FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2046452961> ?p ?o ?g. }

• W2046452961 endingPage "5080" @default.
• W2046452961 startingPage "5072" @default.
• W2046452961 abstract "Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Δ cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed. Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Δ cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed. Copper plays an essential role in the biochemistry of all aerobic organisms (1Linder M.C. Hazegh-Azam M. Am. J. Clin. Nutr. 1996; 63: 7975-8115Google Scholar). This metal functions as a cofactor permitting the facile transfer of electrons in key enzymes including Cu/Zn superoxide dismutase for antioxidant defense, tyrosinase for melanin synthesis, and cytochrome c oxidase for electron transport in the mitochondrial respiratory chain. When copper homeostasis is perturbed, the reactivity of copper with dioxygen may also lead to toxicity (2Henle E.S. Linn S. J. Biol. Chem. 1997; 272: 19095-19098Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 3Valentine J.S. Wertz D.L. Lyons T.J. Liou L.-L. Goto J.J. Gralla E.B. Curr. Opin. Chem. Biol. 1998; 2: 253-262Crossref PubMed Scopus (183) Google Scholar). For this reason, specific mechanisms have evolved for the compartmentalization and trafficking of copper within cells (4Huffman D.L. O'Halloran T.V. Annu. Rev. Biochem. 2001; 70: 677-701Crossref PubMed Scopus (413) Google Scholar).The mechanism of copper ion routing to cytochrome c oxidase (CcO) 1The abbreviations used are: CcOcytochrome c oxidaseIMinner membraneOMouter membraneIMSintermembrane spaceSod1superoxide dismutaseWTwild-type.1The abbreviations used are: CcOcytochrome c oxidaseIMinner membraneOMouter membraneIMSintermembrane spaceSod1superoxide dismutaseWTwild-type. within the mitochondrion is unknown. Bovine CcO consists of 13 polypeptide subunits, three of which (Cox1–Cox3) are encoded by the mitochondrial genome, and the remaining subunits are encoded by the nuclear genome (5Capaldi R.A. Ann. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (512) Google Scholar). The three mitochondrially encoded subunits form the catalytic core of the enzyme. Cox2 requires two copper ions in the binuclear CuA site, and Cox1 requires one copper ion in the CuB site (6Tsukihara 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 (1284) Google Scholar). CcO is localized within the inner mitochondrial membrane. The inner membrane (IM) differs from the outer membrane (OM) in being highly convoluted and folding into tubular cristae. Three internal spaces are created by the double membrane structure. The volume enclosed within the inner membrane is the matrix, which represents about 80% of the total mitochondrial space (7Scorrano L. Ashiya M. Buttle K. Weiler S. Oakes S.A. Mannella C.A. Korsmeyer S.J. Develop. Cell. 2002; 2: 55-67Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). The space between the inner and outer membranes is called the intermembrane space (IMS) and is interrupted by junction points in which the IM and OM are in contact. The IMS is very narrow and is separated from the third space, intracristae space, by tubular cristal junctions (8Frey T.G. Mannella C.A. Trends Biochem. Sci. 2000; 25: 319-324Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 9Mannella C.A. Pfeiffer D.R. Bradshaw P.C. Moraru I.I. Slepchenko B. Loew L.M. Hsieh C. Buttle K. Marko M. IUBMB Life. 2002; 52: 1-7Google Scholar). The bulk of the respiratory complexes, including CcO, exist within the cristae (10Perotti M.E. Anderson W.A. Swift H. J. Histochem. Cytochem. 1983; 31: 351-365Crossref PubMed Scopus (42) Google Scholar). Thus, assembly of functional CcO requires transport of nuclear-encoded subunits across both mitochondrial membranes. It is unclear whether the assembly of newly synthesized subunits occurs within the cristae or on the peripheral surface of the IM.In yeast, several metallochaperones have been identified that shuttle copper ions to sites of utilization (4Huffman D.L. O'Halloran T.V. Annu. Rev. Biochem. 