Yeast Coq5 C-Methyltransferase Is Required for Stability of Other Polypeptides Involved in Coenzyme Q Biosynthesis
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m313712200
ISSN1083-351X
AutoresSuzie W. Baba, Grigory I. Belogrudov, Justine C. Lee, Peter T. Lee, Jeff R. Strahan, Jennifer N. Shepherd, Catherine F. Clarke,
Tópico(s)Biochemical Acid Research Studies
ResumoCoenzyme Q (Q) functions in the electron transport chain of both prokaryotes and eukaryotes. The biosynthesis of Q requires a number of steps involving at least eight Coq polypeptides. Coq5p is required for the C-methyltransferase step in Q biosynthesis. In this study we demonstrate that Coq5p is peripherally associated with the inner mitochondrial membrane on the matrix side. Phenotypic characterization of a collection of coq5 mutant yeast strains indicates that while each of the coq5 mutant strains are rescued by the Saccharomyces cerevisiae COQ5 gene, only the coq5-2 and coq5-5 mutants are rescued by expression of Escherichia coli ubiE, a homolog of COQ5. The coq5-2 and coq5-5 mutants contain mutations within or adjacent to conserved methyltransferase motifs that would be expected to disrupt the catalysis of C-methylation. The steady state levels of the Coq5-2 and Coq5-5 mutant polypeptides are not decreased relative to wild type Coq5p. Two other polypeptides required for Q biosynthesis, Coq3p and Coq4p, are detected in the wild type parent and in the coq5-2 and coq5-5 mutants, but are not detected in the coq5-null mutant, or in the coq5-4 or coq5-3 mutants. The effect of the coq5-4 mutation is similar to a null, since it results in a stop codon at position 93. However, the coq5-3 mutation (G304D) is located just four amino acids away from the C terminus. While C-methyltransferase activity is detectable in mitochondria isolated from this mutant, the steady state level of Coq5p is dramatically decreased. These studies show that at least two functions can be attributed to Coq5p; first, it is required to catalyze the C-methyltransferase step in Q biosynthesis and second, it is involved in stabilizing the Coq3 and Coq4 polypeptides required for Q biosynthesis. Coenzyme Q (Q) functions in the electron transport chain of both prokaryotes and eukaryotes. The biosynthesis of Q requires a number of steps involving at least eight Coq polypeptides. Coq5p is required for the C-methyltransferase step in Q biosynthesis. In this study we demonstrate that Coq5p is peripherally associated with the inner mitochondrial membrane on the matrix side. Phenotypic characterization of a collection of coq5 mutant yeast strains indicates that while each of the coq5 mutant strains are rescued by the Saccharomyces cerevisiae COQ5 gene, only the coq5-2 and coq5-5 mutants are rescued by expression of Escherichia coli ubiE, a homolog of COQ5. The coq5-2 and coq5-5 mutants contain mutations within or adjacent to conserved methyltransferase motifs that would be expected to disrupt the catalysis of C-methylation. The steady state levels of the Coq5-2 and Coq5-5 mutant polypeptides are not decreased relative to wild type Coq5p. Two other polypeptides required for Q biosynthesis, Coq3p and Coq4p, are detected in the wild type parent and in the coq5-2 and coq5-5 mutants, but are not detected in the coq5-null mutant, or in the coq5-4 or coq5-3 mutants. The effect of the coq5-4 mutation is similar to a null, since it results in a stop codon at position 93. However, the coq5-3 mutation (G304D) is located just four amino acids away from the C terminus. While