Glycogen synthase kinase-3 is required for optimal de novo synthesis of inositol

Studies have shown that the inositol biosynthetic pathway and the enzyme glycogen synthase kinase-3 (GSK-3) are targets of the mood-stabilizing drugs lithium and valproate. However, a relationship between these targets has not been previously described. We hypothesized that GSK-3 may play a role in inositol synthesis, and that loss of GSK-3 may lead to inositol depletion, thus providing a mechanistic link between the two drug targets. Utilizing a yeast Saccharomyces cerevisiae gsk-3Delta quadruple-null mutant, in which all four genes encoding homologues of mammalian GSK-3 are disrupted, we tested the hypothesis that GSK-3 is required for de novo inositol biosynthesis. The gsk-3Delta mutant exhibited multiple features of inositol depletion, including defective growth in inositol-lacking medium, decreased intracellular inositol, increased INO1 and ITR1 expression, and decreased levels of phosphatidylinositol. Treatment of wild-type cells with a highly specific GSK-3 inhibitor led to a significant increase in INO1 expression. Supplementation with inositol alleviated the temperature sensitivity of gsk-3Delta. Activity of myo-inositol-3 phosphate synthase, the rate-limiting enzyme in inositol de novo biosynthesis, was decreased in gsk-3Delta. These results demonstrate for the first time that GSK-3 is required for optimal myo-inositol-3 phosphate synthase activity and de novo inositol biosynthesis, and that loss of GSK-3 activity causes inositol depletion.     

A dominant mutation that alters the regulation of INO1 expression in Saccharomyces cerevisiae.

A dominant, single nuclear gene mutation, CSE1, caused inositol auxotrophy in yeast cells. The inositol requirement was marked when choline was present in the medium. Inositol-1-phosphate synthase, the regulatory enzyme of inositol synthesis, is repressed by inositol, or more profoundly by a combination of inositol and choline in the wild type. In CSE1, the level of inositol-1-phosphate synthase was low and was greatly repressed on the addition of choline alone. In accordance with this, INO1 mRNA encoding the enzyme was low even under the depressed conditions and was profoundly decreased by choline in CSE1. But in the wild type, the addition of choline alone had little effect. An INO1-lacZ fusion was constructed and the control of the INO1 promoter in CSE1 was studied. lacZ expression was repressed not only by inositol, but also by choline in CSE1, whereas it was repressed by inositol, but only slightly by choline in the wild type. CSE1 was unlinked to the INO1 structural gene. Thus CSE1 was thought to be a regulatory mutation. Furthermore, when the CDP-choline pathway was mutationally blocked, choline did not affect INO1 expression, indicating that the metabolism of choline via the CDP-choline pathway is required for INO1 repression.     

Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe.

Phospholipid metabolism in the fission yeast Schizosaccharomyces pombe was examined. Three enzymes of phospholipid biosynthesis, cytidine diphosphate diacylglycerol synthase (CDP-DG), phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase, were characterized in extracts of S. pombe cells. Contrary to an earlier report, we were able to demonstrate that CDP-DG served as a precursor for PI and PS biosynthesis in S. pombe. S. pombe is naturally auxotrophic for the phospholipid precursor inositol. We found that S. pombe was much more resistant to loss of viability during inositol starvation than artificially generated inositol auxotrophs of Saccharomyces cerevisiae. The phospholipid composition of S. pombe cells grown in inositol-rich medium (50 microM) was similar to that of S. cerevisiae cells grown under similar conditions. However, growth of S. pombe at low inositol concentrations (below 30 microM) affected the ratio of the anionic phospholipids PI and PS, while the relative proportions of other glycerophospholipids remained unchanged. During inositol starvation, the rate of PI synthesis decreased rapidly, and there was a concomitant increase in the rate of PS synthesis. Phosphatidic acid and CDP-DG, which are precursors to these phospholipids, also increased when PI synthesis was blocked by lack of exogenous inositol. The major product of turnover of inositol-containing phospholipids in S. pombe was found to be free inositol, which accumulated in the medium and could be reused by the cell.     

