1887

Microbiology Society logo

Volume 147, Issue 2

Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions Free

Abstract

Yeast phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1) are allosterically regulated enzymes that catalyse essentially irreversible reactions in glycolysis. Both the synthesis and activity of these enzymes are tightly regulated. To separate experimentally the control of Pf1k and Pyk1 synthesis from their allosteric regulation, a congenic set of , and mutants was constructed in which these wild-type coding regions were driven by alternative promoters. Mutants carrying promoter fusions displayed normal rates of growth, glucose consumption and ethanol production, indicating that the relatively tight regulation of Pyk1 and Pf1k synthesis is not essential for glycolytic control under fermentative growth conditions. Mutants carrying fusions to an enhancer-less version of the promoter ( ) expressed Pyk1 and Pf1k at about 25-fold lower levels than normal. Physiological and metabolic analysis of the double mutant indicated that decreased Pf1k had no significant effect on growth, apparently due to compensatory increases in its positive effector, fructose 2,6-bisphosphate. In contrast, growth rate and glycolytic flux were reduced in the mutant, which had decreased Pyk1 levels. Unexpectedly, the reduced Pyk1 levels caused the flow of carbon to the TCA cycle to increase, even under fermentative growth conditions. Therefore, Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): Glycolysis , MCA, metabolic control analysis , metabolic flux , Pf1k, 6-phosphofructo-1-kinase , phosphofructokinase , Pyk1, pyruvate kinase , pyruvate kinase and yeast physiology
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-2-391
2001-02-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/2/1470391a.html?itemId=/content/journal/micro/10.1099/00221287-147-2-391&mimeType=html&fmt=ahah

