Molecular and General Genetics MGG

, Volume 238, Issue 3, pp 315–324

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

  • T. Lodi
  • I. Ferrero
Original Articles

Abstract

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate pyruvate+ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono-γ-lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.

Key words

Saccharomyces cerevisiae Nuclear gene Mitochondrial enzyme Lactate dehydrogenase Flavoprotein 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Appleby CA, Morton RK (1954) Crystalline cytochrome b2 and lactic dehydrogenase of yeast. Nature 173:749–752Google Scholar
  2. Arrigoni O, Singer TP (1962) Limitation of phenazine methosulfate assay for succinic and related dehydrogenases. Nature 193:1256–1258Google Scholar
  3. Bennetzen JL, Hall BD (1982) Codon selection in yeast. J Biol Chem 257:3026–3031Google Scholar
  4. Bernheim F (1928) The specificity of the dehydrogenases. The separation of the citric acid dehydrase from liver and of the lactic acid dehydrase from yeast. Biochem J 22:1178–1191Google Scholar
  5. Bonneaud N, Ozier-Kalogeropoulos O, Li G, Labouesse M, Minvielle-Sebastia L, Lacroute F (1991) A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7:609–615Google Scholar
  6. Boswell RD, Lesk AM (1988) Sequence comparison and alignment: the measurement and interpretation of sequence similarity. In: Lesk AM (ed) Computational molecular biology: sources and methods for sequence analysis. Oxford University Press, Oxford, UKGoogle Scholar
  7. Casabadan MJ, Martinez-Arias A, Shapira SK, Chou J (1983) β-galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Methods Enzymol 100:293–308Google Scholar
  8. Cassio F, Leao C, van Uden N (1987) Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 53:509–513Google Scholar
  9. Ferrero I, Saccani MG, Rossi C, Viola AM, Puglisi PP (1981) The role of the mitochondrial particle in the regulation of the l- and d-lactate ferricytochrome c oxidoreductases in the yeast Saccharomyces cerevisiae. Microbiologica 4:131–139Google Scholar
  10. Galzy P, Slonimski PP (1957) Variations physiologiques de la levure au cours de la croissance sur l'acide lactique come seule source de carbone. C R Acad Sci 245:2423–2426Google Scholar
  11. Gasser SM, Ohashi A, Daum G, Bohni PC, Gibson J, Reid GA, Yonetani T, Schatz G (1982) Imported mitochondrial proteins cytochrome b2 and cytochrome c1 are processed in two steps. Proc Natl Acad Sci USA 79:267–271Google Scholar
  12. Genga AM, Lodi T, Tassi F, Ferrero T (1983) Mitochondrial NAD, l-lactate dehydrogenase and NAD, d-lactate dehydrogenase in the yeast Saccharomyces cerevisiae. Microbiologica 1:1–8Google Scholar
  13. Gregolin C, Singer TP (1962) Zinc-FAD prosthetic groups of d-lactate cytochrome reductase. Biochim Biophys Acta 57:410–412Google Scholar
  14. Gregolin C, Singer T (1963) The lactic dehydrogenase of yeast. III. D(−)lactic cytochrome c reductase, a zinc-flavoprotein from aerobic yeast. Biochim Biophys Acta 67:201–218Google Scholar
  15. Guiard B (1985) Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast l(+)-lactate cytochrome c oxidoreductase (cytochrome b2). EMBO J 4:3265–3272Google Scholar
  16. Guiard B, Lederer F (1976) Baker's yeast flavocytochrome b2 (l-(+)-lactate dehydrogenase). Eur J Biochem 65:537–542Google Scholar
  17. Hilger F (1973) Construction and analysis of tetraploid yeast sets for gene dosage studies. J Gen Microbiol 75:23–31Google Scholar
  18. Ito H, Fukada Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168Google Scholar
  19. Jacq C, Lederer F (1972) Sur les deux formes moléculaires du cytochrome b2 de S. cerevisiae. Eur J Biochem 25:41–48Google Scholar
  20. Jacq C, Lederer F (1974) Cytochrome b2 from baker's yeast (l-lactate dehydrogenase) a double-headed enzyme. Eur J Biochem 41:311–320Google Scholar
  21. Koshizaka T, Nishikimi M, Ozawa T, Yagi K (1988) Isolation and sequence analysis of a complementary DNA encoding rat liver l-gulono-γ-lactone oxidase, a key enzyme for l-ascorbic acid biosynthesis, J Biol Chem 263:1619–1621Google Scholar
