues for a number of these assembly factors have been identified, and some are known to be involved in disease (9Barrientos A. Barros M.H. Valnot I. Rötig A. Rustin P. Tzagoloff A. Gene (Amst.). 2002; 286: 53-63Crossref PubMed Scopus (157) Google Scholar). In this study, we have investigated the presence of subassemblies of cytochrome c oxidase in fibroblast cultures from patients harboring mutations in the assembly factors SURF1, SCO1, or COX10 to learn more about assembly mechanism of the human enzyme. SURF1 resides in the mitochondrial inner membrane (11Tiranti V. Galimberti C. Nijtmans L. Bovolenta S. Perini M.P. Zeviani M. Hum. Mol. Genet. 1999; 8: 2533-2540Crossref PubMed Scopus (123) Google Scholar, 12Yao J. Shoubridge E.A. Hum. Mol. Genet. 1999; 8: 2541-2549Crossref PubMed Scopus (99) Google Scholar). Mutations in the SURF1 gene are a relatively common cause of mitochondrial disease (13Zhu Z. 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Absence of SURF1 or its yeast homologue, Shy1p, causes a severe deficiency of cytochrome c oxidase but does not completely abolish enzyme activity in patients (13Zhu Z. Yao J. Johns T. Fu K. De B.I. Macmillan C. Cuthbert A.P. Newbold R.F. Wang J. Chevrette M. Brown G.K. Brown R.M. Shoubridge E.A. Nat. Genet. 1998; 20: 337-343Crossref PubMed Scopus (533) Google Scholar, 14Tiranti V. Hoertnagel K. Carrozzo R. Galimberti C. Munaro M. Granatiero M. Zelante L. Gasparini P. Marzella R. Rocchi M. Bayona-Bafaluy M.P. Enriquez J.A. Uziel G. Bertini E. Dionisi-Vici C. Franco B. Meitinger T. Zeviani M. Am. J. Hum. Genet. 1998; 63: 1609-1621Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar), Surf1 knockout mice (16Agostino A. Invernizzi F. Tiveron C. Fagiolari G. Prelle A. Lamantea E. Giavazzi A. Battaglia G. Tatangelo L. Tiranti V. Zeviani M. Hum. Mol. Genet. 2003; 12: 399-413Crossref PubMed Scopus (69) Google Scholar), or yeast shy1 mutants (17Barrientos A. Korr D. Tzagoloff A. EMBO J. 2002; 21: 43-52Crossref PubMed Scopus (139) Google Scholar). This suggests that the protein has a redundant function. The precise role of SURF1 is, however, unknown. SCO1 and SCO2 are the human members of the SCO family of proteins that appear to be widely distributed in aerobic organisms (18Papadopoulou L.C. Sue C.M. Davidson M.M. Tanji K. Nishino I. Sadlock J.E. Krishna S. Walker W. Selby J. Glerum D.M. Coster R.V. Lyon G. Scalais E. Lebel R. Kaplan P. Shanske S. De Vivo D.C. Bonilla E. Hirano M. Dimauro S. Schon E.A. Nat. Genet. 1999; 23: 333-337Crossref PubMed Scopus (485) Google Scholar, 19Mattatall N.R. Jazairi J. Hill B.C. J. Biol. Chem. 2000; 275: 28802-28809Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 20McEwan A.G. Lewin A. Davy S.L. Boetzel R. Leech A. Walker D. Wood T. Moore G.R. FEBS Lett. 2002; 518: 10-16Crossref PubMed Scopus (48) Google Scholar). Like SURF1, SCO1 and SCO2 are mitochondrial inner membrane proteins (21Paret C. Ostermann K. Krause-Buchholz U. Rentzsch A. Rödel G. FEBS Lett. 1999; 447: 65-70Crossref PubMed Scopus (40) Google Scholar, 22Buchwald P. Krummeck G. Rödel G. Mol. Gen. Genet. 