PtdIns(4,5)P(2) and phospholipase C-independent Ins(1,4,5)P(3) signals induced by a nitrogen source in nitrogen-starved yeast cells.

Addition of ammonium sulphate to nitrogen-depleted yeast cells resulted in a transient increase in Ins(1,4,5)P(3), with a maximum concentration reached after 7-8 min, as determined by radioligand assay and confirmed by chromatography. Surprisingly, the transient increase in Ins(1,4,5)P(3) did not trigger an increase in the concentration of intracellular calcium, as determined in vivo using the aequorin method. Similar Ins(1,4,5)P(3) signals were also observed in wild-type cells treated with the phospholipase C inhibitor 3-nitrocoumarin and in cells deleted for the only phospholipase C-encoding gene in yeast, PLC1. This showed clearly that Ins(1,4,5)P(3) was not generated by phospholipase C-dependent cleavage of PtdIns(4,5)P(2). Apart from a transient increase in Ins(1,4,5)P(3), we observed a transient increase in PtdIns(4,5)P(2) after the addition of a nitrogen source to nitrogen-starved glucose-repressed cells. Inhibition by wortmannin of the phosphatidylinositol 4-kinase, Stt4, which is involved in PtdIns(4,5)P(2) formation, did not affect the Ins(1,4,5)P(3) signal, but significantly delayed the PtdIns(4,5)P(2) signal. Moreover, wortmannin addition inhibited the nitrogen-induced activation of trehalase and the subsequent mobilization of trehalose, suggesting a role for PtdIns(4,5)P(2) in nitrogen activation of the fermentable-growth-medium-induced signalling pathway.

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose.

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.