References

  1. Arvantidis A., Heinisch J. J. 1994; Studies on the function of yeast phosphofructokinase subunits by in vitro mutagenesis. J Biol Chem 269:8911–8918
     [Google Scholar]
  2. Avigad G. 1981; Stimulation of yeast phosphofructokinase activity by fructose 2,6-bisphosphate. Biochem Biophys Res Commun 102:985–991 [CrossRef]
     [Google Scholar]
  3. Bañuelos, M., Gancedo, C. & Gancedo, J. M. 1977; Activation of phosphate by yeast phosphofructokinase. J Biol Chem 252:6394–6398
     [Google Scholar]
  4. Bartrons R., Van Schaftingen E., Vissers S., Hers H.-G. 1982; The stimulation of yeast phosphofructokinase by fructose-2,6-bisphosphate. FEBS Lett 143:137–140 [CrossRef]
     [Google Scholar]
  5. Bergmeyer H. U. 1986 Methods in Enzymatic Analysis, 3rd edn. Weinheim: Verlag Chemie;
     [Google Scholar]
  6. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72:248–254 [CrossRef]
     [Google Scholar]
  7. Brown A. J. P. 1994; Measurement of mRNA stability. In Molecular Genetics of Yeast: a Practical Approach pp. 147–159Edited by Johnston J. R. Oxford: Oxford University Press;
     [Google Scholar]
  8. Burke R. L., Tekamp-Olson P., Najarian R. 1983; The isolation, characterisation and sequence of the pyruvate kinase gene of Saccharomyces cerevisiae. J Biol Chem 258:2193–2201
     [Google Scholar]
  9. Church G. M., Gilbert W. 1984; Genomic sequencing Proc . Natl Acad Sci USA 81:1991–1995 [CrossRef]
     [Google Scholar]
  10. Crimmins C. 1995 The significance of genetic regulation in the control of glycolysis in Saccharomyces cerevisiae PhD thesis University of Aberdeen;
     [Google Scholar]
  11. Davies S. E. C., Brindle K. M. 1992; Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae. Biochemistry 31:4729–4735 [CrossRef]
     [Google Scholar]
  12. Dickinson J. R. 1999; Carbon metabolism. In The Metabolism and Molecular Physiology of Saccharomyces cerevisiae pp. 23–55Edited by Dickinson J. R., Schweizer M. London: Taylor & Francis;
     [Google Scholar]
  13. Dickinson J. R., Hewlins M. J. E. 1988; A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae. J Gen Microbiol 134:333–337
     [Google Scholar]
  14. Dickinson J. R., Hewlins M. J. E. 1991; 13C NMR analysis of a developmental pathway mutation in Saccharomyces cerevisiae reveals a cell derepressed for succinate dehydrogenase. J Gen Microbiol 137:1033–1037 [CrossRef]
     [Google Scholar]
  15. Dickinson J. R., Lanterman M. M., Danner D. J., Pearson B. M., Sanz P., Harrison S. J., Hewlins M. J. E. 1997; A 13C nuclear magnetic resonance investigation of the metabolism of leucine to isoamyl alcohol in Saccharomyces cerevisiae. J Biol Chem 272:26871–26878 [CrossRef]
     [Google Scholar]
  16. Feinberg A. P., Vogelstein B. 1983; A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13 [CrossRef]
     [Google Scholar]
  17. Fell D. A. 1984; Phosphofructokinase and glycolytic flux. Trends Biochem Sci 9:515–516
     [Google Scholar]
  18. Fell D. A. 1997 Understanding the Control of Metabolism London: Portland Press;
     [Google Scholar]
  19. Ferguson G. P., McLaggan D., Booth I. R. 1995; Potassium channel activation by glutathione-S-conjugates in Escherichia coli: protection against methylglyoxal is mediated by cytoplasmic acidification. Mol Microbiol 17:1025–1033 [CrossRef]
     [Google Scholar]
  20. Fraenkel D. G. 1982; Carbohydrate metabolism. In The Molecular Biology of the Yeast Saccharomyces: Metabolism and Biosynthesis pp. 1–37Edited by Strathern J. N., Jones E. W., Broach J. R. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  21. Gancedo C., Serrano R. 1989; Energy yielding metabolism. In The Yeasts, vol. 3: Metabolism and Physiology of Yeasts pp. 205–259Edited by Rose A. H., Harrison J. S. London: Academic Press;
     [Google Scholar]
  22. Gietz R. D., Woods R. A. 1998; Transformation of yeast by the lithium acetate/single-stranded carrier DNA/PEG method. In Yeast Gene Analysis: Methods in Microbiology pp. 53–66Edited by Brown A. J. P., Tuite M. F. London: Academic Press;
     [Google Scholar]
  23. Gonzalez B., François J., Renaud M. 1997; A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol. Yeast 13:1347–1356 [CrossRef]
     [Google Scholar]
  24. Heinisch J. 1986; Isolation and characterisation of the two structural genes coding for phosphofructokinase in yeast. Mol Gen Genet 202:75–82 [CrossRef]
     [Google Scholar]
  25. Heinisch J. J., Boles E., Timpel C. 1996; A yeast phosphofructokinase insensitive to the allosteric activator fructose-2,6-bisphosphate. J Biol Chem 271:15928–15933 [CrossRef]
     [Google Scholar]
  26. Hess B., Boiteux A., Kruger J. 1969; Cooperation of glycolytic enzymes. Adv Enzyme Regul 7:149–169 [CrossRef]
     [Google Scholar]
  27. Hoffman C. S., Winston F. 1987; A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272 [CrossRef]
     [Google Scholar]
  28. Hunsley J. R., Suelter C. H. 1969; Yeast pyruvate kinase: kinetic properties. J Biol Chem 244:4819–4822
     [Google Scholar]
