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.

SummaryYeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0. We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STREcontrolled genes and induction of ribosomal protein genes. Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the Gprotein coupled receptor Gpr1 and by the glucosephosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor. The nonmetabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphatestarved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.

SummaryAddition of a nitrogen source to yeast ( Saccharomyces cerevisiae ) cells starved for nitrogen on a glucosecontaining medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STREcontrolled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1 D D D D strain, transport of high concentrations of L -citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for L -glutamate and L -citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.

In cells of the yeast Sacchammyces cerevisiae, trehalase activation, repression of CTTl (catalase), SSA3 (Hsp7O) and other STRE-controlled genes, feedback inhibition of CAMP synthesis and to some extent induction of ribosomal protein genes is controlled by the Ras-adenylate cyclase pathway and by the fermentable-growth-medium-induced pathway (FGM pathway). When derepressed cells are shifted from a non-fermentable carbon source to glucose, the Ras-adenylate cyclase pathway is transiently activated while the FGM pathway triggers a more lasting activation of the same targets when the cells become glucose-repressed. Activation of the FGM pathway is not mediated by CAMP but requires catalytic activity of CAMP-dependent protein kinase (cAPK; Tpkl, 2 or 3). This study shows that elimination of Sch9, a protein kinase with homology to the catalytic subunits of cAPK, affects all target systems in derepressed cells in a way consistent with higher activity of cAPK in wiwo. In witm measurements with trehalase and kemptide as substrates confirmed that elimination of Sch9 enhances cAPK activity about two-to threefold, in both the absence and presence of CAMP. In wiwo it similarly affected the basal and final level but not the extent of the glucose-induced responses in derepressed cells. The reduction in growth rate caused by deletion of SCHS is unlikely to be responsible for the increase in cAPK activity since reduction of growth rate generally leads to lower cAPK activity in yeast. On the other hand, deletion of SCHS abolished the responses of the protein kinase A targets in glucoserepressed cells. Re-addition of nitrogen to cells starved for nitrogen in the presence of glucose failed to trigger activation of trehalase, caused strongly reduced and aberrant repression of m l and SSA3, and failed to induce the upshift in RPL25 expression. From these results three conclusions can be drawn: (1) Sch9 either directly or indirectly reduces the activity of protein kinase A; (2) Sch9 is not required for glucose-induced activation of the Rasadenylate cyclase pathway; and (3) Sch9 is required for nitrogen-induced activation of the FGM pathway. The latter indicates that Sch9 might be the target of the FGM pathway rather than cAPK itself.

SummaryIn the yeast Saccharomyces cerevisiae starvation for nitrogen on a glucose-containing medium causes entrance into G0 and downregulation of all targets of the PKA pathway. Re-addition of a nitrogen source in the presence of glucose causes rapid activation of trehalase and other PKA targets. Trehalase activation upon ammonium re-supplementation is dependent on PKA activity, but not on its regulatory subunit nor is it associated with an increase in cAMP. In nitrogenstarved cells, ammonium transport and activation of trehalase are most active in strains expressing either the Mep2 or Mep1 ammonium permease, as opposed to Mep3. The non-metabolizable ammonium analogue, methylamine, also triggers activation of trehalase when transported by Mep2 but not when taken up by diffusion. Inhibition of ammonium incorporation into metabolism did not prevent signalling. Extensive sitedirected mutagenesis of Mep2 showed that transport and signalling were generally affected in a similar way, although they could be separated partially by specific mutations. Our results suggest an ammonium permease-based sensing mechanism for rapid activation of the PKA pathway. Mutagenesis of Asn246 to Ala in Mep2 abolished transport and signalling with methylamine but had no effect with ammonium. The plant At Amt1;1, At Amt1;2, At Amt1;3 and At Amt2 ammonium transporters sustained transport and trehalase activation to different extents. Specific mutations in Mep2 affected the activation of trehalase differently from induction of pseudohyphal differentiation. We also show that Mep permease involvement in PKA control is different from their role in haploid invasive growth, in which Mep1 sustains and Mep2 inhibits, in a way independent of the ammonium level in the medium.

Addition of ammonium sulphate to nitrogen-depleted yeast cells resulted in a transient increase in Ins(1,4,5)P3, with a maximum concentration reached after 7–8min, as determined by radioligand assay and confirmed by chromatography. Surprisingly, the transient increase in Ins(1,4,5)P3 did not trigger an increase in the concentration of intracellular calcium, as determined in vivo using the aequorin method. Similar Ins(1,4,5)P3 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)P3 was not generated by phospholipase C-dependent cleavage of PtdIns(4,5)P2. Apart from a transient increase in Ins(1,4,5)P3, we observed a transient increase in PtdIns(4,5)P2 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)P2 formation, did not affect the Ins(1,4,5)P3 signal, but significantly delayed the PtdIns(4,5)P2 signal. Moreover, wortmannin addition inhibited the nitrogen-induced activation of trehalase and the subsequent mobilization of trehalose, suggesting a role for PtdIns(4,5)P2 in nitrogen activation of the fermentable-growth-medium-induced signalling pathway.

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.

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