Figure 1.

Overview of experimental perturbations. (a–d) Overview of strategy to identify nitrogen and TOR-regulated phosphorylation sites. Specific pathways that will be inhibited by the indicated perturbation are shown in grey for each panel. (a) Nitrogen stress: a change from a good (glutamate) to a poor (proline) nitrogen source, transiently activates AMPK, which, in turn, inhibits TORC1 to accelerate mitotic entry and cell division continues at reduced cell size. Nitrogen stress does not inhibit TORC2 [7]. Removal of arginine and lysine also generates amino acid stress. (b) The ssp2::ura4+ AMPKα deletion strain ‘freezes’ nitrogen stress signalling prior to TORC1 inhibition and the ensuing increase in mitotic commitment that would otherwise arise from TORC1 inhibition. (c) Addition of Torin1 to glutamate grown cells blocks both TORC1 and TORC2 signalling. (d) To block mitotic commitment as we imposed nitrogen and amino acid stress, the ATP analogue 3BrB-PP1 was added to the cdc2.asM17 mutant (CDK1) [21].