2001; 70: 677-701Crossref PubMed Scopus (413) Google Scholar). Copper insertion into Cu,Zn-superoxide dismutase (Sod1) in yeast requires the function of the Lys7 (CCS) metallochaperone (11Culotta V.C. Klomp L.W.J. Strain J. Casareno R.L.B. Krems B. Gitlin J.D. J. Biol. Chem. 1997; 272: 23469-23472Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 12Gamonet F. Lauquin G.J.M. Eur. J. Biochem. 1998; 251: 716-723Crossref PubMed Scopus (42) Google Scholar, 13Casareno R.L.B. Waggoner D. Gitlin J.D. J. Biol. Chem. 1998; 273: 23625-23628Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 14Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Crossref PubMed Scopus (1345) Google Scholar). Like-wise, Ccc2, a P-type ATPase copper ion transporter, receives copper ions from the Atx1 metallochaperone (15Lin S.-J. Pufahl R.A. Dancis A. O'Halloran T.V.O. Culotta V.C. J. Biol. Chem. 1997; 272: 9215-9220Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 16Huffman D.L. O'Halloran T.V. J. Biol. Chem. 2000; 275: 18611-18614Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Based on the necessity of Lys7 and Atx1 for shuttling copper ions to sites of utilization, the prediction is that CcO would require a specific protein involved in copper shuttling to the mitochondrion.Cox17, a 8-kDa protein conserved in eukaryotes, has several characteristics consistent with a role in copper ion trafficking to the mitochondrion (17Amaravadi R. Glerum D.M. Tzagoloff A. Human Genetics. 1997; 99: 329-333Crossref PubMed Scopus (134) Google Scholar). The initial observation implicating Cox17 in metallation of CcO was the demonstration that the respiratory defect of cells harboring a non-functional cox17–1 mutant was suppressed by the addition of near toxic levels of copper ions in the growth medium (18Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Two subsequent observations consistent with this postulate were the dual localization of Cox17 in both the cytosol and mitochondrial intermembrane space compartments and the Cu(I) binding ability of Cox17 (19Beers J. Glerum D.M. Tzagoloff A. J. Biol. Chem. 1997; 272: 33191-33196Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 20Heaton D.N. George G.N. Garrison G. Winge D.R. Biochem. 2001; 40: 743-751Crossref PubMed Scopus (103) Google Scholar). Three cysteinyl residues present in a Cys-Cys-Xaa-Cys sequence motif are critical for in vivo Cox17 function and Cu(I) ion binding (21Heaton D. Nittis T. Srinivasan C. Winge D.R. J. Biol. Chem. 2000; 275: 37582-37587Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Although these observations suggest that Cox17 plays an important role in copper ion trafficking, the direct role of Cox17 in copper delivery to the mitochondria is unresolved. Cox17 may deliver Cu(I) ions to a mitochondrial OM transporter in analogy to the Atx1 routing of Cu(I) ions to the Ccc2 trans-Golgi translocase. Alternatively, Cox17 may ferry Cu(I) ions directly across the semi-porous mitochondrial OM. If the former is the case, Cox17 may have a secondary function within the IMS. If, however, Cox17 translocates the OM for Cu(I) delivery, we predict that Cox17 must contain a mitochondrial targeting sequence directing import. Proteins imported across the OM are typically synthesized as a precursor protein with an N-terminal import sequence directing import. Such proteins are taken up as unfolded proteins through the TOM complex (22Gaume B. Klaus C. Ungermann C. Guiard B. Neupert W. Brunner M. EMBO J. 1998; 17: 6497-6507Crossref PubMed Scopus (79) Google Scholar, 23Pfanner N. Wiedemann N. Curr. Opin. Cell Biol. 2002; 14: 400-411Crossref PubMed Scopus (77) Google Scholar). Classical mitochondrial import sequences are enriched in cationic