1991; 229: 413-420Crossref PubMed Scopus (72) Google Scholar, 23Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). SCO proteins have a conserved CXXXC motif protruding into the intermembrane space, similar to the CXXXC motif present at the CuA site of MTCO2 (24Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Studies of the yeast SCO protein Sco1p have indicated that the protein binds one Cu+ ion, ligated via the cysteine residues present in the CXXXC motif and a conserved histidine residue (25Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The Bacillus subtilis SCO protein YpmQ is required for the biosynthesis of cytochrome c oxidase but not for the alternative terminal oxidase, menaquinol oxidase, which only contains a CuB site (19Mattatall N.R. Jazairi J. Hill B.C. J. Biol. Chem. 2000; 275: 28802-28809Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This observation together with mutation and co-immunoprecipitation studies in yeast (26Dickinson E.K. Adams D.L. Schon E.A. Glerum D.M. J. Biol. Chem. 2000; 275: 26780-26785Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 27Lode A. Kuschel M. Paret C. Rödel G. FEBS Lett. 2000; 485: 19-24Crossref PubMed Scopus (92) Google Scholar) C+ suggest that SCO proteins transfer copper to the CuA site. An unrelated Cu+-binding protein, COX11, appears to be involved in the provision of copper to the CuB site (28Hiser L. Di Valentin M. Hamer A.G. Hosler J.P. J. Biol. Chem. 2000; 275: 619-623Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 29Carr 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). It is not clear why two SCO proteins are present in human mitochondria. Mutations in SCO2 have been reported in several unrelated patients (18Papadopoulou L.C. Sue C.M. Davidson M.M. Tanji K. Nishino I. Sadlock J.E. Krishna S. Walker W. Selby J. Glerum D.M. Coster R.V. Lyon G. Scalais E. Lebel R. Kaplan P. Shanske S. De Vivo D.C. Bonilla E. Hirano M. Dimauro S. Schon E.A. Nat. Genet. 1999; 23: 333-337Crossref PubMed Scopus (485) Google Scholar, 30Jaksch M. Ogilvie I. Yao J. Kortenhaus G. Bresser H.G. Gerbitz K.D. Shoubridge E.A. Hum. Mol. Genet. 2000; 9: 795-801Crossref PubMed Scopus (193) Google Scholar, 31Jaksch M. Horvath R. Horn N. Auer D.P. Macmillan C. Peters J. Gerbitz K.D. Kraegeloh-Mann I. Muntau A. Karcagi V. Kalmanchey R. Lochmuller H. Shoubridge E.A. Freisinger P. Neurology. 2001; 57: 1440-1446Crossref PubMed Scopus (75) Google Scholar), but only a single family with SCO1 mutations has been described (32Valnot I. Osmond S. Gigarel N. Mehaye B. Amiel J. Cormier-Daire V. Munnich A. Bonnefont J.P. Rustin P. Rotig A. Am. J. Hum. Genet. 2000; 67: 1104-1109Abstract Full Text Full Text PDF PubMed Google Scholar). The patients from this family carried a frameshift mutation on one allele, introducing a premature termination codon, and a missense mutation on the other allele, resulting in a proline to leucine replacement immediately adjacent to the CXXXC motif. Cytochrome c oxidase activity was markedly decreased in patient tissues, but there was still some residual activity, suggesting that the amino acid substitution does not completely eliminate SCO1 function. COX10 is a farnesyltransferase that catalyzes the conversion of heme B (ferric protoporphyrin IX) to heme O (33Saiki K. Mogi T. Ogura K. Anraku Y. J. Biol. Chem. 1993; 268: 26041-26044Abstract Full Text PDF PubMed Google Scholar, 34Tzagoloff A. Nobrega M. Gorman N. Sinclair P. Biochem. Mol. Biol. Int. 1993; 31: 593-598PubMed Google Scholar). Heme O is subsequently converted into heme A in a biosynthetic pathway involving COX15 (35Barros M.H. Carlson C.G. Glerum D.M. Tzagoloff A. FEBS Lett. 2001; 492: 133-138Crossref PubMed Scopus (117) Google Scholar). COX10 and COX15 are considered constituents of the mitochondrial inner membrane. Studies in yeast have indicated that both proteins are necessary for cytochrome c oxidase biosynthesis (36Nobrega M.P. Nobrega F.G. Tzagoloff A. J. Biol. Chem. 1990; 265: 14220-14226Abstract Full Text PDF PubMed Google Scholar, 37Glerum D.M. Muroff I. Jin C. Tzagoloff A. J. Biol. Chem. 1997; 272: 19088-19094Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Three families with COX10 gene mutations have been documented (38Valnot I. Kleist-Retzow J.C. Barrientos A. Gorbatyuk M. Taanman J.W. Mehaye B. Rustin P. Tzagoloff A. Munnich A. Rotig A. Hum. Mol. Genet. 