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Regulation of fitness in yeast overexpressing glycolytic enzymes: parameters of growth and viability

Published online by Cambridge University Press:  14 April 2009

R. F. Rosenzweig
R. F. Rosenzweig
Affiliation:
Department of Biology, Leidy Laboratories, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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Current models predict that large increases over wild-type in the activity of one enzyme will not alter an organism's fitness. This prediction is tested in Saccharomyces cerevisiae through the use of a high copy plasmid that bears one of the following: hexokinase B (HEXB), phosphoglucose isomerase (PGI), phosphofructokinase (PFKA and PFKB), or pyruvate kinase (PYK). Transformants containing these plasmids demonstrate a four to ten-fold increase in enzyme specific activity over either the parent strain or transformants containing the plasmid alone. Haploid and diploid transformants derived from independent backgrounds were grown on both fermentable and non-fermentable carbon sources and evaluated for several components of fitness. These include growth rate under non-limiting conditions, maximum stationary phase density, and viability in extended batch culture. Cell viability is not affected by overproduction of these enzymes. Growth rate and stationary phase density do not differ significantly among strains that overexpress HEXB, PGI or contain the vector alone. PFKA, B transformants show reduced growth rate on glucose in one background only. For these loci the current model is confirmed. By contrast, when grown on glucose, yeast overexpressing PYK demonstrate reduced growth rate and increased stationary phase density in both backgrounds. These effects are abolished in cells containing plasmids with a Tn5 disrupted copy of the PYK gene. Our results are consistent with reports that the PYK locus may exert control over the yeast cell cycle and suggest that it will be challenging to model relations between fitness and activity for multifunctional proteins.

Type
Research Article
Information
Genetics Research , Volume 59 , Issue 1 , February 1992 , pp. 35 - 48
Copyright
Copyright © Cambridge University Press 1992

References

Adams, B. G. (1972). Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. Journal of Bacteriology 111, 308–315.CrossRefGoogle ScholarPubMed
Bach, M.-L. (1984). Ty1-promoted expression of aspartate trans-carbamylase in the yeast Saccharomyces cerevisiae. Molecular and General Genetics 194, 395–401.CrossRefGoogle Scholar
Banuelos, M.Gancedo, C. & Gancedo, J. M. (1977). Activation by phosphate of yeast phosphofructokinase. Journal of Biological Chemistry 252, 6394–6398.CrossRefGoogle ScholarPubMed
Becker, J.-U. & Betz, A. (1972). Membrane transport as controlling pacemaker of glycolysis in Saccharomyces cerevisiae. Biochemica et Biophysica Acta 274, 584–597.CrossRefGoogle Scholar
Bisson, L. F. & Fraenkel, D. G. (1983). Involvement of kinases in glucose and fructose uptake by Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences (USA) 80, 1730–1734.CrossRefGoogle ScholarPubMed
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle ScholarPubMed
Breitenbach-Schmitt, I.Heinisch, J.Schmitt, H. D. & Zimmermann, F. K. (1984). Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation. Molecular and General Genetics 195, 530–535.CrossRefGoogle Scholar
Burke, R. L.Tekamp-Olson, P. & Najarian, R. (1983). The isolation, characterization, and sequence of the pyruvate kinase gene of Saccharomyces cerevisiae. Journal of Biological Chemistry 258, 2193–2201.CrossRefGoogle ScholarPubMed