residues and appear to adopt an amphipathic helix (24Roise D. Theiler F. Horvath S.J. Tomich J.M. Richards J.H. Allison D.S. Schatz G. EMBO J. 1988; 7: 649-653Crossref PubMed Scopus (189) Google Scholar). The lack of a classical mitochondrial import sequence in Cox17 does not preclude translocation across the mitochondrial OM, because a number of proteins including cytochrome c and heme lyase are imported through the TOM complex via non-cleaved internal sequence motifs (25Diekert K. Kispal G. Guiard B. Lill R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11752-11757Crossref PubMed Scopus (106) Google Scholar).Additional proteins implicated in copper ion insertion into CcO include Sco1 and Cox11 in yeast, although both of these proteins are membrane proteins localized within the IM (26Schulze M. Rodel G. Mol. Gen. Genet. 1988; 211: 492-498Crossref PubMed Scopus (98) Google Scholar, 27Rentzsch N. Krummeck-WeiB G. Hofer A. Bartuschka A. Ostermann K. Rodel G. Curr. Genet. 1999; 35: 103-108Crossref PubMed Scopus (80) Google Scholar, 28Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 29Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 30Carr H.S. George G.N. Winge D.R. J. Biol. Chem. 2002; 277: 31237-31242Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Sco1 and Cox11 are implicated in reactions of direct copper transfer to the CuA site in Cox2 and CuB site in Cox1, respectively. CuCox17 may deliver Cu(I) to both Sco1 and Cox11 for subsequent donation to the CuA and CuB sites, respectively. Yeast contains a second Sco1-like molecule, designated Sco2, that is unimportant in the CcO assembly pathway (28Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 31Buchwald P. Krummeck G. Rodel G. Mol. Gen. Genet. 1991; 229: 413-420Crossref PubMed Scopus (71) Google Scholar).Molecular dissection of Cox17 reported here demonstrate that a C-terminal sequence motif is important for mitochondrial uptake and accumulation within the IMS. Furthermore, we show that Cox17 tethered in the IM by Sco2 is functional, suggesting that cycling of Cox17 between the cytosol and the IMS is not essential for function.MATERIALS AND METHODSYeast Strains—A cox17Δ strain (MAT a, ade2–1, his3–1,15, leu2,3,112, trp1–1, ura3–1, ΔCOX17::TRP1) kindly provided by Dr. A. Tzagoloff (18Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) and its isogenic wild-type W303 strain (MAT a, ade2–1, his3–1,15, leu2,3,112, trp1–1, ura3–1) were used for all experiments. Yeast strains were cultured on plates or in liquid medium in either complete medium lacking uracil to ensure maintenance of plasmids or rich media (yeast extract and peptone) with glucose (YPD), glycerol (YPG), or galactose (YPGal) as carbon sources. Copper limiting complete medium contained 0.03 mm bathocuproine sulfonate (Sigma). DNA transformations were performed using a lithium acetate protocol.Vector Construction—All genes were cloned into a yeast low copy vector (YCpRS316) under the control of regulatable MET25 or GAL10 promoters, or under the control of the native COX17 promoter and terminated with either the CYC1 or COX17 terminator (32Mumberg D. Muller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (793) Google Scholar). All constructs used in CYC1 terminator except those used in Fig. 6. Maximum expression with the MET25 promoter occurs in medium lacking methionine, whereas minimal expression occurs in medium containing 0.67 mm methionine (5 × the normal Met level). Maximum expression with the GAL10 promoter occurs in medium containing 2% galactose, limited expression is observed in medium containing 3% glycerol, and minimal expression occurs in medium containing 2% glucose. Deletion mutants, termed Δ9 (lacking residues 1–8) and 59Δ (lacking residues 60–69) were generated by PCR and subcloned into pRS316 as BamHI/SalI fragments. To construct a heterologous IMS targeting sequence, the first 