C-methyltransferase activity is detectable in mitochondria isolated from this mutant, the steady state level of Coq5p is dramatically decreased. These studies show that at least two functions can be attributed to Coq5p; first, it is required to catalyze the C-methyltransferase step in Q biosynthesis and second, it is involved in stabilizing the Coq3 and Coq4 polypeptides required for Q biosynthesis. Ubiquinone, or coenzyme Q (Q), 1The abbreviations used are: Q, coenzyme Q (ubiquinone); QH2, ubiquinol; DDMQH2, demethyl demethoxy ubiquinol; DDMQ, demethyl demethoxy ubiquinone or 2-methoxy-6-polyprenyl-1,4-benzoquinone; DMQH2, demethoxy ubiquinol; DMQ, demethoxy ubiquinone or 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone; DHFB, dihydroxy farnesyl benzoate or 3,4-dihydroxy-5-polyprenyl benzoic acid; DMeQ3, demethyl Q3 or 3-methoxy-4-hydroxy-5-farnesyl benzoic acid; HPLC, high-performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. is a polyprenylated benzoquinone lipid that is a critical component of the electron transport pathways of both eukaryotes and prokaryotes (1Crane F.L. J. Am. Coll. Nutr. 2001; 20: 591-598Crossref PubMed Scopus (711) Google Scholar). Qn consists of a hydrophobic isoprenoid tail and a quinone head group. The tail length (n) varies depending on the organism studied; Saccharomyces cerevisiae contains Q6, Escherichia coli contains Q8, and humans contain Q10. The tail anchors Q in the membrane, while the head group is responsible for the redox chemistry, undergoing reversible redox cycling between the quinone (Q), semiquinone, and hydroquinone (QH2) forms. In eukaryotes Q is primarily associated with the inner mitochondrial membrane and is best known for its role in respiratory metabolism as a member of the electron transport chain shuttling electrons from Complex I (NADH:Q oxidoreductase) and Complex II (succinate:Q oxidoreductase) to Complex III (the cytochrome bc1 complex) (2Dutton P.L. Ohnishi T. Darrouzet E. Leonard M.A. Sharp R.E. Gibney B.R. Daldal F. Moser C.C. Kagan V.E. Quinn P.J. Coenzyme Q: Molecular Mechanisms in Health and Disease. CRC Press, Boca Raton, FL2001: 65-82Google Scholar). QH2 also acts as a lipid-soluble antioxidant, capable of scavenging lipid peroxyl radicals directly, or indirectly, by reducing α-tocopheroxyl radicals (3Ernster L. Forsmark-Andree P. Clin. Investig. 1993; 71: S60-65Crossref PubMed Scopus (255) Google Scholar, 4Kagan V.E. Nohl H. Quinn P.J. Cadenas E. Packer L. Handbook of Antioxidants. Marcel Dekker, New York1996: 157-201Google Scholar). In the plasma membrane, Q participates in a trans-plasma membrane electron transport chain, in which intracellular NADH is oxidized and extracellular ascorbate free radicals are reduced (5Santos-Ocana C. Cordoba F. Crane F.L. Clarke C.F. Navas P. J. Biol. Chem. 1998; 273: 8099-8105Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Recently, Q has been implicated in having a role in the rate of aging in the soil nematode Caenorhabditis elegans (6Larsen P.L. Clarke C.F. Science. 2002; 295: 120-123Crossref PubMed Scopus (213) Google Scholar). Q supplementation is shown to be effective in slowing the progression of Parkinson's disease symptoms (7Shults C.W. Oakes D. Kieburtz K. Beal M.F. Haas R. Plumb S. Juncos J.L. Nutt J. Shoulson I. Carter J. Kompoliti K. Perlmutter J.S. Reich S. Stern M. Watts R.L. Kurlan R. Molho E. Harrison M. Lew M. Arch. Neurol. 2002; 59: 1541-1550Crossref PubMed Scopus (941) Google Scholar) and its effectiveness in treating Huntington's disease is being investigated (8Ferrante R.J. Andreassen O.A. Dedeoglu A. Ferrante K.L. Jenkins B.G. Hersch S.M. Beal M.F. J. Neurosci. 