Burton, R. S. & Feldman, M. (1983). Physiological effects of an allozyme polymorphism: glutamate-pyruvate transaminase and response to hyperosmotic stress in the copepod Tigriopus californicus. Biochemical Genetics 21, 239–251.CrossRefGoogle ScholarPubMed
Carlson, M.Osmond, B. CNeigeborn, L. & Botstein, D. (1984). A suppressor of snf1 mutations causes constitutive high-level invertase synthesis in yeast. Genetics 107,19–32.CrossRefGoogle ScholarPubMed
Carter, A. A. & Watt, W. B. (1989). Adaptation at specific loci. V. Metabolically adjacent enzyme loci may have very distinct experiences of selection pressures. Genetics 119, 913–924.CrossRefGoogle Scholar
Carter, B. L. A. & Sudbury, P. E. (1980). Small-sized mutants of Saccharomyces cerevisiae. Genetics 96, 561–566.CrossRefGoogle ScholarPubMed
Chaput, M.Claes, V.Portetelle, D. I.Cludts, I.Cravador, A.Burny, A.Gras, H. & Tartar, A. (1988). The neurotropic factor neuroleukin is 90 % homologous with phosphohexose isomerase. Nature 332, 454–455.CrossRefGoogle Scholar
Cheah, U. E.Weigand, W. A. & Stark, B. C. (1987). Effects of recombinant plasmid size on cellular processes in Escherichia coli. Plasmid 18, 127–134.CrossRefGoogle ScholarPubMed
Clark, A. G. (1989). Causes and consequences of variation in energy storage in Drosophila melanogaster. Genetics 123, 131–144.CrossRefGoogle ScholarPubMed
Clewell, D. & Helinski, D. (1970). Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry 9, 4428–4440.Google ScholarPubMed
Clifton, D.Weinstock, S. B. & Fraenkel, D. G. (1977). Glycolysis mutants in Saccharomyces cerevisiae. Genetics 88, 1–11.CrossRefGoogle Scholar
Clifton, D. & Fraenkel, D. G. (1981). The gcr (glycolysis regulation) mutation of Saccharomyces cerevisiae. Journal of Biological Chemistry 256, 13074–13078.CrossRefGoogle ScholarPubMed
Coleman, K. G.Steensma, H. Y.Kaback, D. B. & Pringle, J. R. (1986). Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation and characterization of CDC24 gene and adjacent regions of the chromosome. Molecular and Cellular Biology 6, 4516–4525.Google ScholarPubMed
Crabtree, B. & Newsholme, E. A. (1985). A quantitative approach to metabolic control. Current Topics in Cell Regulation 25, 21–76.CrossRefGoogle ScholarPubMed
Cross, F. (1988). DAF1, a mutant gene affecting size control, pheromone response, and cell-cycle kinetics in Saccharomyces cerevisiae. Molecular and Cellular Biology 8, 4675–7684.Google Scholar
Dean, A. M. (1989). Selection and neutrality in lactose operons of Escherichia coli. Genetics 123, 441–454.CrossRefGoogle ScholarPubMed
Dean, A. M.Dykhuizen, D. E. & Hartl, D. L. (1986). Fitness as a function of β-galactosidase activity in Escherichia coli. Genetical Research 481, 1–8.CrossRefGoogle Scholar
De Jong, G. & Scharloo, W. (1976). Environmental determination of selective significance or neutrality of amylase variants in Drosophila melanogaster. Genetics 84, 77–94.CrossRefGoogle ScholarPubMed
Does, A. L. & Bisson, L. F. (1989). Comparison of the glucose uptake kinetics in different yeasts. Journal of Bacteriology 171, 1303–1308.CrossRefGoogle ScholarPubMed
Dykhuizen, D. E. & Hartl, D. L. (1981). Potential for selection among nearly neutral allozymes of 6-phosphogluconate dehydrogenase in Escherichia coli. Proceedings of the National Academy of Sciences (USA) 78, 6344–6348.Google Scholar
Dykhuizen, D. E. & Hartl, D. L. (1983). Functional effects of PGI allozymes in Escherichia coli. Genetics 105, 1–18.CrossRefGoogle ScholarPubMed
Dykhuizen, D. E.Dean, A. M. & Hartl, D. L. (1987). Metabolic flux and fitness. Genetics 115, 25–31.CrossRefGoogle ScholarPubMed
Entian, K.-D. (1988). Glucose repression: a complex regulatory pathway in yeast. Microbiological Science 3, 366–371.Google Scholar
Entian, K.-D. & Zimmermann, F. K. (1980). Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae. Molecular and General Genetics 177, 345–350.CrossRefGoogle ScholarPubMed
