PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
 
J Bacteriol. 1995 April; 177(7): 1817–1823.
PMCID: PMC176811

Sterol uptake induced by an impairment of pyridoxal phosphate synthesis in Saccharomyces cerevisiae: cloning and sequencing of the PDX3 gene encoding pyridoxine (pyridoxamine) phosphate oxidase.

Abstract

Exogenous sterols do not permeate wild-type Saccharomyces cerevisiae in aerobic conditions. However, mutant strain FKerg7, affected in lanosterol synthase, is a sterol auxotroph which is able to grow aerobically in the presence of ergosterol. Viability of this strain depends on the presence of an additional mutation, aux30, that leads to sterol permeability. Cells bearing the aux30 mutation fail to grow in standard yeast nitrogen base medium containing pyridoxine but grow normally if pyridoxine is replaced by either pyridoxal or pyridoxamine. These mutants are characterized by a lack in pyridoxine (pyridoxamine) phosphate oxidase [P(N/M)P oxidase] (EC 1.4.3.5) activity. The pleiotropic phenotype induced by the aux30 mutation includes a strong perturbation in amino acid biosynthesis. Strains bearing the aux30 mutation also display atypic fatty acid, sterol, and cytochrome patterns. Transformation of an aux30 strain with a replicative vector carrying the wild-type PDX3 gene encoding P(N/M)P oxidase restored wild-type fatty acid, sterol, and cytochrome patterns and suppressed exogenous sterol accumulation. It is proposed that sterol permeation of aux30 strains in mainly the consequence of their leaky Hem- character. The amino acid sequence of S. cerevisiae P(N/M)P oxidase inferred from the nucleotide sequence of PDX3 shows a high percentage of homology with the corresponding enzymes from Escherichia coli and Myxococcus xanthus. Several putative Gcn4p binding sequences are present in the PDX3 promoter region, leading to the assumption that transcription of this gene is under the general control of nitrogen metabolism.

