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.
- inositol 1,4,5-trisphosphate
- nitrogen sensing
↵1 Present address: DSM Bakery Ingredients, Technology Cluster, Beijerinck Laboratory, P.O. Box 1, 2600 MA Delft, The Netherlands.
Abbreviations used: groPIns(4,5)P2, glycerophosphoinositol 4,5-bisphosphate; FGM, fermentable-growth-medium-induced; PKB, protein kinase B; TOR, target of rapamycin.
- The Biochemical Society, London ©2001