167 codons of the CYB2 gene were excised from vector pEB15 (33Beasley E.M. Muller S. Schatz G. EMBO J. 1993; 12: 2303-2311Crossref PubMed Scopus (65) Google Scholar) as an EcoRI/BamHI fragment and subcloned in frame to various genes creating fusion proteins termed pCox17, pΔ9, pC57Y, and p59Δ. The IM-tethered Cox17 was constructed by amplifying the first 104 codons of SCO2 by PCR with flanking BamHI sites and inserting it in frame with COX17 or the Δ9 truncate. The resulting fusion protein consisted of the N-terminal mitochondrial import sequence and transmembrane domain of Sco2 fused to Cox17. The head-to-tail dimer of Cox17 was engineered by amplifying the C57Y mutant allele without its stop codon by PCR with flanking BamHI sites. This amplified C57Y variant allele was inserted at the 5′ end of a second mutant allele encoding a C23S/C24S double substitution. Three double mutant alleles encoding F50D/I51D, C57A/C58M, and Y61D/G62D alleles were generated using the Stratagene QuikChange site-directed mutagenesis kit. All mutant genes were sequenced prior to transformation into the cox17Δ strain.Mitochondrial Isolation and Treatment—Mitochondria were isolated according to the method of Glick and Pons (34Glick B.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (285) Google Scholar) in the presence of 0.5 mm phenylmethylsulfonyl fluoride. The mitochondria protein concentration was determined by the Bradford assay (35Bradford N.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213288) Google Scholar). Hypotonic lysis of mitochondria was performed by diluting intact mitochondria (150–250 μg) into 7 volumes of 20 mm Hepes-KOH, pH 6.7, buffer yielding intact mitoplasts. Membrane-associated proteins were solubilized by treatment of the mitoplasts in unbuffered 0.1 m Na2CO3. Inner membrane proteins were solubilized with 1% Triton X-100. Membranes were removed by centrifugation at 12,000 × g for 10 min.Cytochrome c Oxidase Assays—Respiratory function was assayed by growth tests on synthetic complete selective plates (–Ura, complete medium) using the non-fermentable carbon source, 3% glycerol. CcO enzymatic activity in isolated mitochondria was quantified by monitoring the oxidation of 32 μm reduced bovine cytochrome c at 550 nm by mitochondrial samples (5 to 10 μg protein) in 40 mm KH2PO4, pH 6.7, 0.5% Tween 80.Westerns Analysis—Protein (10–50 μg) from the mitochondrial or post-mitochondrial (cytosolic) fraction was electrophoresed on a 15% SDS-PAGE gel system and transferred to nitrocellulose (Bio-Rad Laboratories). The membrane was subsequently probed with appropriate antibodies and proteins were visualized with ECL reagents (Pierce) following the addition of an horseradish peroxidase-conjugated, secondary antibody. The antiserum against Cyb2 (soluble IMS mitochondrial protein) was kindly provided by Dr. Rosemary Stuart. Antisera to the mitochondrial OM porin (Por1) and cytosolic phosphoglycerol kinase (Pgk1) were obtained from Molecular Probes. Antiserum to the mitochondrial IM Sco1 and Cox17 were prepared as described previously (21Heaton D. Nittis T. Srinivasan C. Winge D.R. J. Biol. Chem. 2000; 275: 37582-37587Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 29Nittis T. George G.N. Winge D.R. J. Biol. Chem. 2001; 276: 42520-42526Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). To ensure equal loading of mitochondrial or post-mitochondrial protein into the gel wells, the membranes were stripped by 3 washes in stripping buffer (25 mm glycine, pH 2, 100 mm NaCl, and 0.5% Tween 20) and reprobed with either an anti-Por1 or an anti-Pgk1 antibody.Purification and Analysis of Cox17—Cox17 was expressed as a soluble protein in Escherichia coli and purified as described previously (20Heaton D.N. George G.N. Garrison G. Winge D.R. Biochem. 