Full Text

The Full Text of this article is available as a PDF (299K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • ANDREASEN AA, STIER TJB. Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J Cell Physiol. 1953 Feb;41(1):23–36. [PubMed]
  • Aoyama Y, Yoshida Y. The 14alpha-demethylation of lanosterol by a reconstituted cytochrome P-450 system from yeast microsomes. Biochem Biophys Res Commun. 1978 Nov 14;85(1):28–34. [PubMed]
  • Aoyama Y, Yoshida Y, Sato R, Susani M, Ruis H. Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes. Biochim Biophys Acta. 1981 Jan 26;663(1):194–202. [PubMed]
  • Arndt K, Fink GR. GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5' TGACTC 3' sequences. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8516–8520. [PubMed]
  • BLOOMFIELD DK, BLOCH K. The formation of delta 9-unsaturated fatty acids. J Biol Chem. 1960 Feb;235:337–345. [PubMed]
  • Bonneaud N, Ozier-Kalogeropoulos O, Li GY, Labouesse M, Minvielle-Sebastia L, Lacroute F. A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast. 1991 Aug-Sep;7(6):609–615. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Chambon C, Ladeveze V, Servouse M, Blanchard L, Javelot C, Vladescu B, Karst F. Sterol pathway in yeast. Identification and properties of mutant strains defective in mevalonate diphosphate decarboxylase and farnesyl diphosphate synthetase. Lipids. 1991 Aug;26(8):633–636. [PubMed]
  • Cigan AM, Donahue TF. Sequence and structural features associated with translational initiator regions in yeast--a review. Gene. 1987;59(1):1–18. [PubMed]
  • Del Sal G, Manfioletti G, Schneider C. The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques. 1989 May;7(5):514–520. [PubMed]
  • Dempsey WB. Synthesis of Pyridoxine by a Pyridoxal Auxotroph of Escherichia coli. J Bacteriol. 1966 Aug;92(2):333–337. [PMC free article] [PubMed]
  • Dobson MJ, Tuite MF, Roberts NA, Kingsman AJ, Kingsman SM, Perkins RE, Conroy SC, Fothergill LA. Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 1982 Apr 24;10(8):2625–2637. [PMC free article] [PubMed]
  • Doy CH, Cooper JM. Aromatic biosynthesis in yeast. I. The synthesis of tryptophan and the regulation of this pathway. Biochim Biophys Acta. 1966 Oct 31;127(2):302–316. [PubMed]
  • Duntze W, Manney TR. Two mechanisms of allelic complementation among tryptophan synthetase mutants of Saccharomyces cerevisiae. J Bacteriol. 1968 Dec;96(6):2085–2093. [PMC free article] [PubMed]
  • Ellenberger TE, Brandl CJ, Struhl K, Harrison SC. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. [PubMed]
  • Gollub EG, Liu KP, Dayan J, Adlersberg M, Sprinson DB. Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene. J Biol Chem. 1977 May 10;252(9):2846–2854. [PubMed]
  • Hagen TJ, Shimkets LJ. Nucleotide sequence and transcriptional products of the csg locus of Myxococcus xanthus. J Bacteriol. 1990 Jan;172(1):15–23. [PMC free article] [PubMed]
  • Hamilton R, Watanabe CK, de Boer HA. Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs. Nucleic Acids Res. 1987 Apr 24;15(8):3581–3593. [PMC free article] [PubMed]
  • Haskell BE, Snell EE. Effect of vitamin B6 deficiency on the composition of yeast lipids. Arch Biochem Biophys. 1965 Dec;112(3):494–505. [PubMed]
  • Hata S, Nishino T, Katsuki H, Aoyama Y, Yoshida Y. Two species of cytochrome P-450 involved in ergosterol biosynthesis of yeast. Biochem Biophys Res Commun. 1983 Oct 14;116(1):162–166. [PubMed]
  • Hata S, Nishino T, Komori M, Katsuki H. Involvement of cytochrome P-450 in delta 22-desaturation in ergosterol biosynthesis of yeast. Biochem Biophys Res Commun. 1981 Nov 16;103(1):272–277. [PubMed]
  • Hope IA, Struhl K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell. 1986 Sep 12;46(6):885–894. [PubMed]
  • Kalb VF, Woods CW, Turi TG, Dey CR, Sutter TR, Loper JC. Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA. 1987 Dec;6(6):529–537. [PubMed]
  • Karst F, Lacroute F. Yeast mutant requiring only a sterol as growth supplement. Biochem Biophys Res Commun. 1974 Jul 10;59(1):370–376. [PubMed]
  • Karst F, Lacroute F. Ertosterol biosynthesis in Saccharomyces cerevisiae: mutants deficient in the early steps of the pathway. Mol Gen Genet. 1977 Sep 9;154(3):269–277. [PubMed]
  • Kwon O, Kwok F, Churchich JE. Catalytic and regulatory properties of native and chymotrypsin-treated pyridoxine-5-phosphate oxidase. J Biol Chem. 1991 Nov 25;266(33):22136–22140. [PubMed]
  • Labbe P, Chaix P. Nouvelle technique de réalisation de spectres d'absorption à basse température. Bull Soc Chim Biol (Paris) 1969;51(12):1642–1644. [PubMed]
  • Lam HM, Tancula E, Dempsey WB, Winkler ME. Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations. J Bacteriol. 1992 Mar;174(5):1554–1567. [PMC free article] [PubMed]
