A STUDY OF YEAST PYRUVATE DECARBOXYLASE USING TEMPERATURE SENSITIVE MUTANTS

Abstract

Temperature sensitive (ts) revertants of a yeast glycolytic mutant (pdc1) lacking pyruvate decarboxylase (PDCase) activity were isolated. These ts-revertants were able to grow on media containing glucose as a carbon source only at 23(DEGREES)C and had restored PDCase activity when grown under these conditions. When grown on nonfermentable carbon sources they had no detectable PDCase activity. At 36(DEGREES)C, the ts-revertants were unable to grow on media containing glucose and had no detectable PDCase activity when grown in media containing nonfermentable carbon sources. Thus, PDCase activity has become hexose inducible. Both restoration of growth in media containing glucose and hexose inducibility of PDCase activity were attributable to a single, second site mutation (PDC2). The mutant PDC2 alleles were detectable only in strains containing the original pdc1 mutation. The PDC2 mutations are dominant but they are epistatic to PDC1.;PDCase was purified to homogeneity from wild type and ts-revertant strains. The enzyme from the ts-revertant strains was not any more thermolabile in vitro than the wild type enzyme and its amino acid composition agreed essentially with that of the wild type enzyme. PDCase antibodies were obtained from rabbits immunized with wild type PDCase and used to immunoprecipitate PDCase from crude extracts of wild type and mutant cells. Cyanogen bromide cleavage of immunoprecipitated PDCase in an SDS-polyacrylamide gel demonstrated the similarity in the size of the peptides formed from wild type and ts-revertant enzyme.;In order to assess the effect of growth at the nonpermissive temperature on PDCase synthesis, PDCase was immunoprecipitated from wild type and ts-revertant strains that had been shifted to 36(DEGREES)C while growing in glucose containing media. The rate of synthesis of PDCase, at different times after the temperature shift, was measured by pulse labelling. Synthesis of PDCase in the ts-revertants as measured by immunoprecipitable enzyme rapidly declined after the temperature shift. A less rapid decline in total protein synthesis ensued 30-45 minutes later. The decline in total PDCase activity was not as great or as rapid as the decline in PDCase synthesis which would indicate that the rate of synthesis of PDCase rather than the stability of pre-existing PDCase was affected.;Messenger RNA from ts-revertant cells shifted to 36(DEGREES)C was prepared, added to a cell-free wheat germ translation system and the in vitro translation products immunoprecipitated. The ts-revertant cells were found to have translatable PDCase mRNA up to two hours after being shifted to 36(DEGREES)C, i.e. long after in vivo PDCase synthesis has ceased. Therefore, it did not appear that the mutations had affected PDCase transcription.;A survey of several Saccharomyces species for glucose induction of PDCase revealed that some synthesize PDCase constitutively while others require induction of PDCase by a hexose. The ts-revertants no longer synthesize PDCase constitutively as did their parent and required hexose induction. This could indicate the presence of a hexose induction system in S. cerevisiae which is cryptic due to the presence of a constitutive inducer. Several models are proposed which could explain the significance of the PDC1 and PDC2 genes in the regulation of PDCase synthesis.