2001; 40: 743-751Crossref PubMed Scopus (103) Google Scholar). To evaluate the quaternary structure of Cox17, analytical gel filtration was performed using a 10/30 G-75 Superdex size exclusion column equilibrated with 20 mm phosphate, pH 7, 100 mm NaCl, and 1 mm dithiothreitol. Data were recorded using Unicorn software. Protein luminescence was monitored on a PerkinElmer LS55 luminescence spectrometer and analyzed using PerkinElmer Life Sciences FL1 software. Protein was quantified by amino acid analysis on a Beckman 6300 analyzer after hydrolysis in 5.7 n HCl containing 0.1% phenol in vacuo at 110 °C. The copper concentration of the protein samples was measured using a PerkinElmer Life Sciences Analyst 100 spectrophotometer.RESULTSThe Cox17 polypeptide sequence is conserved in eukaryotes except for the chain termini (Fig. 1). If one of these segments is important for mitochondrial import and not for other functions of the protein, sequence diversity may exist, because mitochondrial targeting sequences do not show high sequence conservation. Amino- and carboxyl-truncation mutations were constructed to test for mitochondrial targeting. The N-terminal truncate lacked the N-terminal nine residues of Cox17 (designated Δ9), whereas the C-terminal truncate lacked the C-terminal ten residues (designated 59Δ) (Fig. 1). The truncated mutants of COX17 under the control of the MET25 promoter were transformed into cox17Δ cells, and the transformants were tested for respiratory growth and cytochrome c oxidase activity. The Δ9 truncate restored wild-type growth on glycerol-containing medium and wild-type cytochrome c oxidase activity (Fig. 2, A and B). In addition, the localization of Δ9 Cox17 in both the mitochondria and cytosolic fractions was similar to that of wild-type Cox17 (Fig. 2C). Thus, the N-terminal segment of Cox17 is not required for mitochondrial targeting.Fig. 1Sequence of yeast Cox17. Residues that are identical with Cox17 molecules from other eukaryotes have an asterisk below, those residues having similar chemical functionalities have a dot below. The positions of the two truncates are labeled with the residue position.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2Complementation of cox17Δ cells by plasmid-borne COX17 (designated WT), or truncate mutants encoding the Δ9 or 59Δ proteins. Wild-type W303 cells are designated 303. The COX17 genes were under the control of the MET25 promoter enabling low expression (5× methionine levels (0.67 mm)) or high expression (no added methionine). A, growth test of transformants cultured with either glucose or glycerol as carbon sources. B, cytochrome c oxidase activity of transformants cultured in medium containing 0.67 mm methionine. Vec, vector. C, Western analysis of Cox17 protein levels in isolated mitochondria or cytosol from transformants cultured in 0.67 mm methionine. Pgk1 is used as a cytosolic marker and Cyb2 as a mitochondrial IMS marker.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In contrast, cells containing the 59Δ truncate were compromised for growth on glycerol-containing medium. Glycerol growth was improved when the 59Δ truncate was overexpressed in medium lacking methionine (Fig. 2A). As expected from the glycerol growth results, CcO activity was minimal under low expression conditions (Fig. 2B). The 59Δ truncate was abundant within the cytosol, but accumulated to a lesser extent in the IMS compared with the WT Cox17 or the Δ9 truncate (Fig. 2C). Under conditions of overexpression the 59Δ truncate accumulates predominantly within the cytosol (data not shown). Consistent with the 59Δ truncate data, the non-functional C57Y mutant of Cox17 also fails to accumulate within the mitochondrial IMS (21Heaton D. Nittis T. Srinivasan C. Winge D.R. J. Biol. Chem. 2000; 275: 37582-37587Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).To test whether defective mitochondrial uptake of the 59Δ truncate was responsible for the slow growth on glycerol medium, DNA