  22. Labeyrie F, Slonimski PP (1964) Mode d'action des lacticodeshydrogénases lilies aux systèmes flavinique et cytochromique. Bull Soc Chim Biol XLIV:1793–1828Google Scholar
  23. Labeyrie F, Slonimski PP, Naslin L (1959) Sur la différence de stéréospecificité entre la deshydrogénase lactique extraite de la levure anaérobie et celle extraite de la levure aéreobie. Biochim Biophys Acta 34:226–265Google Scholar
  24. Lederer F, Cortial S, Becam AM, Haumont PY, Perez L (1985) Complete amino acid sequence of flavocytochrome b2 from baker's yeast. Eur J Biochem 152:419–428Google Scholar
  25. Lodi T, Guiard B (1991) Complex transcriptional regulation of the S. cerevisiae CYB2 gene encoding cytochrome b2. CYPI (HAP1) activator binds to the CYB2 upstream activation site UAS1-B2. Mol Cell Biol 11:3762–3772Google Scholar
  26. Magni G, von Borstel RC (1962) Different rates of spontaneous mutation during mitosis and meiosis in yeast. Genetics 47:1097–1108Google Scholar
  27. Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162Google Scholar
  28. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  29. Mortimer RK, Hawthorne DC (1969) Yeast genetics. In: Rose AH, Harrison JS (eds) The yeasts, vol 1. Academic Press, New YorkGoogle Scholar
  30. Nasmyth KA, Reed SI (1980) Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci 77:2119–2123Google Scholar
  31. Needleman SB, Wunsch CD (1970) A general method applicable to the search for similarities in the amino acids sequence of two proteins J Mol Biol 48:443–453Google Scholar
  32. Nygaard AP (1960) Lactic dehydrogenase in yeast III. A comparative study of the kinetics properties and the stability of two isolated forms of the enzyme. Biochim Biophys Acta 40:85–92Google Scholar
  33. Nygaard AP (1961) d-lactic cytochrome c reductase a flavoprotein from yeast. J Biol Chem 236:1585–1593Google Scholar
  34. Pfanner N, Hartl FU, Neupert W (1988) Import of proteins into mitochondria: a multi-step process. Eur J Biochem 175:205–212Google Scholar
  35. Polakis ES, Bartley W, Meek GA (1965) Changes in the activity of respiratory enzymes during the aerobic growth on different carbon sources. Biochem J 97:298–302Google Scholar
  36. Roise D, Horvath SJ, Tomich JM, Richards JH, Shatz G (1986) A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers. EMBO J 5:1327–1334Google Scholar
  37. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211Google Scholar
  38. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors, Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  39. Sherman F, Steward JW, Parker JH, Inhaber E, Shipman MA, Putterman GJ, Gardisky RL, Margoliash E (1968) The mutational alteration of the primary structure of the yeast iso-1-cytochrome c. J Biol Chem 243:5446–5456Google Scholar
  40. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  41. Slonimski PP (1953) Formation des enzymes respiratoires chez la levure. Masson et Cie Editeurs, ParisGoogle Scholar
  42. Slonimski PP, Acher R, Péré G, Sets A, Somlo M (1963) Elements du système respiratoire et leur régulation: cytochromes et isocytochromes. In: International symposium on mechanisms of regulation of cellular activities in microorganisms, Marseille. CNRS, pp 435–461Google Scholar
  43. Somlo M (1965) Induction des lactico-cytochrome c reductases (d et l-) de la levure aérobie par des lactates (d- et l-). Biochim Biophys Acta 97:183–201Google Scholar
  44. Somlo M (1966) Presence et régulation de la synthèse de la D-LDH chez la levure aérobie. Bull Soc Chim Biol 48:247–276Google Scholar
  45. Somlo M (1967) Etude physiologique des trois lacticodeshydrogénases de la levure. Thèse d'Etat, ParisGoogle Scholar
  46. von Heijne G (1986) Mitochondrial targeting sequences may form amphiphilic helices. EMBO J 5:1335–1342Google Scholar
  47. Zimmerman FK, Schmiedt I, Ten Berge AMA (1969) Dominance and recessiveness at the protein level in mutant and wild type crosses in Saccharomyces cerevisiae. Mol Gen Genet 104:321–330Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • T. Lodi
    • 1
  • I. Ferrero
    • 1
  1. 1.Institute of GeneticsUniversity of ParmaParmaItaly

Personalised recommendations