Molecular and General Genetics MGG

, Volume 241, Issue 5–6, pp 657–666 | Cite as

Characterisation of PDC2, a gene necessary for high level expression of pyruvate decarboxylase structural genes in Saccharomyces cerevlsiae

  • Stefan Hohmann
Original Articles


The regulatory gene PDC2 was identified in a screen for mutations affecting pyruvate decarboxylase activity in yeast. I have cloned and sequenced this gene. The predicted protein of 925 amino acids has no homology to any sequence in the databases. However, the protein sequence is rich in asparagine and serine residues, as is often found for transcriptional regulators. The PDC2 deletion mutant exhibits a phenotype very similar to, but more severe than that of the point mutant: a strongly reduced pyruvate decarboxylase specific activity, slow, respiration-dependent growth on glucose, and accumulation of pyruvate. The activity of other glycolytic enzymes seems to be unaffected by the pdc2Δ mutation. Synthesis of pyruvate decarboxylase is regulated by PDC2 at the transcriptional level. Expression of the major structural gene for pyruvate decarboxylase, PDC1, is strongly reduced in pdc2Δ mutants. Transcription of the generally more weakly expressed PDC5 gene appears to be entirely abolished. However, glucose induction of pyruvate decarboxylase synthesis is unaffected. Thus, PDC2 is either important for a high basal level of PDC gene expression or it plays a positive role in the autoregulation that controls expression of PDC1 and PDC5.

Key words

Saccharomyces cerevisiae Pyruvate decarboxylase Transcription Glucose induction Autoregulation 