encoding the presequence of Cyb2 was fused to the 59Δ truncate. The presequence of Cyb2 is known to target heterologous molecules into the mitochondrial IMS (33Beasley E.M. Muller S. Schatz G. EMBO J. 1993; 12: 2303-2311Crossref PubMed Scopus (65) Google Scholar). Cells with the Cyb2/59Δ Cox17 fusion showed no growth regardless of the expression levels and no oxidase activity (Fig. 3A). In contrast, cells harboring the Cyb2 presequence fused to the N terminus of full-length Cox17 and the Δ9 truncate had wild-type CcO activity (Fig. 3A). The addition of the Cyb2 presequence failed to increase quantities of 59Δ Cox17 in the IMS, even though the fusion of the Cyb2 presequence to WT Cox17 increased the IMS levels of Cox17 considerably (Fig. 3B). As expected, the addition of the Cyb2 presequence to the 59Δ Cox17 did significantly reduce the amount of Cox17 in the cytoplasm. Thus, the failure of the Cyb2/59Δ Cox17 fusion to accumulate within the IMS suggests that the C-terminal motif in Cox17 is important for IMS retention and/or stability.Fig. 3Complementation of cox17Δ cells by plasmid-borne COX17 and truncate mutants encoding the Δ9 or 59Δ proteins fused to the Cyb2 IMS targeting sequence. The letter p in front of the gene designates the Cyb2 presequence. A, cytochrome c oxidase activity of transformants cultured in medium containing 0.67 mm methionine. B, Western analysis of Cox17 protein levels in isolated mitochondria or cytosol in transformants cultured in 0.67 mm methionine. The unprocessed fusion protein is predicted to have a mass of 27 kDa, whereas the IMS processed protein is predicted to be 18 kDa. The observed mass of the Cyb2/Cox17 fusion (pWT) is 18 kDa, consistent with mitochondrial processing. Pgk1 is used as a cytosolic marker and Cyb2 as a mitochondrial IMS marker. Wild-type W303 cells are included for comparison. In lane marked * the protein sample was from a cyb2Δ strain as a control to confirm the Cyb2 western band.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Stability of the CuCox17 protein may be impaired if the C-terminal truncation compromised Cu(I) binding. The 59Δ truncate was expressed in E. coli and purified to evaluate its copper binding properties. The truncate bound Cu(I) normally and exhibited near wild-type Cu(I) luminescence (Fig. 4, inset). The luminescence of CuCox17 arises from the binding of Cu(I) ions within a polycopper cluster (20Heaton D.N. George G.N. Garrison G. Winge D.R. Biochem. 2001; 40: 743-751Crossref PubMed Scopus (103) Google Scholar). Our previous mutational studies suggested that Cox17 function was dependent on the tetrameric structure (20Heaton D.N. George G.N. Garrison G. Winge D.R. Biochem. 2001; 40: 743-751Crossref PubMed Scopus (103) Google Scholar). Cox17 is predicted to exist as a tetrameric molecule within the IMS based on the established equilibrium constant of dimeric verses tetrameric states of wild-type Cox17 and the quantitation of Cox17 levels within the IMS (20Heaton D.N. George G.N. Garrison G. Winge D.R. Biochem. 2001; 40: 743-751Crossref PubMed Scopus (103) Google Scholar). In contrast to wild-type Cox17, the truncate showed an abnormal quaternary structure (Fig. 4). The isolated 59Δ truncate was predominantly monomeric. We demonstrated previously that Cox17 mutants that failed to oligomerize to the tetrameric state were inactive (20Heaton D.N. George G.N. Garrison G. Winge D.R. Biochem. 2001; 40: 743-751Crossref PubMed Scopus (103) Google Scholar). However, not all C-terminal mutants are compromised in oligomerization. The C57Y mutant protein forms predominantly tetrameric species and binds Cu(I) normally (data not shown). Thus, lack of retention within the IMS in C-terminal Cox17 mutants is not primarily due to defective oligomerization or reduced Cu(I) binding. These data are consistent with the C-terminal segment of Cox17 having a role in mitochondrial retention and/or stability.Fig. 4The oligomerization distribution of purified WT Cox17 and the 59Δ truncate (equivalent to 10 μg copper) chromatographed on gel filtration is shown. The elution positions of monomers (M), dimers (D), and tetramers (T) are shown. These different oligomeric states were substantiated previously by sedimentation equilibrium centrifugation (20Heaton D.N. George G.N. Garrison G. Winge D.R. Biochem. 