2002; 22: 1592-1599Crossref PubMed Google Scholar). The proposed biosynthetic pathway of Q was elucidated by the characterization of E. coli and S. cerevisiae Q-deficient mutants (9Jonassen T. Clarke C.F. Kagan V.E. Quinn P.J. Coenzyme Q: Molecular Mechanisms in Health and Disease. CRC Press, Boca Raton, FL2001: 185-208Google Scholar, 10Meganathan R. FEMS Microbiol. Lett. 2001; 203: 131-139Crossref PubMed Google Scholar). In yeast, Q-deficient mutant strains have been placed into eight complementation groups, coq1-coq8 (9Jonassen T. Clarke C.F. Kagan V.E. Quinn P.J. Coenzyme Q: Molecular Mechanisms in Health and Disease. CRC Press, Boca Raton, FL2001: 185-208Google Scholar, 11Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). They are respiratory deficient and hence unable to grow on nonfermentable carbon sources such as glycerol. The E. coli Q-less mutants sort into ten complementation groups, ubiA-ubiH, ubiX, and ispB, and fail to grow on media containing succinate as the sole carbon source (10Meganathan R. FEMS Microbiol. Lett. 2001; 203: 131-139Crossref PubMed Google Scholar). The C-methyltransferase step was identified as being defective in E. coli ubiE mutants which accumulate the Q biosynthetic intermediate DDMQH2 (Fig. 1, compound 3) (12Young I.G. McCann L.M. Stroobant P. Gibson F. J. Bacteriol. 1971; 105: 769-778Crossref PubMed Google Scholar, 13Lee P.T. Hsu A.Y. Ha H.T. Clarke C.F. J. Bacteriol. 1997; 179: 1748-1754Crossref PubMed Scopus (115) Google Scholar). The yeast COQ5 gene product was shown to be required for the C-methyltransferase step converting DDMQH2 to DMQH2 (Fig. 1, compounds 3 and 4, respectively) (14Barkovich 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 (79) Google Scholar). The COQ5 and ubiE genes harbor four sequence motifs common to a wide variety of S-adenosyl-l-methionine-dependent methyltransferase enzymes (13Lee P.T. Hsu A.Y. Ha H.T. Clarke C.F. J. Bacteriol. 1997; 179: 1748-1754Crossref PubMed Scopus (115) Google Scholar, 15Katz J.E. Dlakic M. Clarke S. Mol. Cell. Proteomics. 2003; 2: 525-540Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). These motifs are found in a diverse group of enzymes that share a Class I methyltransferase structure (16Schubert H.L. Blumenthal R.M. Cheng X. Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar). A growing body of genetic evidence suggests that a complex of Coq polypeptides is responsible for Q biosynthesis. First, the same early intermediate, 3-hexaprenyl-4-hydroxybenzoate accumulates in the panel of Q-less yeast mutants (coq3, coq4, coq5, coq6, coq7, and coq8/abc1) (17Poon W.W. Do T.Q. Marbois B.N. Clarke C.F. Mol. Aspects Med. 1997; 18: S121-S127Crossref PubMed Scopus (58) Google Scholar). This is not the case with E. coli ubi mutants, where mutants accumulate the distinct Q-intermediate diagnostic of the blocked step of the Q biosynthetic pathway (18Gibson F. Biochem. Soc. Trans. 1973; 1: 317-326Crossref Google Scholar). Second, the coq7-1 point mutant (G104D), in contrast to the coq7-null mutant, accumulates DMQ, an intermediate only two steps away from Q (Fig. 1, compound 4) (19Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Finally, levels of the Coq3 polypeptide and corresponding O-methyltransferase activity are substantially lower in each of the coq-null mutants (20Hsu A.Y. Do T.Q. Lee P.T. Clarke C.F. Biochim. Biophys. Acta. 2000; 1484: 287-297Crossref PubMed Scopus (68) Google Scholar). Taken together, these data suggest that a multisubunit complex of Coq polypeptides is involved in Q biosynthesis in yeast. This study identifies the Coq5 as a mitochondrial matrix polypeptide peripherally associated with the inner membrane, and demonstrates that Coq5p is essential for the stability and activity of other Coq polypeptides involved in Q biosynthesis. Strains and Growth Media—The S. cerevisiae strains used in this study are listed in Table I. Growth media for yeast (21Burke D. Dawson D. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York2000: 171-181Google Scholar) were prepared as described and included YPD (1% yeast extract, 2% peptone, 2% dextrose), YPG (1% yeast extract, 2% peptone, 3% glycerol), and YPGal (1% yeast extract, 2% peptone, 2% galactose, 0.1% dextrose). E. coli were grown in Luria-Bertani (LB) broth. When required, ampicillin was added to a final concentration of 100 μg/ml. All solid media contained 2% Difco Bacto agar. Yeast and bacteria were grown at 30 °C and 37 °C, respectively.Table IGenotypes and sources of S. cerevisiae strainsStrainGenotypeSourceW303-1AMATα, ade2-1, his3-1,15, leu2-3,112, trp1-1, ura3-1R. RothsteinaDr. Rodney Rothstein, Department of Human Genetics, Columbia University.W303Δcoq5MATα, ade2-1, his3-1,15, leu2-3,112, trp1-1, ura3-1, coq5::HIS3(14Barkovich 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 (79) Google Scholar)aW303Δcoq5MATa, ade2-1, his3-1,15, leu2-3,112 trp1-1, ura3-1, coq5::HIS3(14Barkovich 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 (79) Google Scholar)CH83-B3MATα, coq5-2, ade2-1, his3-1,15, ura3-1(14Barkovich 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 (79) Google Scholar)CH83-B1MATa, coq5-2, ade2-1, his3-1,15, heu2-3,112, trp1-1, ura3-1C83bThe original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. × W303-1ACH256-3AMATa, coq5-3, met6, trp1-1, ura3-1C256bThe original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. × W303-1ACH256-2AMATa, coq5-3, his3-1,15, met6, ura3-1C256bThe original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. × W303-1ACH259-6BMATa, coq5-4, ade2-1, leu2-3,112, met6, ura3-1C259bThe original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. × W303-1ACH259-5DMATα, coq5-4, his3-1,15, leu2-3,112 ura3-1C259bThe original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. × W303-1ACH316-6BMATa, coq5-5, trp1-1, ura3-1C316bThe original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. × W303-1ACH316-1CMATa, coq5-5, his3-1,15, trp1-1, ura3-1C316bThe original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. × W303-1Aa Dr. Rodney Rothstein, Department of Human Genetics, Columbia University.b The original yeast coq5 mutant strains were provided by Dr. Alexander Tzagoloff, Dept. of Biological Sciences, Columbia University. Open table in a new tab Construction of Yeast Expression Plasmids Containing E. coli ubiE— The pUE3 plasmid (13Lee P.T. Hsu A.Y. Ha H.T. Clarke C.F. J. Bacteriol. 1997; 179: 1748-1754Crossref PubMed Scopus (115) Google Scholar) contains the E. coli ubiE ORF fused to an amino-terminal mitochondrial leader sequence of COQ3, and expression is driven by the S. cerevisiae CYC1 promoter. pQM represents the single copy empty vector control for pUE3, and contains only the mitochondrial leader sequence and the CYC1 promoter segment (22Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar). A 1.24-kb BamHI/KpnI fragment of pUE3 was inserted into the multicopy vector pRS426 (23Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), to