Possible involvement of a phosphatidylinositol-type signaling pathway in glucose-induced activation of plasma membrane H(+)-ATPase and cellular proton extrusion in the yeast Saccharomyces cerevisiae.

Addition of glucose to cells of the yeast Saccharomyces cerevisiae causes rapid activation of plasma membrane H(+)-ATPase and a stimulation of cellular H+ extrusion. We show that addition of diacylglycerol and other activators of protein kinase C to intact cells also activates the H(+)-ATPase and causes at the same time a stimulation of H+ extrusion from the cells. Both effects are reversed by addition of staurosporine, a protein kinase C inhibitor. Addition of staurosporine or calmidazolium, an inhibitor of Ca2+/calmodulin-dependent protein kinases, separately, causes a partial inhibition of glucose-induced H(+)-ATPase activation and stimulation of cellular H+ extrusion; together they cause a more potent inhibition. Addition of neomycin, which complexes with phosphatidylinositol 4,5-bisphosphate, or addition of compound 48/80, a phospholipase C inhibitor, also causes near complete inhibition. Diacylglycerol and other protein kinase C activators had no effect on the activity of the K(+)-uptake system and the activity of trehalase and glucose-induced activation of the K(+)-uptake system and trehalase was not inhibited by neomycin, supporting the specificity of the effects observed on the H(+)-ATPase. The results support a model in which glucose-induced activation of H(+)-ATPase is mediated by a phosphatidylinositol-type signaling pathway triggering phosphorylation of the enzyme both by protein kinase C and one or more Ca2+/calmodulin-dependent protein kinases.

TRK2 is not a low-affinity potassium transporter in Saccharomyces cerevisiae.

TRK1 and TRK2 encode proteins involved in K+ uptake in Saccharomyces cerevisiae. A kinetic study of Rb+ influx in trk1 TRK2, trk1 TRK2D, and trk1 trk2 mutants reveals that TRK2 shows moderate affinity for Rb+. K(+)-starved trk1 delta TRK2 cells show a low-affinity component accounting for almost the total Vmax of the influx and a moderate-affinity component exhibiting a very low Vmax. Overexpression of TRK2 in trk1 delta TRK2D cells increases the Vmax of the moderate-affinity component, and this component disappears in trk1 delta trk2 delta cells. In contrast, the low-affinity component of Rb+ influx in trk1 delta TRK2 cells is not affected by mutations in TRK2. Consistent with the different levels of activity of the moderate-affinity Rb+ influx, trk1 delta TRK2 cells grow slowly in micromolar K+, trk1 delta TRK2D cells grow rapidly, and trk1 delta trk2 delta cells fail to grow. The existence of a unique K+ uptake system composed of several proteins is also discussed.

Several routes of activation of the potassium uptake system of yeast.

K+ uptake in yeast is activated by glucose and other fermentable sugars, and by cytoplasmic acidification. In sugar kinase mutants, fermentable sugars and 2-deoxyglucose produced activation if the sugar could be phosphorylated, indicating that phosphorylation of the sugar is sufficient to trigger the activating pathway. Activation by cytoplasmic acidification was mimicked by neomycin, suggesting that a phosphatidylinositol-type pathway could be involved.

Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinositol turnover, rapid post-translational effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggs1 delta strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII.

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1 delta, hxk2 delta grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1 delta, hxk2 delta double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucose-induced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the deletion of HXK2. However, both the ggs1 delta and the ggs1 delta, hk2 delta mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 delta strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the potassium uptake system during fermentation in Saccharomyces cerevisiae.

Fermentable sugars activated the K+ uptake system, increasing the Vmaxs of Rb+, Na+, and Li+ influxes, but sugars did not affect the effluxes of these cations. This activation seems to be a direct effect of fermentation and not the consequence of the H+ pump ATPase activation or internal pH decrease produced by fermentation.