2000; 9: 1245-1249Crossref PubMed Scopus (256) Google Scholar, 39Antonicka H. Leary S.C. Guercin G.H. Agar J.N. Horvath R. Kennaway N.G. Harding C.O. Jaksch M. Shoubridge E.A. Hum. Mol. Genet. 2003; 12: 2693-2702Crossref PubMed Scopus (198) Google Scholar). Patients were either homozygous or compound heterozygous for missense mutations, changing evolutionarily conserved amino acid residues. Their tissues retained some residual cytochrome c oxidase activity, suggesting that the mutated farnesyltransferase was still partially active. Consistent with this view, mutated human COX10 was able to complement the respiratory defect of a yeast cox10 null mutant when present on a high copy plasmid but, unlike wild-type human COX10, not when present on a low copy plasmid (38Valnot I. Kleist-Retzow J.C. Barrientos A. Gorbatyuk M. Taanman J.W. Mehaye B. Rustin P. Tzagoloff A. Munnich A. Rotig A. Hum. Mol. Genet. 2000; 9: 1245-1249Crossref PubMed Scopus (256) Google Scholar). The fibroblast cultures which we investigated were derived from four unrelated patients with SURF1 mutations (40Williams S.L. Taanman J.W. Hansíková H. Houšt'kovát H. Chowdhury S. Zeman J. Houštěk J. Mol. Genet. Metab. 2001; 73: 340-343Crossref PubMed Scopus (22) Google Scholar, 41Williams S.L. Scholte H.R. Gray R.G. Leonard J.V. Schapira A.H. Taanman J.W. Lab. Investig. 2001; 81: 1069-1077Crossref PubMed Scopus (15) Google Scholar), a patient with SCO1 mutations (32Valnot I. Osmond S. Gigarel N. Mehaye B. Amiel J. Cormier-Daire V. Munnich A. Bonnefont J.P. Rustin P. Rotig A. Am. J. Hum. Genet. 2000; 67: 1104-1109Abstract Full Text Full Text PDF PubMed Google Scholar), and a patient with a COX10 mutation (38Valnot I. Kleist-Retzow J.C. Barrientos A. Gorbatyuk M. Taanman J.W. Mehaye B. Rustin P. Tzagoloff A. Munnich A. Rotig A. Hum. Mol. Genet. 2000; 9: 1245-1249Crossref PubMed Scopus (256) Google Scholar). Quantitative immunoblot analysis of native gels revealed residual levels of fully assembled enzyme in all cultures but also some accumulation of subassemblies. The subunit composition of the subassemblies identified in the patient cells provides insights into the temporal positioning of MTCO1, COX4, and COX5A assembly, heme A insertion, and formation of the CuA center within the assembly pathway of human cytochrome c oxidase. Cell Cultures—Fibroblasts were grown from explant cultures of skin biopsies derived from four infants carrying SURF1 mutations (P1–4), one infant carrying SCO1 mutations, one infant carrying a COX10 mutation, and four pediatric controls. All patients harboring SURF1 mutations suffered from Leigh syndrome and died at <4 years of age (40Williams S.L. Taanman J.W. Hansíková H. Houšt'kovát H. Chowdhury S. Zeman J. Houštěk J. Mol. Genet. Metab. 2001; 73: 340-343Crossref PubMed Scopus (22) Google Scholar, 41Williams S.L. Scholte H.R. Gray R.G. Leonard J.V. Schapira A.H. Taanman J.W. Lab. Investig. 