generate pHUE3–1, a multicopy version of pUE3. To construct pS5PE1, pUE3 was digested with BamHI and EcoRI, to remove the DNA segment containing the CYC1 promoter, and a 1.0-kb fragment containing the promoter region of the COQ5 gene was inserted. The COQ5 promoter region (–1000 to –1) was prepared by PCR amplification of the template pBOB (14Barkovich 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 (79) Google Scholar) with the primers pQ5P-1 (–1000 to –980; 5′-CGCGGATCCGCGTTGAAGGGATTCCTTTGAGG-3′) and pQ5P-2 (–21 to –1; 5′-CCGGAATTCCGGTATATCTTTCTTGCTGCGAT-3′). The resulting 1.0-kb PCR product containing the COQ5 promoter was digested with BamHI and EcoRI, and this fragment replaced the CYC1 promoter in pUE3 as described. The 1.86-kb BamHI/KpnI fragment of pS5PE1 was inserted into pRS426 to generate pM5PE1, a multicopy version of pS5PE1. Sequence Analysis—DNA segments containing the protein coding region, and 5′- and 3′-flanking regions of COQ5 alleles were produced by PCR amplification of genomic DNA from yeast strains CH83-B1 (coq5-2), CH256-3A (coq5-3), CH259-5D (coq5-4), and CH316-6B (coq5-5). The 5′-primer pBCOQ5 (5′-CCGTGATACTATCGGCGATA-3′) corresponded to position –400 to –380, and the 3′-primer pTCOQ5 (5′-TGGCTATCACATGGCACAGG-3′) corresponded to +1040 to +1020 relative to the +1 position of the COQ5 coding region. DNA sequence analysis of the PCR product was performed as described previously (13Lee P.T. Hsu A.Y. Ha H.T. Clarke C.F. J. Bacteriol. 1997; 179: 1748-1754Crossref PubMed Scopus (115) Google Scholar). Generation of Antisera Against Coq5p and Western Blot Analysis—A segment of the COQ5 ORF (+91 to +924) encompassing the predicted mature Coq5p was amplified with the primers pHF5 (+91 to +113; 5′-GAAGATCTCAAAGAAGAAGAAGTTAATAGTC-3′) and pHR5 (+924 to +896; 5′-GAAGATCTTTAAAVTTTAATGCCCCAATG-3′). An ∼0.83-kb fragment of COQ5 ORF was digested with BglII and inserted into the BglII site of the expression vector pTrcHisB (Invitrogen) to generate pTHQ5–1. A fusion protein containing a His6 tag at the N terminus was overexpressed in the E. coli strain DH5α. Expression of the recombinant Coq5p was induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside at 37 °C and continued for 3 h. For purification of Coq5p, harvested cells were resuspended in 5 mm imidazole, 0.5 m NaCl, 20 mm Tris-HCl, pH 7.9 buffer containing 6 m urea and cells were disrupted by five freeze-thaw cycles and sonicated. The supernatant obtained after a 1-h centrifugation at 12,000 rpm was applied to a Ni(II)-NTA column (Qiagen). The column was washed with buffer (as above, but containing 20 mm imidazole), and the recombinant Coq5p was eluted with 250 mm imidazole in the above buffer containing 6 m urea. The His6-Coq5 fusion protein was further purified by 15% SDS-PAGE and transferred to an Immobilon P membrane. The electrophoretically pure preparation of the fusion protein was eluted from the membrane and used to generate antiserum in rabbits (Cocalico Biologicals, Inc., Reamstown, PA). The specificity of the antiserum was confirmed by immunoblotting against mitochondria from a wild type strain and a strain containing coq5::HIS3 disruption allele. Western blot analysis was carried out as described (24Belogrudov G. Hatefi Y. Biochemistry. 