2001; 40: 743-751Crossref PubMed Scopus (103) Google Scholar). The inset shows the luminescence of equivalent protein samples. The flat line is the buffer blank. Each sample contained 4 μg Cu(I) per ml.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the Cu(I) binding motif and the C-terminal retention motif can function independently, we constructed a head-to-tail dimer of Cox17 such that each half was compromised for one of these functions (Fig. 5A). Previously, we demonstrated that Cu(I) binding was confined to the C23CXC26 sequence motif near the central segment of the molecule. A C23S/C24S double substitution abolishes Cu(I) binding, but the mutant protein is taken into the mitochondrion normally (21Heaton D. Nittis T. Srinivasan C. Winge D.R. J. Biol. Chem. 2000; 275: 37582-37587Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The non-functional C57Y allele forms a wild-type Cu(I) thiolate cluster but is defective in mitochondrial retention and/or stability (21Heaton D. Nittis T. Srinivasan C. Winge D.R. J. Biol. Chem. 2000; 275: 37582-37587Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). DNA encoding the non-functional C57Y allele was fused to the 5′ end of a mutant COX17 encoding the C23S/C24S double substitution that fails to bind Cu(I). The resulting head-to-tail fusion protein consisted of a N-terminal half containing a functional Cu(I) binding motif and the C-terminal half containing a functional C-terminal retention motif. Transformants of the chimeric gene, under the control of the MET25 promoter in cox17Δ cells, were cultured under low expression conditions were tested for respiratory growth on glycerol-containing medium (Fig. 5B). Whereas neither the mutant C57Y allele nor the mutant C23S/C24S allele was able to support glycerol growth, cells containing the fusion gene were competent for glycerol growth (Fig. 5B). The C57Y mutant is non-functional even if the Cyb2 presequence is fused to its N terminus to direct mitochondrial uptake (Fig. 5B, pC57Y on right). The head-to-tail chimeric protein restores CcO activity to ∼50% of WT levels (Fig. 5C). Western analysis revealed that the fusion protein was stable within the" @default.
• W2046452961 created "2016-06-24" @default.
• W2046452961 creator A5029838553 @default.
• W2046452961 creator A5037024522 @default.
• W2046452961 creator A5052419395 @default.
• W2046452961 date "2004-02-01" @default.
• W2046452961 modified "2023-10-18" @default.
• W2046452961 title "Cox17 Is Functional When Tethered to the Mitochondrial Inner Membrane" @default.
• W2046452961 cites W1573826674 @default.
• W2046452961 cites W1588290914 @default.
• W2046452961 cites W180444040 @default.
• W2046452961 cites W1869259273 @default.
• W2046452961 cites W1963874808 @default.
• W2046452961 cites W1965188640 @default.
• W2046452961 cites W1966483260 @default.
• W2046452961 cites W1974572661 @default.
• W2046452961 cites W1974644558 @default.
• W2046452961 cites W1982870743 @default.
• W2046452961 cites W1984533286 @default.
• W2046452961 cites W1990529395 @default.
• W2046452961 cites W1997819934 @default.
• W2046452961 cites W2000456081 @default.
• W2046452961 cites W2006375756 @default.
• W2046452961 cites W2011444292 @default.
• W2046452961 cites W2013091489 @default.
• W2046452961 cites W2014741617 @default.
• W2046452961 cites W2015025581 @default.
• W2046452961 cites W2015391531 @default.
• W2046452961 cites W2023765770 @default.
• W2046452961 cites W2034345411 @default.