2001; 81: 1069-1077Crossref PubMed Scopus (15) Google Scholar). The patient harboring SCO1 mutations presented with liver failure and encephalopathy and died at age 2 months (32Valnot I. Osmond S. Gigarel N. Mehaye B. Amiel J. Cormier-Daire V. Munnich A. Bonnefont J.P. Rustin P. Rotig A. Am. J. Hum. Genet. 2000; 67: 1104-1109Abstract Full Text Full Text PDF PubMed Google Scholar). The patient harboring a COX10 mutation presented with tubulopathy and leukodystrophy, and died at 2 years of age (38Valnot I. Kleist-Retzow J.C. Barrientos A. Gorbatyuk M. Taanman J.W. Mehaye B. Rustin P. Tzagoloff A. Munnich A. Rotig A. Hum. Mol. Genet. 2000; 9: 1245-1249Crossref PubMed Scopus (256) Google Scholar). The mutations carried by the patients have been reported elsewhere (32Valnot I. Osmond S. Gigarel N. Mehaye B. Amiel J. Cormier-Daire V. Munnich A. Bonnefont J.P. Rustin P. Rotig A. Am. J. Hum. Genet. 2000; 67: 1104-1109Abstract Full Text Full Text PDF PubMed Google Scholar, 38Valnot I. Kleist-Retzow J.C. Barrientos A. Gorbatyuk M. Taanman J.W. Mehaye B. Rustin P. Tzagoloff A. Munnich A. Rotig A. Hum. Mol. Genet. 2000; 9: 1245-1249Crossref PubMed Scopus (256) Google Scholar, 40Williams S.L. Taanman J.W. Hansíková H. Houšt'kovát H. Chowdhury S. Zeman J. Houštěk J. Mol. Genet. Metab. 2001; 73: 340-343Crossref PubMed Scopus (22) Google Scholar, 41Williams S.L. Scholte H.R. Gray R.G. Leonard J.V. Schapira A.H. Taanman J.W. Lab. Investig. 2001; 81: 1069-1077Crossref PubMed Scopus (15) Google Scholar). Control skin biopsies were taken from children younger than 3 years undergoing orthopedic surgery. Informed parental consent, in accordance with the guidelines of the participating institutions, was obtained for all biopsies. The A549 human lung carcinoma cell line was obtained from the European Collection of Cell Cultures (Salisbury, UK). The cell line was depleted of its mtDNA by long term exposure to ethidium bromide to yield cell line A549 ρ0 (42King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1444) Google Scholar). Cells were cultured under standard conditions in medium supplemented with 110 mg/ml sodium pyruvate and 50 μg/ml uridine (43Taanman J.W. Muddle J.R. Muntau A.C. Hum. Mol. Genet. 2003; 12: 1839-1845Crossref PubMed Scopus (71) Google Scholar) to allow the respiratory chain-deficient cells to grow (42King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1444) Google Scholar). Preparation of Mitoplasts and Mitochondria—Mitoplast-enriched pellets were prepared from fibroblasts as described (44Klement P. Nijtmans L.G. Van den Bogert C. Houštěk J. Anal. Biochem. 1995; 231: 218-224Crossref PubMed Scopus (94) Google Scholar), using a solution of 4 mg/ml digitonin. Mitoplasts were washed with phosphate-buffered saline (PBS). 1The abbreviations used are: PBS, phosphate-buffered saline; VDAC, voltage-dependent anion-selective channel; HSP, heat shock protein. Mitochondria were isolated by differential centrifugation of digitonin-treated, homogenized cells (45Bourgeron T. Chretien D. Rötig A. Munnich A. Rustin P. Biochem. Biophys. Res. Commun. 1992; 186: 16-23Crossref PubMed Scopus (53) Google Scholar). Samples were stored at –80 °C. Protein concentrations were determined with the BCA™ protein assay kit (Pierce). Enzyme Activity Assays—Cytochrome c oxidase and citrate synthase activities of mitochondrial preparations were measured spectrophotometrically essentially as described (46Srere P.A. Methods Enzymol. 1969; 13: 3-11Crossref Scopus (2029) Google Scholar, 47Wharton D.C. Tzagoloff A. Methods Enzymol. 