1994; 33: 4571-4576Crossref PubMed Scopus (108) Google Scholar). The primary antibodies were used at the following dilutions: anti-Coq3p, 1:2000; anti-Coq5p, 1:2000; anti-β subunit of F1-ATPase, 1:15,000; anti-Cyt c1, 1:1000; anti-Cyt b2, 1:5000; anti-CCPO, 1:5000; anti-Mge1p, 1:5000; and anti-porin, 1:5000. Horseradish peroxidase-linked secondary antibodies to rabbit IgG (Calbiochem) were used at a 1:1000 dilution. Mitochondrial Localization of Coq5p—Yeast were grown in YPGal media to OD600 of ∼4. Preparation of spheroplasts and cell lysate fractionation were performed as described (25Yaffe M.P. Methods Enzymol. 1991; 194: 627-643Crossref PubMed Scopus (150) Google Scholar). Crude mitochondria were further purified over a linear Nycodenz gradient (26Glick B.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (287) Google Scholar). Proteinase K protection experiments were carried out as described in (27Glick B.S. Brandt A. Cunningham K. Muller S. Hallberg R.L. Schatz G. Cell. 1992; 69: 809-822Abstract Full Text PDF PubMed Scopus (303) Google Scholar) using Nycodenz-purified mitochondria. Mitochondrial subfractionation was carried out by generating mitoplasts (26Glick B.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (287) Google Scholar) followed by sonication (3 × 30 s on ice slurry, 50% duty cycle, 4 output setting; Sonifier W350, Branson Sonic Power Co.). Mitochondrial membrane and soluble fractions were separated by centrifugation (150,000 × g, 60 min, 4 °C). The membrane fraction was washed by repeated centrifugation. Equal aliquots of starting mitochondria, intermembrane space, membrane, and matrix fractions were separated by 15% SDS-PAGE and after transfer to nitrocellulose were subjected to Western blot analysis. In Vitro C-Methyltransferase Assay—Assays of C-methyltransferase activity were performed with 2-farnesyl-6-methoxy-1,4-benzoquinol (compound 3, Fig. 1) as substrate to a final concentration of 50 μm (14Barkovich 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 (79) Google Scholar). The assay was modified so that reactions were similar to those described for the γ-tocopherol methyltransferases (28Koch M. Lemke R. Heise K.P. Mock H.P. Eur. J. Biochem. 2003; 270: 84-92Crossref PubMed Scopus (34) Google Scholar). Each reaction mixture (500 μl) contained 50 mm Tricine-NaOH, pH 7.5, 1 mm MgCl2, 0.5 mm NADH, and 0.2–0.4 mg crude yeast mitochondrial protein. Reactions were started with the addition of S-adenosyl-l-[methyl-3H] methionine to a final concentration of 1 μm (PerkinElmer Life Science Products, 81.5 Ci/mmol) and incubated at 37 °C for 1 h. Assays performed under these conditions with wild-type mitochondria showed linear rates of product formation. Reactions were stopped by addition of 5 μl of glacial acetic acid and oxidized with 25 μl of fresh 1% ammonium cerium(IV) nitrate. Lipids were extracted with 2 ml of 1:2 (v/v) chloroform/methanol and 500 μl of 0.9% (w/v) NaCl and centrifuged at 2500 rpm for 5 min. The organic layer was transferred to a new tube and dried under nitrogen gas, separated by HPLC and assayed for radioactivity (22Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar). In Vitro O-Methyltransferase Assay—Protein concentration was determined with the BCA assay (Pierce) with bovine serum albumin as a standard. Assays of O-methyltransferase activity were performed with 3,4-dihydroxy-5-farnesylbenzoic acid (compound 1, Fig. 1) or 2-farnesyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinol (compound 5, Fig. 1) as substrate (29Poon 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 (95) Google Scholar). The corresponding farnesylated analogs of methylated products (compounds 2 and 6, Fig. 1) served as the respective product standards. Each reaction mixture (250 μl) contained 50 mm sodium phosphate, pH 7.0, 1 mm