Perturbation of the Vacuolar ATPase: A NOVEL CONSEQUENCE OF INOSITOL DEPLETION*

Depletion of inositol has profound effects on cell function and has been implicated in the therapeutic effects of drugs used to treat epilepsy and bipolar disorder. We have previously shown that the anticonvulsant drug valproate (VPA) depletes inositol by inhibiting myo-inositol-3-phosphate synthase, the enzyme that catalyzes the first and rate-limiting step of inositol biosynthesis. To elucidate the cellular consequences of inositol depletion, we screened the yeast deletion collection for VPA-sensitive mutants and identified mutants in vacuolar sorting and the vacuolar ATPase (V-ATPase). Inositol depletion caused by starvation of ino1Δ cells perturbed the vacuolar structure and decreased V-ATPase activity and proton pumping in isolated vacuolar vesicles. VPA compromised the dynamics of phosphatidylinositol 3,5-bisphosphate (PI3,5P₂) and greatly reduced V-ATPase proton transport in inositol-deprived wild-type cells. Osmotic stress, known to increase PI3,5P₂ levels, did not restore PI3,5P₂ homeostasis nor did it induce vacuolar fragmentation in VPA-treated cells, suggesting that perturbation of the V-ATPase is a consequence of altered PI3,5P₂ homeostasis under inositol-limiting conditions. This study is the first to demonstrate that inositol depletion caused by starvation of an inositol synthesis mutant or by the inositol-depleting drug VPA leads to perturbation of the V-ATPase.     

Lithium and valproate decrease the membrane phosphatidylinositol/phosphatidylcholine ratio

Lithium and valproate, two structurally different anti-bipolar drugs, cause decreased intracellular inositol in the yeast Saccharomyces cerevisiae and an in-crease in expression of a structural (INO1) and a regulatory (INO2) gene for phospholipid synthesis that responds to inositol depletion (Vaden, D., Ding, D., Peterson, B., and Greenberg, M.L., 2001, J Biol Chem 276: 15466-15471). We report here that both drugs decrease the relative rate of membrane phosphatidylinositol synthesis and, to a lesser but still significant degree, the steady state relative phosphatidylinositol composition. In addition, both drugs increase the rate of phosphatidylcholine (PC) synthesis. Finally, valproate, but not lithium, increases expression of phosphatidylcholine pathway genes CHO1 and OPI3. The overall effect on membrane phospholipid composition is a reduction in the phosphatidylinositol/phosphatidylcholine ratio by both drugs. Because maintenance of the appropriate phosphatidylinositol/phosphatidylcholine ratio is required for secretory vesicle formation, a decrease in this ratio may have far-reaching implications for understanding the therapeutic mechanisms of action of these drugs.     

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.     

Regulatory Mutations of Inositol Biosynthesis in Yeast: Isolation of Inositol-Excreting Mutants

The enzyme inositol-1-phosphate synthase (I-1-P synthase), product of the INO1 locus, catalyzes the synthesis of inositol-1-phosphate from the substrate glucose-6-phosphate. The activity of this enzyme is dramatically repressed in the presence of inositol. By selecting for mutants which overproduce and excrete inositol, we have identified mutants constitutive for inositol-1-phosphate synthase as well as a mutation in phospholipid biosynthesis. Genetic analysis of the mutants indicates that at least three loci (designated OPI1, OPI2 and OPI4) direct inositol-mediated repression of I-1-P synthase. Mutants of these loci synthesize I-1-P synthase constitutively. Three loci are unlinked to each other and to INO1, the structural gene for the enzyme. A mutant of a fourth locus, OPI3, does not synthesize I-1-P synthase constitutively, despite its inositol excretion phenotype. This mutant is preliminarily identified as having a defect in phospholipid synthesis.     

Lithium and valproate decrease inositol mass and increase expression of the yeast INO1 and INO2 genes for inositol biosynthesis

Bipolar affective disorder (manic-depressive illness) is a chronic, severe, debilitating illness affecting 1-2% of the population. The Food and Drug Administration-approved drugs lithium and valproate are not completely effective in the treatment of this disorder, and the mechanisms underlying their therapeutic effects have not been established. We are employing genetic and molecular approaches to identify common targets of lithium and valproate in the yeast Saccharomyces cerevisiae. We show that both drugs affect molecular targets in the inositol metabolic pathway. Lithium and valproate cause a decrease in intracellular myo-inositol mass and an increase in expression of both a structural (INO1) and a regulatory (INO2) gene required for inositol biosynthesis. The opi1 mutant, which exhibits constitutive expression of INO1, is more resistant to inhibition of growth by lithium but not by valproate, suggesting that valproate may inhibit the Ino1p-catalyzed synthesis of inositol 1-phosphate. Consistent with this possibility, growth in valproate leads to decreased synthesis of inositol monophosphate. Thus, both lithium and valproate perturb regulation of the inositol biosynthetic pathway, albeit via different mechanisms. This is the first demonstration of increased expression of genes in the inositol biosynthetic pathway by both lithium and valproate. Because inositol is a key regulator of many cellular processes, the effects of lithium and valproate on inositol synthesis have far-reaching implications for predicting genetic determinants of responsiveness and resistance to these agents.     

The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes.

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.