  • Lam HM, Winkler ME. Characterization of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations. J Bacteriol. 1992 Oct;174(19):6033–6045. [PMC free article] [PubMed]
  • Lewis TA, Taylor FR, Parks LW. Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae. J Bacteriol. 1985 Jul;163(1):199–207. [PMC free article] [PubMed]
  • Lewis TL, Keesler GA, Fenner GP, Parks LW. Pleiotropic mutations in Saccharomyces cerevisiae affecting sterol uptake and metabolism. Yeast. 1988 Jun;4(2):93–106. [PubMed]
  • Lipman DJ, Pearson WR. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. [PubMed]
  • Lorenz RT, Parks LW. Regulation of ergosterol biosynthesis and sterol uptake in a sterol-auxotrophic yeast. J Bacteriol. 1987 Aug;169(8):3707–3711. [PMC free article] [PubMed]
  • Lorenz RT, Rodriguez RJ, Lewis TA, Parks LW. Characteristics of sterol uptake in Saccharomyces cerevisiae. J Bacteriol. 1986 Sep;167(3):981–985. [PMC free article] [PubMed]
  • Manney TR. Evidence for chain termination by super-suppressible mutants in yeast. Genetics. 1968 Dec;60(4):719–733. [PubMed]
  • Marcireau C, Guilloton M, Karst F. In vivo effects of fenpropimorph on the yeast Saccharomyces cerevisiae and determination of the molecular basis of the antifungal property. Antimicrob Agents Chemother. 1990 Jun;34(6):989–993. [PMC free article] [PubMed]
  • McCombie WR, Adams MD, Kelley JM, FitzGerald MG, Utterback TR, Khan M, Dubnick M, Kerlavage AR, Venter JC, Fields C. Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues. Nat Genet. 1992 May;1(2):124–131. [PubMed]
  • Nagai J, Katsuki H, Nishikawa Y, Nakamura I, Kamihara T. Effects of thiamine and pyridoxine on the content and composition of sterols in Saccharomyces carlsbergensis 4228. Biochem Biophys Res Commun. 1974 Sep 23;60(2):555–560. [PubMed]
  • Nakamura I, Nishikawa Y, Kamihara T, Fukui S. Respiratory deficiency in Saccharomyces carlsbergensis 4228 caused by thiamine and its prevention by pyridoxine. Biochem Biophys Res Commun. 1974 Jul 24;59(2):771–776. [PubMed]
  • Nes WR, Dhanuka IC, Pinto WJ. Evidence for facilitated transport in the absorption of sterols by Saccharomyces cerevisiae. Lipids. 1986 Jan;21(1):102–106. [PubMed]
  • Nishikawa Y, Kamihara T, Fukui S. Thiamine-induced alteration in sterol composition of Saccharomyces carlsbergensis 4228. Biochim Biophys Acta. 1978 Oct 25;531(1):86–95. [PubMed]
  • Nishikawa Y, Nakamura I, Kamihara T, Fukui S. Effects of thiamine and pyridoxine on the composition of fatty acids in Saccharomyces carlsbergensis 4228. Biochem Biophys Res Commun. 1974 Jul 24;59(2):777–780. [PubMed]
  • OGUR M, ST. JOHN R, NAGAI S. Tetrazolium overlay technique for population studies of respiration deficiency in yeast. Science. 1957 May 10;125(3254):928–929. [PubMed]
  • Pfeifer K, Arcangioli B, Guarente L. Yeast HAP1 activator competes with the factor RC2 for binding to the upstream activation site UAS1 of the CYC1 gene. Cell. 1987 Apr 10;49(1):9–18. [PubMed]
  • Poulson R, Polglase WJ. Aerobic and anaerobic coproporphyrinogenase activities in extracts from Saccharomyces cerevisiae. J Biol Chem. 1974 Oct 25;249(20):6367–6371. [PubMed]
  • Poulson R, Polglase WJ. The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae. J Biol Chem. 1975 Feb 25;250(4):1269–1274. [PubMed]
  • Raleigh EA, Murray NE, Revel H, Blumenthal RM, Westaway D, Reith AD, Rigby PW, Elhai J, Hanahan D. McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res. 1988 Feb 25;16(4):1563–1575. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Shinabarger DL, Keesler GA, Parks LW. Regulation by heme of sterol uptake in Saccharomyces cerevisiae. Steroids. 1989 Mar-May;53(3-5):607–623. [PubMed]
  • Struhl K. Molecular mechanisms of transcriptional regulation in yeast. Annu Rev Biochem. 1989;58:1051–1077. [PubMed]
  • Thorsness M, Schafer W, D'Ari L, Rine J. Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Dec;9(12):5702–5712. [PMC free article] [PubMed]
  • Trocha PJ, Sprinson DB. Location and regulation of early enzymes of sterol biosynthesis in yeast. Arch Biochem Biophys. 1976 May;174(1):45–51. [PubMed]
  • Turi TG, Loper JC. Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11). J Biol Chem. 1992 Jan 25;267(3):2046–2056. [PubMed]
  • Waterston R, Martin C, Craxton M, Huynh C, Coulson A, Hillier L, Durbin R, Green P, Shownkeen R, Halloran N, et al. A survey of expressed genes in Caenorhabditis elegans. Nat Genet. 1992 May;1(2):114–123. [PubMed]
  • Xu SH, Norton RA, Crumley FG, Nes WD. Comparison of the chromatographic properties of sterols, select additional steroids and triterpenoids: gravity-flow column liquid chromatography, thin-layer chromatography, gas-liquid chromatography and high-performance liquid chromatography. J Chromatogr. 1988 Oct 28;452:377–398. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)