Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose

Abstract

The systems which control the levels of the gluconeogenic enzymes in Saccharomyces cerevisiae have been bypassed to ascertain their physiological significance. The coding regions of the genes FBP1 and PCK1, which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, have been put under the control of the promoter of ADC1 (alcohol dehydrogenase I), a gene not repressed by glucose, and introduced into yeast in multicopy plasmids. The transformed yeast cells show high levels of the gluconeogenic enzymes during growth on glucose. Generation time and growth yield of yeast expressing either fructose-1,6-bisphosphatase or phosphoenolpyruvate carboxykinase are not significantly different from those of the wild-type strain. For a strain expressing both enzymes the increase in generation time is about 20% and the decrease in growth yield around 30%. The concentration of ATP is about 1.5 mM in the growing cells of the different strains. The extent of in vivo cycling was measured by 13C NMR in cell-free extracts from yeast growing on [6-13C]glucose. Cycling between fructose-6-phosphate and fructose-1,6-bisphosphate is <2%, most likely due to the very strong inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate. Cycling between phosphoenolpyruvate and pyruvate is low, but a precise figure could not be obtained due to poor equilibration of label between carbons 2 and 3 of oxaloacetate.