COQ9, a New Gene Required for the Biosynthesis of Coenzyme Q in Saccharomyces cerevisiae
2005; Elsevier BV; Volume: 280; Issue: 36 Linguagem: Inglês
10.1074/jbc.m503277200
ISSN1083-351X
AutoresAlisha Johnson, Peter Gin, Beth N. Marbois, Edward J. Hsieh, Mian Wu, Mário H. Barros, Catherine F. Clarke, Alexander Tzagoloff,
Tópico(s)Plant biochemistry and biosynthesis
ResumoCurrently, eight genes are known to be involved in coenzyme Q6 biosynthesis in Saccharomyces cerevisiae. Here, we report a new gene designated COQ9 that is also required for the biosynthesis of this lipoid quinone. The respiratory-deficient pet mutant C92 was found to be deficient in coenzyme Q and to have low mitochondrial NADH-cytochrome c reductase activity, which could be restored by addition of coenzyme Q2. The mutant was used to clone COQ9, corresponding to reading frame YLR201c on chromosome XII. The respiratory defect of C92 is complemented by COQ9 and suppressed by COQ8/ ABC1. The latter gene has been shown to be required for coenzyme Q biosynthesis in yeast and bacteria. Suppression by COQ8/ABC1 of C92, but not other coq9 mutants tested, has been related to an increase in the mitochondrial concentration of several enzymes of the pathway. Coq9p may either catalyze a reaction in the coenzyme Q biosynthetic pathway or have a regulatory role similar to that proposed for Coq8p. Currently, eight genes are known to be involved in coenzyme Q6 biosynthesis in Saccharomyces cerevisiae. Here, we report a new gene designated COQ9 that is also required for the biosynthesis of this lipoid quinone. The respiratory-deficient pet mutant C92 was found to be deficient in coenzyme Q and to have low mitochondrial NADH-cytochrome c reductase activity, which could be restored by addition of coenzyme Q2. The mutant was used to clone COQ9, corresponding to reading frame YLR201c on chromosome XII. The respiratory defect of C92 is complemented by COQ9 and suppressed by COQ8/ ABC1. The latter gene has been shown to be required for coenzyme Q biosynthesis in yeast and bacteria. Suppression by COQ8/ABC1 of C92, but not other coq9 mutants tested, has been related to an increase in the mitochondrial concentration of several enzymes of the pathway. Coq9p may either catalyze a reaction in the coenzyme Q biosynthetic pathway or have a regulatory role similar to that proposed for Coq8p. Biosynthesis of coenzyme Q (ubiquinone) in eukaryotes occurs in mitochondria. Eight genes designated COQ1–8 have been shown to be involved in the biosynthesis of this lipoid component of the electron transport chain of Saccharomyces cerevisiae (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). The products of these genes have been localized to the inner membrane (2Poon W.W. Barkovich R.J. Hsu A.Y. Frankel A. Lee P.T. Shepherd J.N. Myles D.C. Clarke C.F. J. Biol. Chem. 1999; 274: 21665-21672Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 3Jonassen T. Proft M. Randez-Gil F. Schultz J.R. Marbois B.N. Entian K.-D. Clarke C.F. J. Biol. Chem. 1998; 273: 3351-3357Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 4Gin P. Clarke C.F. J. Biol. Chem. 2005; 280: 2676-2681Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 5Belogrudov G.I. Lee P.T. Jonassen T. Hsu A.Y. Gin P. Clarke C.F. Arch. Biochem. Biophys. 2001; 392: 48-58Crossref PubMed Scopus (54) Google Scholar, 6Baba S.W. Belogrudov G.I. Lee J.C. Lee P.T. Strahan J. Shepherd J.N. Clarke C.F. J. Biol. Chem. 2004; 279: 10052-10059Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7Gin P. Hsu A.Y. Rothman S.C. Jonassen T. Lee P.T. Tzagoloff A. Clarke C.F. J. Biol. Chem. 2003; 278: 25308-25316Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Do T.Q. Hsu A.Y. Jonassen T. Lee P.T. Clarke C.F. J. Biol. Chem. 2001; 276: 18161-18168Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and, in some cases, were inferred to be present in a complex (4Gin P. Clarke C.F. J. Biol. Chem. 2005; 280: 2676-2681Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9Hsu A.Y. Do T.Q. Lee P.T. Clarke C.F. Biochim. Biophys. Acta. 2000; 1484: 287-297Crossref PubMed Scopus (66) Google Scholar, 10Marbois B.N. Gin P. Faull K.F. Poon W.W. Lee P.T. Strahan J. Shepherd J.N. Clarke C.F. J. Biol. Chem. 2005; 280: 20231-20238Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The chemical intermediates detected in some mutants have revealed that the pathways in bacteria and in this yeast are identical up to the formation of 3-hexaprenyl-4-hydroxybenzoic acid (HHB) 1The abbreviations used are: HHB, 3-hexaprenyl-4-hydroxybenzoic acid; HA, hemagglutinin; GST, glutathione S-transferase; HPLC, high performance liquid chromatography.1The abbreviations used are: HHB, 3-hexaprenyl-4-hydroxybenzoic acid; HA, hemagglutinin; GST, glutathione S-transferase; HPLC, high performance liquid chromatography. (11Olson R.E. Rudney H. Vitam. Horm. 1983; 40: 1-43Crossref PubMed Scopus (144) Google Scholar, 12Gibson F. Biochem. Soc. Trans. 1973; 1: 317-326Crossref Google Scholar), at which point they diverge for the next three steps, but then converge again in the last stages of biosynthesis (11Olson R.E. Rudney H. Vitam. Horm. 1983; 40: 1-43Crossref PubMed Scopus (144) Google Scholar, 12Gibson F. Biochem. Soc. Trans. 1973; 1: 317-326Crossref Google Scholar).A hallmark of most yeast coq mutants is the accumulation of HHB when the biochemical block occurs at any step subsequent to the formation of this early intermediate (5Belogrudov G.I. Lee P.T. Jonassen T. Hsu A.Y. Gin P. Clarke C.F. Arch. Biochem. Biophys. 2001; 392: 48-58Crossref PubMed Scopus (54) Google Scholar, 6Baba S.W. Belogrudov G.I. Lee J.C. Lee P.T. Strahan J. Shepherd J.N. Clarke C.F. J. Biol. Chem. 2004; 279: 10052-10059Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7Gin P. Hsu A.Y. Rothman S.C. Jonassen T. Lee P.T. Tzagoloff A. Clarke C.F. J. Biol. Chem. 2003; 278: 25308-25316Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Do T.Q. Hsu A.Y. Jonassen T. Lee P.T. Clarke C.F. J. Biol. Chem. 2001; 276: 18161-18168Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Poon W.W. Marbois B.N. Faull K.F. Clarke C.F. Arch. Biochem. Biophys. 1995; 320: 305-314Crossref PubMed Scopus (46) Google Scholar, 14Poon W.W. Do T.Q. Marbois B.N. Clarke C.F. Mol. Aspects Med. 1997; 18: s121-s127Crossref PubMed Scopus (56) Google Scholar). This may indicate that 1) most intermediates of the pathway are unstable and degraded; 2) the pathway is highly regulated; or 3) the enzymes are organized in a complex, which is highly sensitive to mutations in any one of its components. The latter possibility is supported by recent evidence indicating an interdependence of some Coq proteins for their stability (4Gin P. Clarke C.F. J. Biol. Chem. 2005; 280: 2676-2681Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Most of the COQ gene products have been related to specific reactions of the eukaryotic pathway based on their homology to the bacterial counterparts (6Baba S.W. Belogrudov G.I. Lee J.C. Lee P.T. Strahan J. Shepherd J.N. Clarke C.F. J. Biol. Chem. 2004; 279: 10052-10059Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7Gin P. Hsu A.Y. Rothman S.C. Jonassen T. Lee P.T. Tzagoloff A. Clarke C.F. J. Biol. Chem. 2003; 278: 25308-25316Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The reactions catalyzed by the products of COQ4 and COQ8/ABC1, however, still need to be clarified (5Belogrudov G.I. Lee P.T. Jonassen T. Hsu A.Y. Gin P. Clarke C.F. Arch. Biochem. Biophys. 2001; 392: 48-58Crossref PubMed Scopus (54) Google Scholar, 8Do T.Q. Hsu A.Y. Jonassen T. Lee P.T. Clarke C.F. J. Biol. Chem. 2001; 276: 18161-18168Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar).In the course of analyzing the biochemical defects of respiratory-deficient pet mutants of S. cerevisiae, we have identified a new gene that, when mutated, produces a phenotype similar to that of coenzyme Q mutants. This gene is defined by complementation group G61 of our pet mutant collection (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar) and has been named COQ9. Homologs of COQ9 are present in a wide range of different eukaryotes, but not in bacteria, indicating that its function is specific to coenzyme Q