Glucose-induced calcium influx in budding yeast involves a novel calcium transport system and can activate calcineurin.

Glucose addition to glucose-starved Saccharomyces cerevisiae cells triggers a quick and transient influx of calcium from the extracellular environment. In yeast at leasttwodifferent carrier systems were identified: a high affinity system, requiring Cch1 channel, and a low affinity system. Here we report that another calcium transport system exists in yeast, not yet identified, that can substitute the two known systems whenthey are inactivated. This systemwascalled GIC (for Glucose Induced Calcium) system and it is a high affinity calcium transport system, magnesium-sensitive but nickel-resistant. Moreover, GIC transport is sensitive to gadolinium and nifedipine, but it is not sensitive to inhibition by verapamil, which conversely behaves as an agonist on glucose response. GIC transport is fully functional in conditions when calcineurin is active, a serine/threonine specificity phosphatase involved in the regulation of calcium homeostasis and in many other cellular phenomena such as tolerance to high concentrations of Na+ and Li+, response to pheromones and gene transcription regulation. Here it is reported for the first time that calcineurin can also be activated by nutrients: the activation of Crz1 transcription factor by calcineurin was observed in derepressed cells after addition of glucose in the presence of extracellular calcium

Glucose-induced calcium influx in budding yeast involves a novel calcium transport system and can activate calcineurin.

Glucose addition to glucose-starved Saccharomyces cerevisiae cells triggers a quick and transient influx of calcium from the extracellular environment. In yeast at leasttwodifferent carrier systems were identified: a high affinity system, requiring Cch1 channel, and a low affinity system. Here we report that another calcium transport system exists in yeast, not yet identified, that can substitute the two known systems whenthey are inactivated. This systemwascalled GIC (for Glucose Induced Calcium) system and it is a high affinity calcium transport system, magnesium-sensitive but nickel-resistant. Moreover, GIC transport is sensitive to gadolinium and nifedipine, but it is not sensitive to inhibition by verapamil, which conversely behaves as an agonist on glucose response. GIC transport is fully functional in conditions when calcineurin is active, a serine/threonine specificity phosphatase involved in the regulation of calcium homeostasis and in many other cellular phenomena such as tolerance to high concentrations of Na+ and Li+, response to pheromones and gene transcription regulation. Here it is reported for the first time that calcineurin can also be activated by nutrients: the activation of Crz1 transcription factor by calcineurin was observed in derepressed cells after addition of glucose in the presence of extracellular calcium

Possible involvement of a phosphatidylinositol-type signaling pathway in glucose-induced activation of plasma membrane ATPase and cellular proton in the yeast Sacchamyces 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

Glucose induced activation of the plasma membrane ATPase in Fusarium oxysporum.

Addition of glucose and other sugars to derepressed cells of the fungus Fusarium oxysporum var. Zini triggered activation of the plasma membrane H+-ATPase within 5 min. Glucose was the best activator while galactose and lactose had a lesser effect. The activation was not prevented by previous addition of cycloheximide and it was fully reversible when the glucose was removed. The activation process in uiuo also caused changes in the kinetic properties of the enzyme. The non-activated enzyme had an apparent K, of about 3.2 mM for ATP whereas the activated enzyme showed an apparent K,,, of 0.26 mM. In addition, the pH optimum of the H+-ATPase changed from 6.0 to 7.5 upon activation. The activated enzyme was more sensitive to inhibition by vanadate. When F. oxysporum was cultivated in media containing glucose as the major carbon source, enhanced M+-ATPase activity was largely confined to the period corresponding to the lag phase, i.e. just before the start of acidification of the medium. This suggests that the activation process might play a role in the onset of extracellular acidification. Addition of glucose to F. oxysporum var. Zini cells also caused an increase in the cAMP level. No reliable increase could be demonstrated for the other sugars. Addition of proton ionophores such as DNP and CCCP at pH 5-0 caused both a large increase in the intracellular level of cAMP and in the activity of the plasma membrane H+- ATPase. Inhibition of the DNP-induced increase in the cAMP level by acridine orange also resulted in inhibition of the activation of plasma membrane H+-ATPase. These results suggest a possible causal relationship between the activity of F. oxysporum var. Zini plasma membrane H+-ATPase and the intracellular level of CAMP

Effect of substrate and pH on the activity of proteases from Fusarium oxysporum var. lini.

