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 chromat ography. Surprisingly, the transient increase in Ins(1,4,5)P-3 did not trig ger an increase in the concentration of intracellular calcium, as determine d in vivo using the aequorin method. Similar Ins(1,4,5)P-3 signals were als o observed in wild-type cells treated with the phospholipase C inhibitor 3- nitrocoumarin and in cells deleted for the only phospholipase C-encoding ge ne 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 tran sient increase in Ins(1,4,5)P-3, 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-ki nase, Stt4, which is involved in PtdIns(4,5)P-2 formation, did not affect t he Ins(1,4,5)P-3 signal, but significantly delayed the PtdIns(4,5)P-2 signa l. Moreover, wortmannin addition inhibited the nitrogen-induced activation of trehalase and the subsequent mobilization of trehalose, suggesting a rol e for PtdIns(4,5)P-2 in nitrogen activation of the fermentable-growth-mediu m-induced signalling pathway.