biosynthesis in mitochondria. Suppression of a coq9 mutant by COQ8/ABC1 (8Do T.Q. Hsu A.Y. Jonassen T. Lee P.T. Clarke C.F. J. Biol. Chem. 2001; 276: 18161-18168Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 16Bousquet I. Dujardin G. Slonimski P.P. EMBO J. 1991; 10: 2023-2031Crossref PubMed Scopus (93) Google Scholar) suggests that the products of these two genes may be functionally related.EXPERIMENTAL PROCEDURESStrains and Media—The strains of yeast used in this study are listed in Table I. The respiratory-deficient mutants of complementation group G61 were derived from S. cerevisiae strain D273-10B/A1 or D273-10B/A21 by mutagenesis with nitrosoguanidine or ethylmethane sulfonate (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). The following media were used routinely to grow yeast: 1% yeast extract, 2% peptone, and 2% glucose; 1% yeast extract, 2% peptone, and 2% galactose; and 1% yeast extract, 2% ethanol, 2% peptone, and 3% glycerol.Table IGenotypes and sources of yeast strainsStrainGenotypeSourceW303-1AMATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1R. RothsteinaR. Rothstein, Department of Human Genetics, Columbia University, New York.W303-1BMATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1R. RothsteinaR. Rothstein, Department of Human Genetics, Columbia University, New York.C92MATα met6 coq9-1Ref. 1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google ScholarC92/UL1MATα leu2-3,112 ura3-1 coq9-1C92 × W303-1AC92/UL3MATα leu2-3,112 ura3-1 coq9-1C92 × W303-1AC92/UL1/T2MATα leu2-3,115 ura3-1 coq9-1 + pG61/T2This studyE40MATα met6 coq9-2This studyE40/UL1MATα leu2-3,112 ura3-1 coq9-2E40 × W303-1AE270MATα met6 coq9-3This studyaE270/UL1MATα leu2-3,112 ura3-1 coq9-3E270 × W303-1AE680MATα met6 coq9-4This studyE680/UL1MATα leu2-3,112 ura3-1 coq9-4E680 × W303-1AE820MATα met6 coq9-5This studyE820/UL1MATα leu2-3,112 ura3-1 coq9-5E820 × W303-1AaW303ΔCOQ9MATa ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1This studycoq9::URA3W303ΔCOQ9MATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1This studycoq9::URA3W303ΔCOQ5MATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1Ref. 17Barkovich R.J. Shtanko A. Shepherd J.A. Lee P.T. Myles D.C. Tzagoloff A. Clarke C.F. J. Biol. Chem. 1997; 272: 9182-9188Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholarcoq5::HIS3aW303ΔCOQ8MATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1Ref. 8Do T.Q. Hsu A.Y. Jonassen T. Lee P.T. Clarke C.F. J. Biol. Chem. 2001; 276: 18161-18168Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholarcoq8::HIS3W303ΔCOQ8,9MATα ade2-1 his3-1,15 leu2-3,112 trp1-1 ura3-1aW303ΔCOQ8 × W303ΔCOQ9coq8::HIS3 coq9::URA3a R. Rothstein, Department of Human Genetics, Columbia University, New York. Open table in a new tab Cloning of COQ9 and COQ8 —Recombinant plasmids containing COQ9 (pG61/T2) and COQ8/ABC1 (pG61/T3) were isolated by transformation of C92/UL1 with a yeast genomic library consisting of partial Sau3A fragments of yeast nuclear DNA cloned in the yeast/Escherichia coli shuttle plasmid YEp24 (18Botstein D. Davis R.W. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces cerevisiae: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 607-636Google Scholar). Approximately 5 × 108 cells were transformed with 50 μg of the library DNA by the method of Schiestl and Gietz (19Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1766) Google Scholar).Disruption of COQ9 —The linear KpnI fragment containing COQ9 was transferred to YEp352K. This plasmid is identical to YEp352 (20Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1078) Google Scholar) except that it has only the KpnI site of the multiple cloning sequence. This construct was linearized at the SacI site inside the COQ9 coding sequence and ligated to a 1-kb SacI fragment containing the yeast URA3 gene. The linear KpnI fragment with the disrupted allele was substituted for the wild-type gene in strain W303 by the one-step gene replacement procedure (21Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2015) Google Scholar).Construction of Hybrid Genes Expressing Coq9p Tagged with