• W2046452961 cites W2039168627 @default.
• W2046452961 cites W2039276913 @default.
• W2046452961 cites W2045303124 @default.
• W2046452961 cites W2051891646 @default.
• W2046452961 cites W2059725953 @default.
• W2046452961 cites W2069473876 @default.
• W2046452961 cites W2080040488 @default.
• W2046452961 cites W2083545218 @default.
• W2046452961 cites W2087813537 @default.
• W2046452961 cites W2093477805 @default.
• W2046452961 cites W2135747052 @default.
• W2046452961 cites W2154778632 @default.
• W2046452961 cites W2169805130 @default.
• W2046452961 cites W2176573833 @default.
• W2046452961 cites W4293247451 @default.
• W2046452961 cites W60529302 @default.
• W2046452961 doi "https://doi.org/10.1074/jbc.m311772200" @default.
• W2046452961 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14615477" @default.
• W2046452961 hasPublicationYear "2004" @default.
• W2046452961 type Work @default.
• W2046452961 sameAs 2046452961 @default.
• W2046452961 citedByCount "102" @default.
• W2046452961 countsByYear W20464529612012 @default.
• W2046452961 countsByYear W20464529612013 @default.
• W2046452961 countsByYear W20464529612014 @default.
• W2046452961 countsByYear W20464529612015 @default.
• W2046452961 countsByYear W20464529612016 @default.
• W2046452961 countsByYear W20464529612017 @default.
• W2046452961 countsByYear W20464529612018 @default.
• W2046452961 countsByYear W20464529612019 @default.
• W2046452961 countsByYear W20464529612020 @default.
• W2046452961 countsByYear W20464529612021 @default.
• W2046452961 countsByYear W20464529612022 @default.
• W2046452961 countsByYear W20464529612023 @default.
• W2046452961 crossrefType "journal-article" @default.
• W2046452961 hasAuthorship W2046452961A5029838553 @default.
• W2046452961 hasAuthorship W2046452961A5037024522 @default.
• W2046452961 hasAuthorship W2046452961A5052419395 @default.
• W2046452961 hasConcept C12554922 @default.
• W2046452961 hasConcept C159382241 @default.
• W2046452961 hasConcept C185592680 @default.
• W2046452961 hasConcept C28859421 @default.
• W2046452961 hasConcept C65871279 @default.
• W2046452961 hasConcept C74897846 @default.
• W2046452961 hasConcept C75385678 @default.
• W2046452961 hasConcept C86803240 @default.
• W2046452961 hasConcept C95444343 @default.
• W2046452961 hasConceptScore W2046452961C12554922 @default.
• W2046452961 hasConceptScore W2046452961C159382241 @default.
• W2046452961 hasConceptScore W2046452961C185592680 @default.
• W2046452961 hasConceptScore W2046452961C28859421 @default.
• W2046452961 hasConceptScore W2046452961C65871279 @default.
• W2046452961 hasConceptScore W2046452961C74897846 @default.
• W2046452961 hasConceptScore W2046452961C75385678 @default.
• W2046452961 hasConceptScore W2046452961C86803240 @default.
• W2046452961 hasConceptScore W2046452961C95444343 @default.
• W2046452961 hasIssue "7" @default.
• W2046452961 hasLocation W20464529611 @default.
• W2046452961 hasOpenAccess W2046452961 @default.
• W2046452961 hasPrimaryLocation W20464529611 @default.
• W2046452961 hasRelatedWork W17309769 @default.
• W2046452961 hasRelatedWork W1999438797 @default.
• W2046452961 hasRelatedWork W2017875929 @default.
• W2046452961 hasRelatedWork W2053030473 @default.
• W2046452961 hasRelatedWork W2085424464 @default.
• W2046452961 hasRelatedWork W2145509877 @default.
• W2046452961 hasRelatedWork W2168843989 @default.
• W2046452961 hasRelatedWork W2886583284 @default.

• next