1967; 10: 245-250Crossref Scopus (1330) Google Scholar). Each reported value is the mean of 4–6 independent measurements. Histochemical staining for cytochrome c oxidase activity in native gels was performed as described previously (48Zerbetto E. Vergani L. Dabbeni-Sala F. Electrophoresis. 1997; 18: 2059-2064Crossref PubMed Scopus (254) Google Scholar). Primary Antibodies—We used mouse monoclonal antibodies raised against human MTCO1 or MTCO2 (49Taanman J.-W. Burton M.D. Marusich M.F. Kennaway N.G. Capaldi R.A. Biochim. Biophys. Acta. 1996; 1315: 199-207Crossref PubMed Scopus (54) Google Scholar), yeast MTCO3 (50Taanman J.-W. Capaldi R.A. J. Biol. Chem. 1993; 268: 18754-18761Abstract Full Text PDF PubMed Google Scholar), and bovine COX4, COX5A, COX5B, COX6B, or COX6C (49Taanman J.-W. Burton M.D. Marusich M.F. Kennaway N.G. Capaldi R.A. Biochim. Biophys. Acta. 1996; 1315: 199-207Crossref PubMed Scopus (54) Google Scholar, 51Taanman J.-W. Hall R.E. Tang C. Marusich M.F. Kennaway N.G. Capaldi R.A. Biochim. Biophys. Acta. 1993; 1225: 95-100Crossref PubMed Scopus (45) Google Scholar). In addition, we used a rabbit polyclonal antiserum raised against an oligopeptide corresponding to the N terminus of bovine COX6A1 (51Taanman J.-W. Hall R.E. Tang C. Marusich M.F. Kennaway N.G. Capaldi R.A. Biochim. Biophys. Acta. 1993; 1225: 95-100Crossref PubMed Scopus (45) Google Scholar). All the above antibodies have been developed in the laboratory of Dr. R. A. Capaldi (University of Oregon); the monoclonal antibodies are available through Molecular Probes (Eugene, OR). We also used mouse monoclonal antibodies raised against the flavoprotein subunit of succinate:ubiquinone oxidoreductase (SDHA; Molecular Probes) (52Marusich M.F. Robinson B.H. Taanman J.W. Kim S.J. Schillace R. Smith J.L. Capaldi R.A. Biochim. Biophys. Acta. 1997; 1362: 145-159Crossref PubMed Scopus (97) Google Scholar), the voltage-dependent anion-selective channel (VDAC; monoclonal 31HL, Calbiochem), and mitochondrial heat shock protein 70 (HSP70; Alexis Biochemicals, San Diego, CA). Immunoblot Analysis—Gel electrophoresis and blotting was carried out with the Mini Protean® 3 System (Bio-Rad). For immunoblot analysis of one-dimensional denaturing gels, mitochondrial fractions were dissociated in 50 mm Tris·HCl (pH 6.8), 12% glycerol, 4% SDS, 2% β-mercaptoethanol, and 0.01% bromphenol blue for 30 min at 37 °C and resolved on either 7.5, 12.5, or 15% polyacrylamide, 0.1% SDS, 5.5 m urea gels run according to Laemmli (53Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206602) Google Scholar). For immunoblot analysis of one-dimensional native gels, mitochondrial or mitoplast fractions were solubilized with n-dodecyl-β-d-maltoside, as described elsewhere (11Tiranti V. Galimberti C. Nijtmans L. Bovolenta S. Perini M.P. Zeviani M. Hum. Mol. Genet. 1999; 8: 2533-2540Crossref PubMed Scopus (123) Google Scholar, 54Schägger H. Bentlage H. Ruitenbeek W. Pfeiffer K. Rotter S. Rother C. Bottcher-Purkl A. Lodemann E. Electrophoresis. 1996; 17: 709-714Crossref PubMed Scopus (32) Google Scholar), and resolved on blue native 8–16% polyacrylamide gels as devised by Schägger (55Schägger H. Electrophoresis. 