Physiological Effects of Seven Different Blocks in Glycolysis inSaccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae mutants unable to grow and ferment glucose have been isolated. Of 45 clones isolated, 25 had single enzyme defects of one of the following activities: phosphoglucose isomerase (pgi), phosphofructokinase (pfk), triosephosphate isomerase (tpi), phosphoglycerate kinase (pgk), phosphoglyceromutase (pgm), and pyruvate kinase (pyk). Phosphofructokinase activities in crude extracts of the pfk mutant were only 2% of the wild-type level. However, nornal growth on glucose medium and normal fermentation of glucose suggested either that the mutant enzyme was considerably more active in vivo or, alternatively, that 2% residual activity was sufficient for normal glycolysis. All other mutants were moderately to strongly inhibited by glucose. Unusually high concentrations of glycolytic metabolites were observed before the reaction catalyzed by the enzyme which was absent in a given mutant strain when incubated on glucose. This confirmed at the cellular level the location of the defect as determined by enzyme assays. With adh (lacks all three alcohol dehydrogenase isozymes) and pgk mutants, accumulation of the typical levels of hexosephosphates was prevented when respiration was blocked with antimycin A. A typical feature of all glycolytic mutants described here was the rapid depletion of the intracellular adenosine 5'-triphosphate pool after transfer to glucose medium. No correlation of low or high levels of fructose-1,6-bisphosphate with the degree of catabolite repression and inactivation could be found. This observation does not support the concept that hexose metabolites are directly involved in these regulatory mechanisms in yeast.

A family of hexosephosphate mutases in Saccharomyces cerevisiae

“…Crude extracts were prepared using glass beads for breaking the cells as described by Ciriacy and Breitenbach (1979). Protein was determined as described by Zamenhoff (1957) using bovine serum albumin as a standard.…”

TKL2, a second transketolase gene of Saccharomyces cerevisiae

“…Yeast crude extracts were prepared according to Ciriacy and Breitenbach (1979) using 50 mM imidazol buffer, pH 7.6. Specific transketolase activity was determined according to Kochetov (method B.…”

Characterization of mutations that overcome the toxic effect of glucose on phosphoglucose isomerase less strains of Saccharomyces cerevisiae

“…Yeast mutants blocked in any of the steps of the glycolytic pathway do not grow on glucose; moreover, glucose produces an inhibition of growth on other permissive carbon sources [1,2]. This effect has been particularly well documented in the case of mutants lacking phosphoglucose isomerase (pgi).…”

“…While Escherichia coli mutants affected in the phosphoglucose isomerase step grow on glucose through the pentose phosphate pathway [3], yeast mutants affected in the same step do not grow on glucose [4][5][6]. These mutants grow on rich media with fructose or ethanol as carbon sources, but addition of small amounts of glucose arrests growth without affecting viability [2,4]. Addition of glucose to yeast pgi mutants produces accumulation of glucose-6-phosphate and a decrease in ATP content [2,4,7].…”