Hemagglutinin (HA) and Coq8p Tagged with Glutathione S-Transferase (GST) at Their C Termini, Respectively—The COQ9 coding sequence plus 359 nucleotides of the 5′-untranslated region was amplified with primers 5′-ggcggatccgggaggccatgcccaactttc and 5′-ggcaagctttcaagcgtagtctgggacgtcgtatgggtaccctcctccacccctaactaattgagatttg. The product was digested with a combination of BamHI and HindIII and cloned in YIp351 (20Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1078) Google Scholar), yielding pG61/ST8. Following linearization at the ClaI site of the LEU2 gene, the plasmid was used to transform the W303ΔCOQ9 mutant containing the disrupted coq9 allele. Insertion of the plasmid into the chromosomal leu2 locus of the mutant yielded leucine-independent and respiratory-competent transformants (W303ΔCOQ9/ST8).Similarly, COQ8 was amplified with primers 5′-ggctctagactcaatcgcatccccgtaaacg and 5′-ggcctgcag-aactttataggcaaaaatctc. The resultant 2-kb product containing 446 nucleotides of the 5′-untranslated region and the entire coding sequence minus the termination codon was digested with a combination of XbaI and PstI and cloned in YIp352-GST. (This plasmid was made by cloning the sequence coding for glutathione reductase into YIp352 (20Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1078) Google Scholar).) The plasmid obtained from this ligation (pG75/ST3) contained COQ8 fused in-frame to the GST sequence at the 3′-end of the gene. pG75/ST3 was linearized at the NcoI site in the URA3 gene of the plasmid and targeted (21Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2015) Google Scholar) to the ura3 allele in the chromosomal DNA of aW303ΔCOQ8. The resultant strain (aW303ΔCOQ8/ST3) was respiratory-competent, indicating that the presence of the GST tag does not interfere with the activity of the protein.Lipid Extraction and Reverse-phase HPLC with Electrochemical Detection—Lipid extracts were prepared from 1 mg of mitochondrial protein in 350 μl of water in borosilicate centrifuge tubes (50 ml, polytetrafluoroethylene-lined caps). Coenzymes Q4 and Q6 were obtained from Sigma. Coenzyme Q4 (750 ng in 25.9 μl of ethanol) was added to provide an internal standard; glass beads (1 g) were added; and the sample was vortexed for 2 min. Methanol (9 ml) and petroleum ether (6 ml) were added, the sample was vortexed 30 s and then incubated on a rotary shaker for 12–16 h in the dark (4 °C, 220 rpm). Following centrifugation at 900 × g for 10 min, the upper organic layer was transferred to a 10-ml glass tube and dried under a stream of N2. The remaining pellet and lower phase were re-extracted by vortexing two additional times with 4 ml of petroleum ether for 30 s and shaking at 220 rpm for 1 h in the dark at 4 °C. The organic phases obtained by centrifugation at 900 × g for 10 min were pooled and dried under N2, and the lipid residue was resuspended in 300 μl of 9:1 (v/v) methanol/ethanol. Aliquots of 10 μl were injected with a Gilson Model 234 autosampler (samples maintained at 6 °C) and separated by reverse-phase HPLC on a BetaBasic 18 column (150 × 4.6 mm, Thermo Electron Corp.) at a flow rate of 1 ml/min with 98:2 methanol/water and 10 mm lithium perchlorate. Quinones were quantified with a Coulochem II electrochemical detector (ESA Biosciences, Inc.), a pre-column electrode (E, +700 mV), a 5010 analytical cell with two electrons (E1, –700 mV; and E2, +650 mV), and Gilson Unipoint Version 1.71 software by measuring the area under the peaks with coenzyme Q4 and Q6 standards. Three-point external standard curves were created for coenzymes Q4 (168.7, 84.4, and 21.1 ng) and Q6 (123.2, 61.6, and 15.4 ng). Each sample and standard were injected three times, and the peak areas were calculated to obtain the mean ± S.D. The amount of coenzyme Q6 was corrected for recovery of the coenzyme Q4 internal standard.Labeling, Lipid Extraction, and HPLC Separation of Yeast Whole Cell Extracts—These procedures were modified from those described previously (13Poon W.W. Marbois B.N. Faull K.F. Clarke C.F. Arch. Biochem. Biophys. 