Fig. 4.

Pib2 vacuolar targeting promotes TORC1 reactivation. (A) Localization of the indicated targeting constructs. Vacuoles were stained with FM4-64. (B) Schematic of chimeric Pib2 vacuolar and endosomal targeting constructs. Each construct consists of the targeting protein and yEGFP fused to the N-terminus of Pib2. (C) Rapamycin exposure and recovery assay of Δpib2 cells expressing the indicated targeting constructs. These were performed as in Fig. 1. (D) Representative western blot from a glutamine response assay (n=4). Cells were nitrogen starved for 2 h in SD −N. For stimulation, cells were treated with SD −N supplemented with 3 mM glutamine and incubated for indicated times prior to lysis. TORC1 activation was assessed by phosphorylation of Rps6 under the indicated conditions. Total Rps6 and Pgk1 are shown as loading controls. (E) Quantification of D (mean±s.d.; n=4). Values were normalized to the corresponding Pgk1 loading control. Differences were assessed by two-way ANOVA (P<0.0001). Differences between constructs at each time point as determined by Tukey's post-hoc multiple comparisons are indicated (*P=0.0007, **P<0.0001). Images in A,C are representative of three experiments.