  29. Johnston M., Carlson M. 1992; Regulation of carbon and phosphate utilization. In The Molecular and Cellular Biology of the Yeast Saccharomyces, vol. 2: Gene Expression pp. 193–281Edited by Jones E. W., Pringle J. R., Broach J. R. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  30. Kacser H., Burns J. A. 1973; The control of flux. Symp Soc Exper Biol 27:65–104
     [Google Scholar]
  31. Kacser H., Burns J. A. 1979; Molecular democracy: who shares the controls?. Biochem Soc Trans 7:1149–1160
     [Google Scholar]
  32. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
     [Google Scholar]
  33. Meijer M. M. C., Boostra J., Verkleij A. J., Verrips C. T. 1998; Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux. J Biol Chem 273:24102–24107 [CrossRef]
     [Google Scholar]
  34. Mellor J. E., Dobson M. J., Roberts N. A., Kingsman A. J., Kingsman S. M. 1985; Factors affecting heterologous gene expression in Saccharomyces cerevisiae. Gene 33:215–226 [CrossRef]
     [Google Scholar]
  35. Moore P. A., Bettany A. J. E., Brown A. J. P. 1990a; The expression of a yeast glycolytic gene is subject to dosage compensation. Gene 89:85–92 [CrossRef]
     [Google Scholar]
  36. Moore P. A., Bettany A. J. E., Brown A. J. P. 1990b; Multiple copies of the pyruvate kinase gene inhibit yeast cell growth. J Gen Microbiol 136:2359–2366 [CrossRef]
     [Google Scholar]
  37. Moore P. A., Bettany A. J. E., Brown A. J. P. 1990c; The yeast pyruvate kinase gene is regulated at multiple levels. In Post-Transcriptional Control of Gene Expression pp. 421–432Edited by McCarthy J. E. G., Tuite M. F. Berlin: Springer;
     [Google Scholar]
  38. Moore P. A., Sagliocco F. A., Wood R. C. M., Brown A. J. P. 1991; Yeast glycolytic mRNAs are differentially regulated. Mol Cell Biol 11:5330–5337
     [Google Scholar]
  39. Ogden J. E., Stanway C., Kim S., Mellor J., Kingsman A. J., Kingsman S. M. 1986; Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences. Mol Cell Biol 6:4335–4343
     [Google Scholar]
  40. Piper P. W., Curran B., Davies M. W., Hirst K., Lockheart A., Ogden J. E., Stanway C., Kingsman A. J., Kingsman S. M. 1988; A heat shock element in the phosphoglycerate kinase gene promoter of yeast. Nucleic Acids Res 16:1333–1348 [CrossRef]
     [Google Scholar]
  41. Planta R., Brown A. J. P., Cadahia J. L.13 other authors 1999; Yeast functional analysis reports: transcript analysis of 250 novel yeast genes from chromosome XIV. Yeast 15:329–350 [CrossRef]
     [Google Scholar]
  42. Postma E., Verduyn C., Scheffers W. A., van Dijken J. P. 1989; Enzymic analysis of the Crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae. Appl Environ Microbiol 55:468–477
     [Google Scholar]
  43. Racker E. 1947; Spectrophotometric measurement of hexokinase and phosphohexokinase activity. J Biol Chem 167:843–854
     [Google Scholar]
  44. Reibstein D., den Hollander J. A., Pilkis S. J., Shulman R. G. 1986; Studies on the regulation of yeast phosphofructo-1-kinase: its role in aerobic and anaerobic glycolysis. Biochemistry 25:219–227 [CrossRef]
     [Google Scholar]
  45. Schaaff I., Heinisch J., Zimmermann F. K. 1989; Overproduction of glycolytic enzymes in yeast. Yeast 5:285–290 [CrossRef]
     [Google Scholar]
  46. Sikorski R. S., Hieter P. 1989; A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27
     [Google Scholar]
  47. Strathern J. N., Mastrangelo M., Rinckel L. A., Garfinkel D. J. 1994; Ty insertional mutagenesis. In Molecular Genetics of Yeast: a Practical Approach pp. 111–119Edited by Johnston J. R. Oxford: Oxford University Press;
     [Google Scholar]
  48. Teusink B., Baganz F., Westerhoff H. V., Oliver S. G. 1998; Metabolic control analysis as a tool in the elucidation of the function of novel genes. In Yeast Gene Analysis: Methods in Microbiology pp. 297–336Edited by Brown A. J. P., Tuite M. F. London: Academic Press;
     [Google Scholar]
  49. Thomas B. J., Rothstein R. 1989; The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptional regulation gene. Genetics 123:725–738
     [Google Scholar]
  50. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354 [CrossRef]
     [Google Scholar]
  51. Van Schaftingen E., Lederer B., Bartrons R., Hers H.-G. 1982; A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Eur J Biochem 129:191–195 [CrossRef]
     [Google Scholar]
  52. Wicksteed B. L., Collins I., Dershowitz A., Stateva L. I., Green R. P., Oliver S. G., Brown A. J. P., Newlon C. S. 1994; A physical comparison of chromosome III in six strains of Saccharomyces cerevisiae. Yeast 10:39–57 [CrossRef]
     [Google Scholar]
  53. Ye L., Kruckelberg A. L., Berden J. A., van Dam K. 1999; Growth and glucose repression are controlled by glucose transport in Saccharomyces cerevisiae cells containing only one glucose transporter. J Bacteriol 181:4671–4675
     [Google Scholar]
  54. Yun S. L., Aust A. E., Suelter C. H. 1976; A revised preparation of yeast (Saccharomyces cerevisiae) pyruvate kinase. J Biol Chem 251:124–128
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-2-391
Loading
Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions
Microbiology 147, 391 (2001); https://doi.org/10.1099/00221287-147-2-391
/content/journal/micro/10.1099/00221287-147-2-391
/content/journal/micro/10.1099/00221287-147-2-391
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error