1995; 320: 305-314Crossref PubMed Scopus (46) Google Scholar). Yeast cells (4–6 g, wet weight) were collected after overnight growth in 600 ml of minimal medium (2% galactose and 0.1% dextrose) containing 6.34 μCi of 4-[U-14C]hydroxybenzoic acid (450 mCi/mmol; American Radiolabeled Chemicals, Inc. St. Louis, MO). Lipid extracts of cell pellets were prepared as described (13Poon W.W. Marbois B.N. Faull K.F. Clarke C.F. Arch. Biochem. Biophys. 1995; 320: 305-314Crossref PubMed Scopus (46) Google Scholar), dried completely under N2 gas, and stored at –20 °C. The dried lipids were resuspended in 50 μl of 100% acetonitrile and vortexed vigorously, and the tubes were centrifuged briefly to remove a small amount of a white precipitate. The clear supernatant (25 μl) was injected into an HPLC system as described (13Poon W.W. Marbois B.N. Faull K.F. Clarke C.F. Arch. Biochem. Biophys. 1995; 320: 305-314Crossref PubMed Scopus (46) Google Scholar) with the following modifications. A reversephase BetaBasic column (2.1 × 100 mm, Thermo Electron Corp.) was developed with a gradient of solvent A (50:50 acetonitrile/H2O and 0.01% trifluoroacetic acid) and solvent B (99:1 acetonitrile/H2O and 0.01% trifluoroacetic acid) at a flow rate of 400 μl/min. At the time of injection, solvent A/B was 50:50; and at 2 min, the percentage of solvent B was altered linearly so that solvent B was 100% at 9 min and remained at 100% until 25 min. The retention time for coenzyme Q6 measured at 272 nm was 22 min, with a delay time of 0.37 min due to the fraction collector. Fractions were collected every minute, and the radioactivity was measured by scintillation counting in 10 ml of ScintiVerse (Fisher).Miscellaneous Methods—Standard methods were used for plasmid manipulations (22Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar) and DNA sequencing (23Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52357) Google Scholar). Yeast mitochondria were prepared by the method of Faye et al. (24Faye G. Kujawa C. Fukuhara H. J. Mol. Biol. 1974; 88: 185-203Crossref PubMed Scopus (180) Google Scholar) except that Zymolyase 20T (MP Biomedicals, Aurora, OH) instead of Glusulase was used to obtain spheroplasts. Spectral analyses of mitochondrial cytochromes and measurements of respiratory enzymes were performed as described previously (25Tzagoloff A. Akai A. Needleman R. J. Biol. Chem. 1975; 250: 8228-8235Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined by the method of Lowry et al. (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar).RESULTSPhenotype of coq9 Mutants—C92 is one of seven independent isolates assigned to complementation group G61 of a pet mutant collection (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). The mutants grow on glucose, but not ethanol and/or glycerol, as a carbon source, indicating a defect in respiration. The respiratory deficiency of G61 mutants stems from recessive mutations in a nuclear gene, as they are complemented by a ρ0 strain with a normal complement of nuclear genes but lacking mitochondrial DNA.The spectra of mitochondria from C92 indicated reduced concentrations of cytochromes b and a (Fig. 1). This is also evident from the NADH-cytochrome c reductase and cytochrome oxidase activities of the mitochondria, both of which were reduced in the mutants (Table II). The NADH-cytochrome c reductase activity in the mutant was <10% of that measured in the wild-type strain, even though the presence of cytochrome b in the spectrum of the mutant mitochondria indicated a substantial amount of the cytochrome bc1 complex. This phenotype could be explained by a defect in NADH dehydrogenase, the cytochrome bc1 complex, or coenzyme Q6, all of which participate in the reduction of cytochrome c. The restoration of NADH-cytochrome c reductase by addition of exogenous coenzyme Q2 (Table II) excluded a defect in either NADH dehydrogenase or the cytochrome bc1 complex; rather, it pointed to a deficiency in coenzyme Q as the explanation for the respiratory defect.Table IINADH-cytochrome c reductase and cytochrome oxidase activities of mitochondriaStrainNADH-cytochrome c reductaseCytochrome