Entian, K.-D. & Frohlich, K.-U.. (1984). Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression. Journal of Bacteriology 158, 29–35.CrossRefGoogle ScholarPubMed
Entian, K.-D.Kopetzki, E.Frohlich, K.-U. & Mecke, D. (1984). Cloning of hexokinase PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isozyme PII for glucose repression in yeasts. Molecular and General Genetics 198, 50–54.CrossRefGoogle ScholarPubMed
Flint, H. J.Tateson, R. W.Barthelmess, I. B.Porteous, D. J.Donachie, W. D. & Kacser, H. (1981). Control of flux in the arginine pathway. Biochemical Journal 200, 231–246.CrossRefGoogle ScholarPubMed
Folk, W. R. & Berg, P. (1971). Duplication of the structural gene for glycyl-transfer RNA synthetases in Escherichia coli. Journal of Molecular Biology 58, 595–610.CrossRefGoogle Scholar
Fraenkel, D. G. (1982). Carbohydrate metabolism in yeast. In The Molecular Biology of the Yeast Saccharomyces cerevisiae (ed. Strathern, J.Young, D. and Broach, J.). New York: Cold Spring Harbor Laboratory, Cold Spring Harbor.Google Scholar
Fraenkel, D. G. (1986). Mutants in glucose metabolism. Annual Review of Biochemistry 55, 317–337.CrossRefGoogle ScholarPubMed
Gancedo, J. M. & Gancedo, C. (1986). Catabolite repression mutants of yeast. FEMS Microbiological Reviews 32, 179–187.Google Scholar
Garfinkel, D.Garfinkel, L.Pring, M.Green, S. B. & Chance, B. (1970). Computer applications to biochemical kinetics. Annual Review of Biochemistry 39, 473–498.CrossRefGoogle ScholarPubMed
Gascon, S.Neumann, N. P. & Lampen, J. O. (1968). Comparative study of the properties of the internal and external invertases from yeast. Journal of Biological Chemistry 243, 1573–1577.CrossRefGoogle ScholarPubMed
Hall, B. G. (1981). Changes in the substrate specificities of an enzyme during directed evolution of new functions. Biochemistry 20, 4042–4049.CrossRefGoogle ScholarPubMed
Hames, B. D. (1981). An introduction to polyacrylamide gel electrophoresis. In Gel Electrophoresis of Proteins: A Practical Approach (ed. Hames, B. D. and Rickwood, D.). Oxford: IRL Press.Google Scholar
Hartl, D. L. & Dykhuizen, D. E. (1985). The neutral theory and the molecular basis of preadaptation. In Population Genetics and Molecular Evolution (ed. Ohta, T. and Aoki, K.), pp. 107–124. Tokyo: Japan Scientific Societies Press.Google Scholar
Hartl, D. L.Dykhuizen, D. E. & Dean, A. M. (1985). Limits to adaptation: the evolution of selective neutrality. Genetics 111, 655–674.CrossRefGoogle ScholarPubMed
Hartwell, L. H. (1973). Three additional genes required for DNA synthesis in Saccharomyces cerevisiae. Journal of Bacteriology 115, 966–974.CrossRefGoogle ScholarPubMed
Heinisch, J. (1986). Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast. Molecular and General Genetics 202, 75–82.CrossRefGoogle ScholarPubMed
Heinrich, R. & Rappoport, T. (1974). A linear steady-state treatment of enzymatic chains. European Journal of Biochemistry 42, 89–95.CrossRefGoogle ScholarPubMed
Herrero, P.Fernandez, R. & Moreno, F. (1989). The hexokinase PII isozyme of Saccharomyces cerevisiae is a protein kinase. Journal of General Microbiology 135, 1209–1216.Google ScholarPubMed
Holmes, D. S. & Quigley, M. (1981). A rapid boiling method for the preparation of bacterial plasmids. Analytical Biochemistry 114, 193–197.CrossRefGoogle ScholarPubMed
Hunsley, J. R. & Suelter, C. H. (1969). Yeast pyruvate kinase. II. Kinetic properties. Journal of Biological Chemistry 244, 4819–4822.CrossRefGoogle ScholarPubMed
Ito, H.Fukudu, Y.Murata, K. & Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. Journal of Bacteriology 153, 163–168.CrossRefGoogle ScholarPubMed
