Parallel changes in catabolite repression of haem biosynthesis and cytochromes in repression-resistant mutants of Saccharomyces cerevisiae.

Effects of three mutant genes, CAT1-2d, cat2-1 and hex2-3, on catabolite repression of mitochondrial cytochromes and the first two enzymes of haem biosynthesis were compared. The CAT1-2d mutation gave no resistance to glucose, whereas cat2-1 endowed both cytochromes and 5-aminolaevulinate dehydratase with resistance, but did not alter the effect of glucose on 5-aminolaevulinate synthase. The hex2-3 mutation caused repression resistance of cytochromes and of the two haem biosynthetic enzymes. hex2-3 strains also accumulated intracellular 5-aminolaevulinate. Co-inheritance of the latter traits, sensitivity to maltose inhibition and ability to grow on raffinose in the presence of 2-deoxyglucose, demonstrated that the pleiotropic phenotype is a function of the single gene hex2-3. Revertants which grew on maltose regained sensitivity to deoxyglucose and exhibited normal sensitivity of cytochromes and haem biosynthesis enzymes to repression. Addition of the hex1-18 mutation, which renders cytochromes resistant to repression, to a cat2-1 strain did not produce the same effect on 5-aminolaevulinate synthase as hex2-3. It is concluded that repression patterns of haem and cytochrome biosynthesis are substantially affected by hex2-3 and cat2-1 but not by CAT1-2d.

Regulation of porphyrin biosynthesis in yeast. Level of delta-aminolevulinic acid in porphyrin mutants of Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants bearing mutations at the cyc4 locus are partially deficient in cytochrome synthesis. Although the mutation is not in the structural gene for delta-aminolevulinic acid (Alv) synthase, the mutants are deficient in Alv synthesis in vivo as indicated by abnormally low intracellular Alv concentrations. The cyc4 mutation causes cells to grow very slowly in minimal glucose medium, but not in yeast extract-peptone-glucose medium. A simple nutritional defect caused by the cyc4 mutation is not involved because cytochrome deficiency is enhanced by growing cyc4 cells in yeast extract-peptone medium. A regulatory role for CYC4 is indicated. Evidence for negative feed-back control of Alv synthase by heme is provided by the observation of enhanced intracellular Alv accumulation in yeast mutants partially deficient in decarboxylation of uroporphyrinogen and coproporphyrinogen, respectively.

In situ assay for 5-aminolevulinate dehydratase and application to the study of a catabolite repression-resistant Saccharomyces cerevisiae mutant.

To facilitate the study of the effects of carbon catabolite repression and mutations on 5-aminolevulinate dehydratase (EC 4.2.1.24) from Saccharomyces cerevisiae, a sensitive in situ assay was developed, using cells permeabilized by five cycles of freezing and thawing. Enzymatic activity was measured by colorimetric determination of porphobilinogen with a modified Ehrlich reagent. For normal strains, porphobilinogen production was linear for 15 min, and the reaction rate was directly proportional to the permeabilized cell concentration up to 20 mg (dry weight) per ml. The reaction exhibited Michaelis-Menten-type kinetics, and an apparent Km of 2.6 mM was obtained for 5-aminolevulinic acid. This value is only slightly higher than the value of 1.8 mM obtained for the enzyme assayed in cell extracts. The in situ assay was used to assess catabolite repression-dependent changes in 5-aminolevulinate dehydratase during batch culture on glucose medium. In normal S. cerevisiae cells, the enzyme is strongly repressed as long as glucose is present in the medium. In contrast, a strain bearing the hex2-3 mutation exhibits derepressed levels of enzyme activity during growth on glucose. Synthesis of cytochromes by this strain is also resistant to catabolite repression. Similar studies employing a strain containing the glc1 mutation, which enhances porphyrin accumulation, did not reveal any significant phenotypic change in catabolite regulation of 5-aminolevulinate dehydratase.

Regulation of energy metabolism in yeast. Inheritance of a pleiotropic mutation causing defects in metabolism of energy reserves, ethanol utilization and formation of cytochrome a.a3.

The recessive, nuclear gene mutation glc1, which causes glycogen deficiency in Saccharomyces cerevisiae, is highly pleiotropic. Studies of the inheritance of glc1 revealed two classes of phenotypic characteristics: I. Traits invariably associated with the mutant gene and II. Traits whose expressions require the presence of glc1 and one or more additional genes. Class I traits include glycogen deficiency and the loss of capacity to accumulate trehalose in nonproliferating conditions. Traits in the second class include a decreased rate of growth on ethanol medium, a deficiency in cytochrome a.a3 and an enhanced accumulation of pigment, probably a metalloporphyrin. Constructed strains containing both glc1 and the constitutive maltose fermentation gene MAL4c can accumulate trehalose but not glycogen during growth on glucose. However, accumulated trehalose is degraded when cells are exposed to nonproliferating conditions. It is proposed that the glc1 mutation affects a regulatory system, probably involving a protein kinase and/or protein phosphatase, which regulates glycogen synthase and trehalase. Independent regulation of trehalose synthesis by a system controlled by MAL4c is indicated.

Modulation of cytochrome biosynthesis in yeast by antimetabolite action of levulinic acid.

Levulinic acid, a competitive inhibitor of delta-aminolevulinic acid dehydratase, was used to inhibit cytochrome biosynthesis in growing yeast cells. In Saccharomyces cerevisiae the antimetabolite acts by inhibiting delta-aminolevulinic acid dehydratase in vivo, causing an accumulation of intracellular delta-aminolevulinic acid and simultaneous decreases in all classes of mitochondrial cytochromes. Changes in cellular cytochrome content with increasing levulinic acid concentration suggested the existence of different regulatory patterns in S. cerevisiae and Candida utilis. In C. utilis, cytochrome a.a3 formation is very resistant to the antimetabolite action of levulinic acid. In this aerobic yeast, cytochrome c+c1 is the most sensitive to levulinic acid, and cytochrome b exhibits intermediate sensitivity.

Two forms of trehalase activity in mice.

Trehalase activities from mouse serum and kidney have been compared on ion exchange chromatography (DEAE-cellulose and SP-Sephadex). The two activities showed a distinct behaviour. This was confirmed by polyacrylamide gel electrophoresis and thermal lability, suggesting the presence of at least two forms of the hydrolytic enzyme trehalase in mice.