oxidase-Coenzyme Q2+Coenzyme Q2μmol cytochrome c oxidized or reduced per min/mg proteinW303-1A2.15 ± 0.082.21 ± 0.072.23 ± 0.07C920.21 ± 0.021.90 ± 0.050.51 ± 0.04aW303ΔCOQ90.14 ± 0.011.67 ± 0.020.87 ± 0.01 Open table in a new tab This was further tested by analyzing the coenzyme Q6 content of mitochondria from the wild-type and mutant strains. Lipid extracts of mitochondria were separated by reversephase chromatography on a C18 column. On this column, coenzyme Q6 eluted as a symmetric peak at 15.7 min. The coenzyme Q6 content of mitochondria from the wild-type W303-1A strain was determined to be 3.33 μg/mg of protein (Fig. 2A). No coenzyme Q6 peak was detected in similar extracts from the point mutant C92 or the coq9 null mutant aW303ΔCOQ9 (see below). The absence of coenzyme Q6 in C92 and aW303ΔCOQ9 is consistent with the requirement of exogenous coenzyme Q2 for the NADH-cytochrome c reductase activity of their mitochondria (Table II).Fig. 2Assays of coenzyme Q6 in wild-type and mutant mitochondria. A, mitochondria were prepared from the wild-type strain W303-1A, the coq9 null mutant aW303ΔCOQ9, the point mutant C92, and the C92 point mutant harboring COQ8/ABC1 on a multicopy plasmid (C92/UL1/T3). Lipid extracts were obtained from 1 mg of mitochondria and mixed with coenzyme Q4 (750 ng) as an internal standard prior to extraction, and one-thirtieth of the extract was analyzed by HPLC/electrochemical detector as described under "Experimental Procedures." The elution of coenzymes Q4 and Q6 and demethoxy-coenzyme Q4 (DMQ6) is indicated. The concentrations of coenzyme Q6 are reported as μg/mg of mitochondrial protein and are averages of three determinations with the ranges indicated. B, shown are the results from the analysis of U-14C-labeled coenzyme Q and coenzyme Q intermediates. Lipid extracts from 4-[U-14C]hydroxybenzoic acid-labeled yeast were separated by reversephase HPLC, and the radioactivity was determined as described under "Experimental Procedures." White bars, wild-type strain W303-1A; black bars, coq9 null mutant aW303ΔCOQ9. A coenzyme Q6 standard eluted in fraction 22.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The block in coenzyme Q6 synthesis was confirmed by in vivo labeling of the wild-type strain and the coq9 null mutant with 4-[U-14C]hydroxybenzoic acid. The wild-type strain incorporated the precursor mainly into coenzyme Q6, although some of the label was also present in a polar material eluting at 16 min. This radiolabeled compound was presumed to be HHB based on our previous characterization of lipid extracts from cells labeled under these conditions (13Poon W.W. Marbois B.N. Faull K.F. Clarke C.F. Arch. Biochem. Biophys. 1995; 320: 305-314Crossref PubMed Scopus (46) Google Scholar). In that study, HHB was identified by purification, chemical derivation, and confirmation of its structure by mass spectrometry analyses. HHB is an early intermediate in coenzyme Q biosynthesis and has been shown to be a predominant intermediate in wild-type yeast (13Poon W.W. Marbois B.N. Faull K.F. Clarke C.F. Arch. Biochem. Biophys. 1995; 320: 305-314Crossref PubMed Scopus (46) Google Scholar). In contrast, there was no detectable radioactivity at the position of coenzyme Q6 in the coq9 mutant, and the elution of radiolabeled material at 16 min is consistent with the presence of HHB, indicating that the lesion occurs after the prenylation step (Fig. 2B).Cloning of COQ9 —C92/UL1, a derivative of C92 with the leu2 and ura3 markers, was transformed with a yeast genomic plasmid library. Restriction analysis of the nuclear DNA inserts in plasmids isolated from respiratory-competent transformants indicated three distinct and non-overlapping regions of DNA. The genes responsible for conferring respiration in C92/UL1 were identified by testing the ability of different regions of the cloned nuclear DNA inserts to restore the ability of the mutant to grow on glycerol. The results of the subcloning indicated that rescue of the mutant by the plasmid designated pG61/T2 depended on the unknown reading frame YLR201c (Fig. 3), whereas