Jensen, R.Sprague, G. F. & Herskowitz, I. (1983). Regulation of yeast mating-type interconversion: feedback control of the HO gene expression by the yeast mating-type locus. Proceedings of the National Academy of Sciences (USA) 80, 3935.CrossRefGoogle Scholar
Kacser, H. & Burns, J. A. (1973). The control of flux. Symposia of the Society of Experimental Biology 27, 65–104.Google ScholarPubMed
Kacser, H. & Burns, J. A. (1979). Molecular democracy: who shares the controls? Biochemical Society Transactions 7, 1149–1160.CrossRefGoogle ScholarPubMed
Kacser, H. & Burns, J. A. (1981). The molecular basis of dominance. Genetics 97, 639–666.CrossRefGoogle ScholarPubMed
Kacser, H. & Beeby, R. (1984). Evolution of catalytic proteins: On the origin of enzyme species by means of natural selection. Journal of Molecular Evolution 20, 38–51.CrossRefGoogle ScholarPubMed
Kamerud, J. Q. & Roon, R. J. (1986). Asparaginase II of Saccharomyces cerevisiae: Selection of four mutations that cause derepressed enzyme synthesis. Journal of Bacteriology 165, 293–296.CrossRefGoogle ScholarPubMed
Kawasaki, G. & Fraenkel, D. G. (1982). Cloning of yeast glycolysis genes by complementation. Biochemical and Biophysical Research Communications 108, 1107–1112.CrossRefGoogle ScholarPubMed
Koehn, R. K.Milkman, R. & Mitton, J. B. (1976). Population genetics of marine pelecypods. IV. Selection, migration and genetic differentiation in the blue mussel, Mytilus edulis. Evolution 30, 3–32.CrossRefGoogle ScholarPubMed
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle ScholarPubMed
Laurie-Ahlberg, C.Williamson, J. H.Cochrane, B. J.Wilton, A. N & Chaslow, F. I. (1981). Autosomal factors with correlated effects on the activities of the Glucose 6-phosphate and 6-phosphogluconate dehydrogenases in Drosophila melanogaster. Genetics 99, 127–150.CrossRefGoogle ScholarPubMed
Lin, C.Hacking, A. J. & Aguilar, J. (1976). Experimental models of acquisitive evolution. BioScience 26, 548–555.CrossRefGoogle Scholar
Maitra, P. K. & Lobo, Z. (1971). A kinetic study of glycolytic enzyme synthesis in yeast. Journal of Biological Chemistry 246, 475–488.CrossRefGoogle ScholarPubMed
Maniatis, T.Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor.Google Scholar
McDonald, J. F. (1983). The molecular basis of adaptation: a critical review of relevant ideas and observations. Annual Review of Ecology and Systematics 14, 77–102.CrossRefGoogle Scholar
McKnight, G. L.Cardillo, T. S. & Sherman, F. (1981). An extensive deletion causing overproduction of yeast iso-2-cytochrome c. Cell 25, 409–419.CrossRefGoogle ScholarPubMed
Michels, C. & Romanowski, A. (1980). Pleiotropic glucose repression-resistant mutation in Saccharomyces carlsbergensis. Journal of Bacteriology 143, 674–679.CrossRefGoogle Scholar
Michels, C. A.Hahnenberger, K. M. & Sylvestre, Y. (1983). Pleitropic mutations regulating resistance to glucose repression in Saccharomyces carlsbergensis are allelic to the structural gene for hexokinase B. Journal of Bacteriology 153, 574–578.CrossRefGoogle Scholar
Middleton, R. J. & Kacser, H. (1983). Enzyme variation, metabolic flux and fitness: alcohol dehydrogenase in Drosophila melanogaster. Genetics 105, 633–650.CrossRefGoogle ScholarPubMed
Moore, P. A.Bettany, A. J. E. & Brown, A. J. P. (1990). Expression of a glycolytic gene is subject to dosage limitation. Gene 89, 85–92.CrossRefGoogle ScholarPubMed
Mortlock, R. P. (1982). Metabolic acquisitions through laboratory selection. Annual Review of Microbiology 36, 259–284.CrossRefGoogle ScholarPubMed
Nash, R.Tokiwa, G.Anand, S.Erickson, K. & Futcher, A. B. (1988). The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO Journal 7, 4335–4346.CrossRefGoogle ScholarPubMed
Perlman, P. & Mahler, H. R. (1974). Derepression of mitochondria and their enzymes in yeast. Archives of Biochemistry and Biophysics 162, 248.CrossRefGoogle ScholarPubMed
