GENETIC REGULATION OF THE BIOSYNTHESIS AND UTILIZATION OF INOSITOL IN YEAST
GREENBERG, MIRIAM LEBA
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This dissertation involves both genetic and biochemical approaches to the analysis of regulation of a major biosynthetic enzyme in the yeast Saccharomyces cerevisiae. The enzyme, inositol-1-phosphate synthase, catalyzes the conversion of glucose-6-phosphate to inositol-1-phosphate, which is then converted to inositol by phosphatase activity. Previously, it has been shown that inositol-1-phosphate synthase is repressed 50-fold in wild type yeast grown in media containing 50 uM inositol. In order to study the regulation of this enzyme, I have selected regulatory mutants using a procedure based on secretion of inositol into the growth medium by putative mutants.;The selection procedure can be summarized as follows: Mutagenized cells were replicated onto inositol-deficient plates which had been spread with a lawn of a strain auxotrophic for inositol and containing the adel marker conferring a red phenotype to the strain. Mutagenized colonies which secrete inositol into the medium support growth of the auxotrophic lawn, resulting in a red ring of growth around secretor colonies.;Genetic analysis of the mutants isolated by this procedure revealed the following information: At least three complementing loci are involved in the negative control of the synthase enzyme. These loci are unlinked to each other and unlinked as well to the structural gene for the enzyme. The mutant phenotype in two of the mutants is recessive, while secretor mutations in a third locus are codominant with the wild type phenotype. All of these mutants synthesize I-1-P synthase constitutively, as shown by immunoprecipitation of enzyme in crude extracts of cells grown in the presence of inositol. Furthermore, all of these mutants showed elevated endogenous levels of inositol, as seen in the rate of synthesis of the phospholipid phosphatidylinositol.;Biochemical analysis of one of the mutants confirmed that the nature of the mutation was regulatory, and not in the structural gene for the enzyme. Immunoprecipitation of crude extracts with antibody directed against purified synthase showed that a protein which reacts with the antibody is present in the mutant during both repressing and depressing conditions, in contrast to the wild type which synthesizes the enzyme only when derepressed. Assay of inositol-1-phosphate synthase activity in crude extracts of the mutant verified synthase activity in cells grown under both repressing and derepressing conditions. Synthase purified from this mutant was characterized with respect to molecular weight, thermolability and affinity for substrates glucose-6-phosphate and NAD. These analyses indicated that purified mutant synthase was identical to the wild type enzyme.;Evidence is presented for the isolation of an inositol-secreting mutant defective in phospholipid biosynthesis. Genetic analysis indicates linkage of this mutation to the IN04 locus. Quantitation of membrane phospholipids in this mutant showed an accumulation of the precursors phosphatidyl mono- and di-methyl ethanolamine.;In order to pinpoint the level at which regulation of the synthase occurs, the rates of synthesis and degradation of the wild type enzyme were quantitated during repressing conditions. These experiments revealed that synthesis of the enzyme is repressed within minutes after the addition of as little as 1 uM inositol to growing wild type cultures. Determination of the half-life of the synthase mRNA was made using the rna1 mutant ts136, defective in transport of RNA from the nucleus to the cytoplasm. The mRNA half-life of I-1-P synthase was shown to be 15 minutes. This is consistent with the hypothesis that regulation of I-1-P synthase occurs at a transcriptional or post-transcriptional level.
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