ZnSO4, and 1 mg of crude yeast mitochondrial protein. Assays with compound 5 as substrate also contained 1 mm NADH. The final concentration of substrate per reaction was 50 μm. Reactions were started with the addition of S-adenosyl-l-[methyl-3H]methionine to a final concentration of 0.43 μm (PerkinElmer Life Science Products, 63.8 Ci/mmol) and incubated at 37 °C for 30 min. Reactions were stopped by addition of 5 μl glacial acetic acid. The lipids were extracted twice with 1 ml of 5:2 (v/v) hexane/ethanol, dried under nitrogen. HPLC and assays for radioactivity were performed as described above. RNA Isolation and Northern Analysis—Total RNA was isolated from the yeast strains listed in Table I. Yeast were grown in YPGal media and harvested in mid-log phase (OD600 = 1.0). RNA was isolated by the hot phenol method as described (30Chanfreau G. Rotondo G. Legrain P. Jacquier A. EMBO J. 1998; 17: 3726-3737Crossref PubMed Scopus (140) Google Scholar). RNA was resolved by electrophoresis on a 1% formaldehyde gel and subsequently transferred to a Hybond-N+ charged nylon membrane (Amersham Biosciences). The membrane was rinsed in 2× SSC (1× SSC: 0.15 m sodium chloride, 0.014 m sodium citrate, pH 7.0), UV-treated, and prehybridized in hybridization buffer containing 7% SDS, 0.5 m sodium phosphate, pH 7.2, 1 mm EDTA, and 1% bovine serum albumin, at 65 °C for 20 min. Hybridization was performed at 65 °C overnight in hybridization buffer containing both the CHC1 and one of the COQ radiolabeled probes. Membranes were washed once with 2× SSC, 0.1% SDS at 65 °C for 15 min and once with 0.2× SSC, 0.1% SDS at 65 °C for 15 min, and placed under a phosphorimager screen overnight and analyzed with a PhosphorImager (Molecular Dynamics, version 4.0). Four radiolabeled probes were used. A DNA segment containing 440 bp of the COQ5 ORF (924 bp) was amplified from pRSHA5–1 by PCR with primers pHF5 and pSW5–2 (+507 to +482; 5′-CTTGAAATATTTTCCTTGTTCCAT-3′). A DNA segment containing the COQ3 ORF (951 bp) and 650 bp upstream was amplified from pRS12A2–2.5Sma (31Clarke C.F. Williams W. Teruya J.H. J. Biol. Chem. 1991; 266: 16636-16644Abstract Full Text PDF PubMed Google Scholar) by PCR with primers pBC3–1 (–650 to –631; 5′-TAAATTTCTGAGCTCGCCCCCGGGTATTTCATTTG-3′) and pBC3–2 (5′CGCGGGATCCATTCAGTCTCTGAATAGCCA-3′) generating a product of 420 bp. A DNA segment containing the COQ4 ORF (1008 bp) was amplified from pTAP4 (COQ4) 2M. Gulmezian and C. F. Clarke, unpublished results. by PCR with primers pHF4 (+87 to +107; 5′-CGGGATCCGTACACCTTAGGCTCATTAAT-3′) and pHR4 (+902 to +925; 5′-CGGGATCCTTAGATCCGGAGGAGTGTTA-3′) generating a 851-bp product. A clathrin heavy chain specific probe was prepared from pCHCe200 (32Payne G.S. Hasson T.B. Hasson M.S. Schekman R. Mol. Cell. Biol. 1987; 7: 3888-3898Crossref PubMed Scopus (56) Google Scholar) by PCR with primers pSWCHC1–1 (+120 to +149; 5′-GAATAAATACAAAGAGAATTAAGAAAAGTA-3′) and pSWCHC1–2 (+892 to +865; 5′-GTTTTTGTTAGCCACAAAATTGATAAT-3′), generating a 672 bp clathrin (CHC1) DNA product. The resulting PCR products were separated on a 1% agarose gel and purified using a DNA purification kit (Qiagen). The purified PCR products were labeled by random priming with [α-32P]dCTP (3000 Ci/mmol, PerkinElmer Life Science Products) and the Prime-It RmT Random Primer Labeling Kit (Stratagene). Unincorporated nucleotides were eliminated with the NucTrap push column (Stratagene). A Subset of the Yeast coq5 Mutant Strains Are Rescued by E. coli ubiE—Previous work showed that a yeast strain harboring the coq5-2 