Place, A. R. & Powers, D. A. (1979). Genetic variation and relative efficiencies: Lactate dehydrogenase B allozymes of Fundulus heteroclitus. Proceedings of the National Academy of Sciences (USA) 76, 2354–2358.CrossRefGoogle ScholarPubMed
Polakis, E. S. & Bartley, W. (1965). Changes in enzyme activities in Saccharomyces cerevisiae during aerobic growth on different carbon sources. Biochemical Journal 97, 284–297.CrossRefGoogle ScholarPubMed
Rosenzweig, R. F. (1991). Physiological and Fitness Phenotypes of Yeast Overexpressing Glyctolytic Enzymes. Ph.D. Thesis, University of Pennsylvania, Philadelphia, PA.Google Scholar
Savageau, M. (1972). The behavior of intact biochemical control systems. Current Topics in Cellular Regulation 6, 63–130.CrossRefGoogle Scholar
Savageau, M. (1976). Biochemical Systems Analysis: A Study of Function and Design in Molecular Biology. Massachusetts: Addison-Wesley, Reading.Google Scholar
Schaaf, I.Heinisch, J. & Zimmermann, F. K. (1989). Overproduction of glycolytic enzymes in yeast. Yeast 5, 285–290.CrossRefGoogle Scholar
Schmidheini, T.Sperisen, P.Paravicini, G.Hutter, R. & Braus, G. (1989). A single point mutation results in a constitutively activated and feedback resistant chorismate mutase of Saccharomyces cerevisiae. Journal of Bacteriology 171, 1245–1253.CrossRefGoogle Scholar
Sherman, F.Fink, G. R. & Hicks, J. B. (1986). Methods in Yeast Genetics. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor.Google Scholar
Sinha, P. & Maitra, P. K. (1977). Mutants of Saccharomyces cerevisiae having structurally altered pyruvate kinase. Molecular and General Genetics 158, 171–177.CrossRefGoogle Scholar
Sokal, R. R. & Rohlf, F. J. (1981). Biometry. San Francisco: W. H. Freeman.Google Scholar
Sprague, G. F. (1977). Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity. Journal Bacteriology 130, 232–241.CrossRefGoogle ScholarPubMed
Stewart, F.Porteous, D. J.Flint, H. J. & Kacser, H. (1986). Control of the flux in the arginine pathway of Neurospora crassa: Effects of co-ordinate changes of enzyme concentration. Journal of General Microbiology 132, 1159–1166.Google Scholar
Thompson, L. W. & Krawiec, S. (1983). Acquisitive evolution of ribitol dehydrogenase in Klebsiella pneumoniae. Journal of Bacteriology 154, 1027–1031.CrossRefGoogle ScholarPubMed
Walsh, R. B.Kawasaki, G. & Fraenkel, D. G. (1983). Cloning of genes that complement yeast hexokinase and glucokinase mutants. Journal of Bacteriology 154, 1002–1004.CrossRefGoogle ScholarPubMed
Watt, W. B. (1985 a). Bioenergetics and evolutionary genetics: opportunities for a new synthesis. American Naturalist 125, 118–143.CrossRefGoogle Scholar
Williamson, V. M.Cox, D.Young, E. T.Russel, D. W. & Smith, M. (1983). Transposable elements associated with constitutive expression of alcohol dehydrogenase II expression. Molecular and Cellular Biology 3, 20–31.Google ScholarPubMed
Wistow, G. & Piatorgsky, J. (1987). Recruitment of enzymes as lens proteins. Science 236, 1554–1556.CrossRefGoogle Scholar
Wittenburg, C.Sugimoto, K. & Reed, S. I. (1990). Gl specific cyclins of S. cerevisiae: Cell-cycle periodicity, regulation by mating pheromone, and association with the p34cdc28 protein kinase. Cell 62, 225–237.CrossRefGoogle Scholar
Yamamoto, M.Jones, J. M.Senghas, E.Gawron-Burke, C. & Clewell, D. B. (1987). Generation of Tn5 insertions in streptococcal conjugative plasmids. Applied and Environmental Microbiology 57, 1069–1072.CrossRefGoogle Scholar
Zund, P. & Lebek, G. (1980). Generation time prolonging R plasmids: correlation between increases in the generation time of Escherichia coli caused by R plasmids and their molecular size. Plasmid 3, 65–69.CrossRefGoogle ScholarPubMed