Fig 6. Whi2 suppression of TORC1 activity requires Psr1 and Psr2.
(A, C, E) Growth of indicated yeast strains spotted in parallel in 5-fold serial dilutions on both control/high (SCCSH) to verify equal cell numbers and low amino acid plates (SCME). Representative of ≥3 independent experiments is shown. (B) Immunoblot analysis for TORC1 activity assessed with anti-phospho-RpsS6 in indicated strains after switching from control/high (SCCSH) to low amino acid media (SCME). Equal loading was achieved primarily by optical density-matched cultures and monitored with anti-Pgk. Corresponding bars below show quantification of TORC1 activity (the ratio of phospho-Rps6/Pgk). Values were normalized to the average value of WT at time zero, and presented in the bar graph as means ± SD (n = 4–5 independent experiments). **P < 0.01; *P < 0.05; n.s., not significant, compared to the respective WT control using a two-tailed T test. (D) Immunoblot analysis for TORC1 activity assessed with anti-phospho-RpsS6 in indicated strains after switching from control/high (SCCSH) to low amino acid media (SCME). Equal loading was achieved primarily by optical density-matched cultures and monitored with anti-Pgk. Anti-HA was used to detect N-terminal HA-tagged Whi2, Psr1, and Psr2 expressed from the PGK1 promoter in plasmids. Corresponding bars below show quantification of TORC1 activity (the ratio of phospho-Rps6/Pgk). Values were normalized to the average value of Δpsr1Δpsr2 transformed with empty vector at time zero, and presented in the bar graph as means ± SD (n = 4 independent experiments). **P < 0.01; *P < 0.05; n.s., not significant, compared to the respective vector control using a two-tailed T test. (F) Immunoblot analysis was performed as for panel D for expressed Psr1 in wild type and Δwhi2. Corresponding bars below show quantification of TORC1 activity (the ratio of phospho-Rps6/Pgk). Values were normalized to the average value of WT transformed with empty vector at time zero, and presented in the bar graph as means ± SD (n = 4 independent experiments). **P < 0.01; *P < 0.05; n.s., not significant, compared to the respective vector control using a two-tailed T test. (G) Group data for the effect of Psr1 on TORC1 activity in WT versus Δwhi2 from panel F. The ratios of TORC1 activity between indicated strains transformed with HA-Psr1 and empty vector were presented at each time point for 4 independent experiments. (H) Psr1 is co-immunoprecipitated with Whi2. HA-Psr1 was co-expressed with Flag-Whi2 or an empty vector in the WT (BY4741) strain. Anti-Flag immunoprecipitates were analyzed on immunoblots with anti-HA and anti-Flag.