Control of inositol biosynthesis in Saccharomyces cerevisiae; inositol-phosphate synthetase mutants

Abstract

Inositol-requiring mutants of Saacharomyces cerevisiae were tested in cell extracts for the ability to convert glucose-6-phosphate to inositol-phosphate (IP synthetase) and inositol (IP phosphatase). Mutants representing any one of 10 unlinked loci conferring the inositol requirement were unable to synthesize either compound in an assay with glucose-6-phosphate as the substrate. These resSlts indicate that the mutants lack IP synthetase activity and that at least 10 genes control the conversion of glucose-6-phosphate to inositol-phosphate. In addition, a mutation known to be unlinked with the inol locus interacts with a leaky inol allele and may play a role in the regulation of IP synthetase. This mutation causes a 47% reduction in wild-type IP synthetase activity and, when combined in a haploid strain with the leaky inol allele, it reduces IP synthetase activity to a level below that which is growth supporting. Wild-type and IP synthetase-deficient strains were tested for reduced nicotinamide adenine dinucleotide (NADH) accumulation, since NAD+ is required in the conversion of glucose-6-phosphate to inositol. No detectable accumulation of NADH was observed in the wild-type strain, presumably because the NADH generated is rapidly oxidized during subsequent partial reactions of IP synthetase. Mutants representing three different loci accumulate NADH and may, therefore, lack the NADH-mediated reductase activity of IP synthetase. Other mutants tested fail to accumulate NADH and may, therefore, lack the NAD+-mediated oxidase activity of IP synthetase. Phospholipid synthesis was studied by 32P pulse labeling in one mutant under conditions of inositol supplementation and starvation. Starved cells incorporate 32P into phospholipids normally for 2 h, followed by a period in which the rate of phosphatidylinositol synthesis decreases and the rate of phosphatidylcholine synthesis increases. After 5 to 6 h of starvation, all cellular phospholipid synthesis ceases.

Control of inositol biosynthesis in Saccharomyces cerevisiae: properties of a repressible enzyme system in extracts of wild-type (Ino+) cells

“…The availability of mutants specifically lacking the capacity to synthesize inositol may prove instrumental in providing an approach toward understanding the mechanism through which inositol biosynthesis is controlled and coordinated with the demand for inositol in the synthesis of phospholipids. In the following communication (9), an attempt is made to correlate some parameters of inositol biosynthesis with the metabolism of inositol in membrane phospholipids through the biochemical characterization of inositol-requiring mutants.…”

“…Since 10 unlinked loci w be represented among inositol-re tants in S. cerevisiae, the genetic e gests that a large number of poly be involved in these reactions (10) The present work concerns the tion of inositol biosynthetic enzym( cell extracts of a wild-type (Ino+) cerevisiae. Two reactions in ino thesis are described which presui spond to the reactions catalyzed iI nisms by IP synthetase and IP phc addition, evidence is presented tha biosynthetic pathway is regulated mediated repression of enzyme sy accompanying paper (9) (10) ove are cataserved as the source of wild-type enzyme. nes (7).…”

Vitamin requirements and biosynthesis in Saccharomyces cerevisiae

“…In organisms capable of synthesizing myo-inositol, it is formed from glucose-6-phosphate via two enzyme-catalyzed reactions. The genes involved in the S. cerevisiae inositol biosynthesis pathway were discovered by complementation of inositol-requiring mutants (Culbertson, Donahue, & Henry, 1976; Figure 5a). First, L-myo-inositol 1-phosphate is generated from glucose-6-phosphate by L-myo-inositol 1-phosphate synthase (Ino1; Donahue & Henry, 1981 (Henry, Kohlwein, & Carman, 2012).…”

Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opi1 mutant

“…The enzyme inositol-1-phosphate synthase, which is the gene product of the INOI locus (11,20), is repressed by exogenous inositol (8,11). The ino2 and ino4 mutants, originally isolated as inositol auxotrophs (10,12), are unable to derepress inositol-1-phosphate synthase despite the presence of a nonmutated copy of its structural gene, INOI (9,11,20). Thus, the IN02 and IN04 genes were identified as regulatory genes whose wild-type gene product is required for the expression of the INOI gene product (11,23).…”