1995; 16: 763-770Crossref PubMed Scopus (71) Google Scholar). For immunoblot analysis of two-dimensional native/denaturing gels, n-dodecyl-β-d-maltoside-solubilized mitochondrial fractions were first separated on blue native 8–16% polyacrylamide gels, followed by separation of single sample lanes on 13.5% polyacrylamide, 0.1% SDS, 5.5 m urea gels. First dimension gel lanes were soaked in 50 mm Tris·HCl (pH 6.8), 1% SDS, 1% β-mercaptoethanol for 15 min, followed by two 10-min soaks in 50 mm Tris·HCl (pH 6.8), 1% SDS. Gel strips were mounted horizontally in the Mini Protean® 3 System, and second dimension gels were poured around them, with a 3% polyacrylamide stacking gel surrounding the first dimension gel strip. Proteins were electrotransferred from the gels onto Immobilon™-P poly(vinylidene difluoride) membranes (Millipore, Bedford, MA) (56Capaldi R.A. Marusich M.F. Taanman J.-W. Methods Enzymol. 1996; 260: 117-132Crossref Scopus (125) Google Scholar). Blots were air-dried overnight, rinsed three times with methanol to remove residual dye, rinsed once with PBS, and incubated in PBS, 10% nonfat dry milk for 1 h to saturate protein-binding sites. Subsequently, blots were incubated with primary antibodies in PBS, 0.3% Tween 20 for 2 h. After three 10-min washes in PBS, 0.3% Tween 20, blots were incubated with either rabbit anti-mouse (Dakocytomation, Ely, UK) or goat anti-rabbit (Bio-Rad) IgG horseradish peroxidase conjugate for 1 h. To enhance detection, blots of two-dimensional gels were washed twice in PBS, 0.3% Tween 20, and incubated with a peroxidase-labeled anti-peroxidase mouse monoclonal antibody (Dakocytomation) for 1 h. All blots were washed three times in PBS, 0.3% Tween 20 and twice in PBS. Immunoreactive material was visualized by chemiluminescence (Western Lightning™ Chemiluminescence Reagent Plus, PerkinElmer Life Sciences). Exposures of films to the blots were chosen such that signals of the patient samples were within the linear range of the diluted control samples. Signals were quantified using the NIH Scion Image application. All blotting experiments were repeated with independently isolated mitochondrial samples. Duplicate experiments yielded consistent results. Purification of MTCO1—Human heart cytochrome c oxidase was a kind gift of Dr. A. O. Muijsers (University of Amsterdam). The enzyme was dissociated in 3% SDS, and subunits were resolved under denaturing conditions on a Bio-Gel® P-60 (Bio-Rad) gel filtration column (57Steffens G. Buse G. Hoppe Seylers. Z. Physiol. Chem. 1976; 357: 1125-1137Crossref PubMed Scopus (95) Google Scholar). 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Metab. 2001; 73: 340-343Crossref PubMed Scopus (22) Google Scholar, 41Williams S.L. Scholte H.R. Gray R.G. Leonard J.V. Schapira A.H. Taanman J.W. Lab. Investig. 2001; 81: 1069-1077Crossref PubMed Scopus (15) Google Scholar). To establish the residual cytochrome c oxidase activity of the fibroblast mitochondrial preparations used in this study, we determined the cytochrome c oxidase activity relative to the activity of the mitochondrial marker enzyme citrate synthase. The relative cytochrome c oxidase activities of the preparations from the SCO1-deficient patient and the COX10-deficient patient were 12 and 10% of control values, respectively. The relative activities of the preparations from the four SURF1-deficient patients P1–4 were 14, 13, 8, and 12% of control values, respectively. Immunoblots of One-dimensional Denaturing Gels—For detection of cytochrome c oxidase subunits on immunoblots, the same mitochondrial preparations as used in the activity assays were separated on SDS-polyacrylamide gels and transferred onto Immobilon™-P membranes. Samples were carefully balanced on the basis of the immunoblot signal of the mitochondrial inner membrane protein SDHA. Equal loading was verified with a
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