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  1. Bennetzen JL, Hall BD (1982) Codon selection in yeast. J Biol Chem 257:3026–3031Google Scholar
  2. Bitter GA, Chang KKH, Egan KM (1991) A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter. Mol Gen Genet 231:22–32Google Scholar
  3. Boles E, Heinisch J, Zimmermann FK (1993) Different signals control the activation of glycolysis in the yeast Saccharomyces cerevisiae. Yeast 9:761–770Google Scholar
  4. Brindle PK, Holland JP, Willett CE, Innis MA, Holland MJ (1990) Multiple factors bind the upstream activation site of the yeast enolase genes ENO1 and ENO2: ABF1 protein, like repressor activator protein RAP1, binds cis-acting sequences which modulate repression or activation of transcription. Mol Cell Biol 10:4872–4885Google Scholar
  5. Broach JR (1983) Construction of high copy yeast vectors using 2 μm circle sequences. Methods Enzymol 101:307–325Google Scholar
  6. Butler G, McConnell DJ (1988) Identification of an upstream activation site in the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae. Curr Genet 14:405–412Google Scholar
  7. Butler G, Dawes IW, McConnell DJ (1990) TUF factor binds to the upstream region of the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae. Mol Gen Genet 223:449–456Google Scholar
  8. Chambers A, Tsang JSH, Stanway C, Kingsman AT, Kingsman SM (1989) Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1. Mol Cell Biol 9:5516–5524Google Scholar
  9. Chambers A, Stanway C, Tsang JSH, Henry Y, Kingsman AJ, Kingsman SM (1990) ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeast glycolytic genes PGK1 and PYK1. Nucleic Acids Res 18:5393–5399Google Scholar
  10. Chasman DI, Lue NF, Buchman AR, LaPointe JW, Lorch Y, Kornberg RD (1990) A yeast protein that influences the chromatin structure of UASG and functions as a powerful auxiliary gene activator. Genes Dev 4:503–514Google Scholar
  11. Clifton D, Fraenkel DG (1981) The gcr (glycolysis regulation) mutation in Saccharomyces cerevisiae. J Biol Chem 256:13074–13078Google Scholar
  12. Denis CL, Ferguson J, Young ET (1981) A positive regulatory gene is required for accumulation of functional messenger RNA for the glucose repressible alcohol dehydrogenase from Saccharomyces cerevisiae. J Mol Biol 148:355–368Google Scholar
  13. Fraenkel DG (1982) Carbohydrate metabolism. In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces: metabolism and gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 1–37Google Scholar
  14. Gancedo C, Serrano R (1989) Energy yielding metabolism. In: Rose AH, Harrison JS (eds) The Yeasts, vol 3, 2nd edn. Academic Press, New York, pp 205–259Google Scholar
  15. Gietz RD, Sugino A (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534Google Scholar
  16. Guthrie C, Fink GR (1990) Guide to yeast genetics and molecular biology. Academic Press, New YorkGoogle Scholar
  17. Hohmann S (1991a) Structure and expression of yeast pyruvate decarboxylase structural genes. In: Bisswanger H, Ullrich J (eds) Biochemistry and physiology of thiamin diphosphate enzymes. Verlag Chemie, Weinheim, pp 106–114Google Scholar
  18. Hohmann S (1991b) Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae. J Bacteriol 173:7963–7969Google Scholar
  19. Hohmann S (1991c) PDC6, a weakly expressed pyruvate decarboxylase gene from yeast, is activated when fused spontaneously under the control of the PDC1 promoter. Curr Genet 20:373–378Google Scholar
  20. Hohmann S (1992) Der Kohlenhydrat Stoffwechsel der Hefe Saccharomyces cerevisiae. Teil 1. Genetik Bioforum 15:4–9Google Scholar
  21. Hohmann S, Cederberg H (1990) Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDC1 and PDC5. Eur J Biochem 188:615–621Google Scholar
  22. Hohmann S, Huse K, Valentin E, Mbonyi K, Thevelein JM, Zimmermann FK (1992) Glucose-induced regulatory defects in the Saccharomyces cerevisiae byp1 growth initiation mutant and identification of MIG1 as partial suppressor. J Bacteriol 174:4183–4188Google Scholar
  23. Hohmann S, Neves MJ, de Koning W, Alijo R, Ramos J, Thevelein JM (1993) The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII. Curr Genet 23:281–289Google Scholar
  24. Hosaka K, Kodaki T, Yamashita S (1989) Cloning and characterization of the yeast CK1 gene encoding choline kinase and its expression in Escherichia coli. J Biol Chem 264:2053–2059Google Scholar
  25. Huie MA, Scott EW, Drazinic CM, Lopez MC, Hornstra IK, Yang TP, Baker HV (1992) Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae. Mol Cell Biol 12:2690–2700Google Scholar
  26. Kellermann E, Hollenberg CP (1988) The glucose- and ethanoldependent regulation of PDC1 from Saccharomyces cerevisiae are controlled by two distinct promoter regions. Curr Genet 14:337–344Google Scholar
  27. Kellermann E, Seeboth PG, Hollenberg CP (1986) Analysis of the primary structure and promoter function of a pyruvate decarboxylase gene (PDC1) from Saccharomyces cerevisiae. Nucleic Acids Res 14:8963–8977Google Scholar
  28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  29. Maitra PK, Lobo Z (1971) A kinetic study of glycolytic enzyme synthesis in yeast. J Biol Chem 246:475–488Google Scholar
  30. Myers AM, Tzagoloff A, Kinney DM, Lusty CJ (1986) Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene 45:299–310Google Scholar
  31. Nasmyth KA, Tatchell K (1980) The structure of transposable yeast mating type loci. Cell 19:753–764Google Scholar
  32. Nishizawa M, Suzuki Y, Nogi Y, Matsumoto K, Fukasawa T (1990) Yeast Gall1 protein mediates the transcriptional activation signal of two different factors, Gal4 and general regulatory factor I/repressor/activator site binding protein 1/translation upstream factor. Proc Natl Acad Sci USA 887:5373–5377Google Scholar
  33. Rothstein RJ (1983) One-step gene disruption. Methods Enzymol 101:202–211Google Scholar
  34. Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics. A laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  35. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  36. Schaaff I, Green JBA, Gozalbo D, Hohmann S (1989) A deletion of the PDC1 for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations. Curr Genet 15:75–81Google Scholar
  37. Schmitt HD, Zimmermann FK (1982) Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis. J Bacteriol 151:1146–1152Google Scholar
  38. Schmitt HD, Ciriacy M, Zimmermann FK (1983) The synthesis of yeast pyruvate decarboxylase is regulated by large variations in the messenger RNA level. Mol Gen Genet 192:247–252Google Scholar
  39. Scott EW, Baker HV (1993) Concerted action of the transcriptional activators REB1, RAP1 and GCR1 in the high-level expression of the glycolytic gene TP1. Mol Cell Biol 13:534–550Google Scholar
  40. Seeboth PG, Bohnsack K, Hollenberg CP (1990) pdc1 o mutants of Saccharomyces cerevisiae give evidence for an additional structural PDC gene: cloning of PDC5, a gene homologous to PDC1. J Bacteriol 172:678–685Google Scholar
  41. Seehaus T (1986) Molekulargenetische Untersuchungen des Pyruvat Decarboxylase Systems bei Saccharomyces cerevisiae. Ph D thesis, Technische Hochschule DarmstadtGoogle Scholar
  42. Sharp PM, Cowe E (1991) Synonymous codon usage in Saccharomyces cerevisiae. Yeast 7:657–678Google Scholar
  43. Sherman F, Fink GR, Hicks JB (1986) Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  44. Struhl K (1989) Molecular mechanisms of transcriptional regulation in yeast. Annu Rev Biochem 58:1051–1077Google Scholar
  45. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039Google Scholar
  46. Uemura H, Fraenkel DG (1990) gcr2, a new mutation affecting glycolytic gene expression in Saccharomyces cerevisiae. Mol Cell Biol 10:6389–6396Google Scholar
  47. Uemura H, Jigami Y (1992a) Role of GCR2 in transcriptional activation of yeast glycolytic genes. Mol Cell Biol 12:3834–3842Google Scholar
  48. Uemura H, Jigami Y (1992b) GCR3 encodes an acidic protein that is required for expression of glycolytic genes in Saccharomyces cerevisiae. J Bacteriol 174:5526–5532Google Scholar
  49. Wills C (1990) Regulation of sugar and ethanol metabolism in Saccharomyces cerevisiae. Crit Rev Biochem Mol Biol 25:245–280Google Scholar
  50. Wright APH, Png H-L, Hartley BS (1989) Identification, cloning and characterization of a new gene required for full pyruvate decarboxylase activity. Curr Genet 15:171–176Google Scholar
  51. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 33:103–119Google Scholar
  52. Zamenhoff S (1957) Preparation and assay of deoxyribo-nucleic acid from animal tissue. Methods Enzymol 3:696–704Google Scholar
  53. Zaret KS, Sherman F (1982) DNA sequence required for efficient transcription termination in yeast. Cell 28:563–573Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Stefan Hohmann
    • 1
  1. 1.Laboratorium voor Moleculaire Celbiologie, Instituut voor PlantkundeKatholieke Universiteit LeuvenLeuven-Heverlee, FlandersBelgium

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