Fig. 5.

The Pib2 helE region and FYVE domain are essential for TORC1 reactivation. (A) Localization of the indicated yEGFP–Pib2 mutants. Vacuoles were stained with FM4-64. Quantification (mean±s.d.) of the data. Data for Pib2, Pib2 ΔhelE, and Pib2 ΔFYVE were the same as in Fig. 2B. Following a ROUT outlier analysis (Q=0.1%), a one-way ANOVA was conducted to determine differences in vacuolar localization (F=180.9, P<0.0001). Constructs significantly different from WT Pib2, as assessed by Tukey post-hoc multiple comparisons test, are indicated (*P<0.0001). A total of 107–181 cells were quantified for each construct. (B) Rapamycin exposure and recovery assays of Δpib2 cells expressing the indicated Pib2 helE region mutants. These were performed as in Fig. 1. (C) Rapamycin exposure and recovery assays of Δpib2 cells expressing the indicated Pib2 FYVE domain mutants. These were performed as in Fig. 1. Images in B,C are representative of three experiments.