in pG61/T3, the gene responsible for conferring respiration is COQ8/ABC1 (8Do T.Q. Hsu A.Y. Jonassen T. Lee P.T. Clarke C.F. J. Biol. Chem. 2001; 276: 18161-18168Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 16Bousquet I. Dujardin G. Slonimski P.P. EMBO J. 1991; 10: 2023-2031Crossref PubMed Scopus (93) Google Scholar). Complementation tests with subclones from the third plasmid, pG61/T4, identified the multicopy suppressor as DTD1 (data not shown), the gene for d-Tyr-tRNATyr deacylase (27Soutourina J. Blanquet S. Plateau P. J. Biol. Chem. 2000; 275: 11626-11630Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), which was previously shown to counter the suppressor activity of sup45-2 in yeast (28Benko A.L. Vaduva G. Martin N.C. Hopper A.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 61-66Crossref PubMed Scopus (48) Google Scholar). The mechanism by which DTD1 rescues the coq9 mutant has not been further studied. Two other mutants from complementation group G61 (E40 and E270) were also tested and found to be complemented by YLR201c. However, the respiratory deficiencies of E40 and E270 were not rescued by either COQ8/ABC1 or pG61/T4 (Fig. 4). The ability of C92/UL1 to grow normally on non-fermentable substrates when transformed with YLR201c and the lack of complementation of C92/UL1 by W303ΔCOQ9, a mutant carrying a deletion of YLR201c, strongly suggest that the restoration of respiration by this gene is due to complementation. In contrast to YLR201c, COQ8/ABC1 was ascertained to be a suppressor based on its specificity for only one of the three mutant alleles tested and complementation of C92 and W303ΔCOQ9 by a coq8/abc1 mutant. These observations imply that the growth defect of C92 and other G61 mutants stems from mutations in reading frame YLR201c. This reading frame has been named COQ9 in keeping with the earlier convention for naming genes involved in coenzyme Q biosynthesis in yeast (1Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar).Fig. 3Restriction maps of the nuclear DNA inserts in pG61/T2 and pG61/T3. E, EcoRI; B, BamHI; G, BglII; K, KpnI; S, SacI; H, HindIII; X, XbaI. The fragments used to obtain the different subclones are denoted by the shorter bars above the restriction maps. The arrows designate the locations and directions of transcription of COQ9/YLR102c and COQ8/ABC1. Complementation and lack thereof by the subclones are indicated by the plus and minus signs, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Suppression of the C92 allele by COQ8. Serial dilutions of the following strains were spotted on 1% yeast extract, 2% peptone, and 2% glucose (YPD) and on 1% yeast extract, 2% ethanol, 2% peptone, and 3% glycerol (YEPG): W303-1A (wild-type (WT) strain), C92/UL3 (coq9 point mutant), C92/UL3/T2 (coq9 mutant transformed with COQ9 on a high copy plasmid), C92/UL3/T3 (coq9 mutant transformed with COQ8/ABC1 on a high copy plasmid), and C92/UL3/T4 (coq9 mutant transformed with DTD1 on a high copy plasmid). E40/UL1 and aE270/UL1 are two other independent coq9 point mutants that were also transformed with COQ9 (T2), COQ8 (T3), and DTD1 (T4) on multicopy plasmids. The plates were photographed after incubation at 30 °C for 2 days.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Disruption of COQ9 —COQ9 was disrupted with the yeast URA3 gene at the internal SacI site. The resultant strain was respiratory-deficient (not shown), and the spectrum of mitochondria disclosed a partial pleiotropy similar to that of C92, resulting in some reduction of cytochromes a and b (Fig. 1). Assays of respiration in the null mutant confirmed that the NADH-cytochrome c reductase and succinate and NADH oxidase activities of the mutant mitochondria were reconstituted by addition of coenzyme Q2 (Fig. 5 and Table II).Fig. 5NADH and succinate oxidase activities of mitochondria from the wild-type strain and a coq9 null mutant. Mitochondria from the wild-type (WT) strain W303-1B and from the coq9 null mutant W303ΔCOQ9 were used to measure succinate and NADH oxidase activit
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