The results obtained in this work suggest that both the pH (through selective inhibition) and the carbon source (through repression and acidification or alkalinization of the medium) may play an important role in the distribution of extracellular proteases in Fusarium oxysporum var. lini

Glucose-induced activation of plasma membrane H+-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.

Addition of glucose-related fermentable sugars or pro,tonophores to derepressed cells of the yeast Saccharomyces ceret'isiae causes a 3- to 4-fold activation of the plasma membrane H +-A'fPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP .~jnthesis, incohati~a at the restrictive temperature reduced the extent of H+-ATPase activation, Incubation of nontemperature- sensitive strains, however, at such temperatures also caused reduction of H +-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H+-ATPase aCtivation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H +-A'l'Pase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAlVlP-protein kinase A signaling pathway is not required for glucose activation of the H*-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phnsphorylating enzymes hexokinase Pl and Pll and glucokinase showed that activation of the H+-ATPase with glucose or fructose was completely dependent on the presence cf a kinase able m phnsphorylate the sugar. These and other data concerning the role of init,:al sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H+-ATPase have a common initiation point

Glucose-induced activation of plasma membrane H+-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.

Addition of glucose-related fermentable sugars or pro,tonophores to derepressed cells of the yeast Saccharomyces ceret'isiae causes a 3- to 4-fold activation of the plasma membrane H +-A'fPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP .~jnthesis, incohati~a at the restrictive temperature reduced the extent of H+-ATPase activation, Incubation of nontemperature- sensitive strains, however, at such temperatures also caused reduction of H +-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H+-ATPase aCtivation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H +-A'l'Pase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAlVlP-protein kinase A signaling pathway is not required for glucose activation of the H*-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phnsphorylating enzymes hexokinase Pl and Pll and glucokinase showed that activation of the H+-ATPase with glucose or fructose was completely dependent on the presence cf a kinase able m phnsphorylate the sugar. These and other data concerning the role of init,:al sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H+-ATPase have a common initiation point

Yeast genes YOL002C and Sul1 are involved in neomycine resistance.

In previous studies we suggested the importance of the control of plasma membrane H+-ATPase by a phosphatidylinositol-like pathway for cellular proton extrusion in Saccharomyces cerevisiae (Brandão et al. 1994; Coccetti et al. 1998). The observations that provided the model above include the inhibition of the glucose-induced activation of the plasma membrane H+-ATPase as well as the inhibition of the glucose-induced external acidification by neomycin, a known inhibitor of the phosphatidylinositol turnover in eukaryotic cells. In this work, using two libraries, we isolated two yeast clones that were able to prevent the inhibition of glucose-induced activation of the H+-ATPase by neomycin. We show that the YOL002C gene, which encodes a protein of unknown function, and the SUL1 gene, which is a sulphate transporter belonging to the major facilitator superfamily, suppress growth inhibition by neomycin. However, they are not required for glucose-induced activation of the plasma membrane H+-ATPase. The resistance of the clones to neomycin is probably related to the level and/or activity of proteins functioning as drug extrusion pumps

The PLC1 encoded phospholipase C in the yeast Saccharomyces cerevisiae is essential for glucose-induced phosphatidylinositol turnover and activation of plasma membrane H -ATPase.

Addition of glucose to glucose-deprived cells of the yeast Saccharomyces cerevisiae triggers rapid turnover of phosphatidylinositol, phosphatidylinositol-phosphate and phosphatidylinositol 4,5-bisphosphate. Glucose stimulation of PI turnover was measured both as an increase in the specific ratio of 32P-labeling and as an increase in the level of diacylglycerol after addition of glucose. Glucose also causes rapid activation of plasma membrane H.-ATPase. We show that in a mutant lacking the PLC1 encoded phospholipase C, both processes were strongly reduced. Compound 48/80, a known inhibitor of mammalian phospholipase C, inhibits both processes. However, activation of the plasma membrane H.- ATPase is only inhibited by concentrations of compound 48/80 that strongly inhibit phospholipid turnover. Growth was inhibited by even lower concentrations. Our data suggest that in yeast cells, glucose triggers through activation of the PLC1 gene product a signaling pathway initiated by phosphatidylinositol turnover and involved in activation of the plasma membrane H.-ATPase