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.
Similar content being viewed by others
References
Appleby CA, Morton RK (1954) Crystalline cytochrome b2 and lactic dehydrogenase of yeast. Nature 173:749–752
Arrigoni O, Singer TP (1962) Limitation of phenazine methosulfate assay for succinic and related dehydrogenases. Nature 193:1256–1258
Bennetzen JL, Hall BD (1982) Codon selection in yeast. J Biol Chem 257:3026–3031
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 22:1178–1191
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 7:609–615
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, UK
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 100:293–308
Cassio F, Leao C, van Uden N (1987) Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae. Appl Environ Microbiol 53:509–513
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 4:131–139
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 245:2423–2426
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 79:267–271
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 1:1–8
Gregolin C, Singer TP (1962) Zinc-FAD prosthetic groups of d-lactate cytochrome reductase. Biochim Biophys Acta 57:410–412
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 67:201–218
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 4:3265–3272
Guiard B, Lederer F (1976) Baker's yeast flavocytochrome b2 (l-(+)-lactate dehydrogenase). Eur J Biochem 65:537–542
Hilger F (1973) Construction and analysis of tetraploid yeast sets for gene dosage studies. J Gen Microbiol 75:23–31
Ito H, Fukada Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Jacq C, Lederer F (1972) Sur les deux formes moléculaires du cytochrome b2 de S. cerevisiae. Eur J Biochem 25:41–48
Jacq C, Lederer F (1974) Cytochrome b2 from baker's yeast (l-lactate dehydrogenase) a double-headed enzyme. Eur J Biochem 41:311–320
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 263:1619–1621
Labeyrie F, Slonimski PP (1964) Mode d'action des lacticodeshydrogénases lilies aux systèmes flavinique et cytochromique. Bull Soc Chim Biol XLIV:1793–1828
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 34:226–265
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 152:419–428
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 11:3762–3772
Magni G, von Borstel RC (1962) Different rates of spontaneous mutation during mitosis and meiosis in yeast. Genetics 47:1097–1108
Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Mortimer RK, Hawthorne DC (1969) Yeast genetics. In: Rose AH, Harrison JS (eds) The yeasts, vol 1. Academic Press, New York
Nasmyth KA, Reed SI (1980) Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci 77:2119–2123
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 48:443–453
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 40:85–92
Nygaard AP (1961) d-lactic cytochrome c reductase a flavoprotein from yeast. J Biol Chem 236:1585–1593
Pfanner N, Hartl FU, Neupert W (1988) Import of proteins into mitochondria: a multi-step process. Eur J Biochem 175:205–212
Polakis ES, Bartley W, Meek GA (1965) Changes in the activity of respiratory enzymes during the aerobic growth on different carbon sources. Biochem J 97:298–302
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 5:1327–1334
Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors, Proc Natl Acad Sci USA 74:5463–5467
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 243:5446–5456
Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, New York
Slonimski PP (1953) Formation des enzymes respiratoires chez la levure. Masson et Cie Editeurs, Paris
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 435–461
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 97:183–201
Somlo M (1966) Presence et régulation de la synthèse de la D-LDH chez la levure aérobie. Bull Soc Chim Biol 48:247–276
Somlo M (1967) Etude physiologique des trois lacticodeshydrogénases de la levure. Thèse d'Etat, Paris
von Heijne G (1986) Mitochondrial targeting sequences may form amphiphilic helices. EMBO J 5:1335–1342
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 104:321–330
Author information
Authors and Affiliations
Additional information
Communicated by W Gajewski
Rights and permissions
About this article
Cite this article
Lodi, T., Ferrero, I. Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase. Molec. Gen. Genet. 238, 315–324 (1993). https://doi.org/10.1007/BF00291989
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00291989