mutation was rescued for growth on glycerol media by expressing E. coli ubiE, a functional homologue of COQ5 (14Barkovich 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 (79) Google Scholar). The rescuing plasmid used in these studies, pUE3, contained the E. coli ubiE coding region fused in-frame to a mitochondrial leader sequence, and expression was driven by the yeast CYC1 promoter. To examine whether E. coli ubiE could completely substitute for yeast COQ5, a panel of coq5 mutants, including a coq5-null mutant (coq5Δ) were transformed with pUE3 and tested for the ability to grow on YPG media, which contains glycerol as a nonfermentable carbon source. As shown in Fig. 2, the E. coli ubiE gene restored growth of the yeast coq5-2 and coq5-5 mutants, but not of coq5Δ, coq5-3, or coq5-4 mutant yeast. These data indicate that the E. coli ubiE gene rescues only a subset of the yeast coq5 mutant strains. Identical results were obtained following transformation with pHUE3-1, a multicopy version of pUE3, and when ubiE was expressed from the yeast COQ5 promoter, present on either single copy (pS5PE1) or multicopy (pM5PE1) plasmids (Table II). Conversely, each of the coq5 mutants is rescued when transformed with plasmids containing the yeast COQ5 gene (Table II).Table IIA subset of yeast coq5 mutant strains are rescued by E. coli ubiEPlasmidPromoterMito leaderCopy numberGeneRelevant genotypecoq5ΔaStrain αW303Δcoq5.coq5-2bStrain CH83-B1.coq5-3cStrain CH256-3A.coq5-4dStrain CH259-5D.coq5-5eStrain CH316-6B.pQM1CYC1COQ3low------pG17-T1COQ5COQ5lowCOQ5+++++pUE3CYC1COQ3lowubiE-+--+pHUE3-1CYC1COQ3highubiE-+--+pS5PE1COQ5COQ3lowubiE-+--+pM5PE1COQ5COQ3highubiE-+--+a Strain αW303Δcoq5.b Strain CH83-B1.c Strain CH256-3A.d Strain CH259-5D.e Strain CH316-6B. Open table in a new tab Complementation Analysis of coq5 Mutants—Complementation analysis was carried out with the yeast coq5 mutant strains. As shown in Table III, diploids resulting from the mating of coq5-3 and coq5-2, or coq5-3 and coq5-5 mutants, were able to grow on YPG media, while diploids produced in all other crosses were defective for growth on YPG. These results suggest that at least two categories of coq5 mutant alleles are present in the panel of coq5 mutants: one that is defective in the C-methyltransferase step (and can be rescued by E. coli ubiE), and another that is defective in a secondary function of Coq5p.Table IIIComplementation of coq5-3 by only the coq5-2 and coq5-5 mutantsα W303Δcoq5 (Δcoq5)αCH83-B3 (coq5-2)αCH259-5D (coq5-4)αCH316-6B (coq5-5)Δcoq5----aW303Δcoq5coq5-2----aCH83-B1coq5-3-+-+aCH256-3Acoq5-4----aCH259-6Bcoq5-5----aCH316-4C Open table in a new tab Sequence Analysis of coq5 Mutant Alleles—To identify the defect in each of the coq5 mutants, DNA segments encompassing the COQ5 coding region plus 400 bp of 5′-flanking and 100 bp of 3′-flanking sequence were amplified from yeast strains bearing the coq5-2, coq5-3, coq5-4, or coq5-5 alleles. Sequence analysis revealed that each coq5 mutant allele harbored a unique nucleotide substitution, resulting in the following amino acid changes: G199D in coq5-2; G304D in coq5-3; W93STOP in coq5-4; and G121R in coq5-5 (Fig. 3). The early stop mutation present in coq5-4 results in a phenotype similar to the coq5Δ mutant. As with the coq5Δ mutant strain, coq5-4 is neither rescued by E. coli ubiE, nor does it complement the coq5-2 or coq5-5 mutants. The G199D mutation in coq5-2 affects an invariant glycine residue present in a conserved regio
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