Fig. 6
VPA inhibits TORC1 but not TORC2 at 1 mM. (A) Cultures were treated with VPA (1 and 2 mM), rapamycin (0.4, 0.8, and 1.2 µM), or vehicles (control; C) for 30 min. Cell lysates were collected and assayed by SDS-PAGE and WB analyses, probing with PP-Rps6 (S232/S233) [TORC1 dependent phosphorylation], P-Rps6 (S232) [either TORC1 or TORC2 dependent phosphorylation], and Rps6 antibodies. Experiment was performed in both WT and npr2∆, the latter has constitutively active TORC1. (B) Diagram of TORC1 and C2 dependent phosphorylation of Rps6. S232 is phosphorylated indirectly in response to either TORC1 or TORC2 activity whereas S233 phosphorylation is dependent on TORC1 activity. The PP-Rps6 antibody binds only if both sites are phosphorylated and the P-Rps6 antibody interacts if either TORC1 or TORC2 is active. (C) Multiple residues on the c-terminus of Sch9 are phosphorylated directly by TORC1. Yeast exogenously expressing c-terminally tagged SCH9-3xHA were treated with cycloheximide (CHX; 100 µM), VPA (V; 1 mM), rapamycin (R; 400 nM), or vehicles (C) for 1 h, using culture medium containing a mixture of 16 amino acids (SC16D) (as our typical SC9D medium did not produce any observable Sch9 c-terminus phosphorylation). The c-terminus of Sch9-3xHA was chemically cleaved in cell lysates, which were then assayed by SDS-PAGE and WB analysis. The membrane was probed with anti-HA antibodies; the multi-phosphorylated Sch9 c-terminus (+ P) migrates slower than its dephosphorylated counterpart. The membrane was reprobed with an antibody that recognizes the TORC1 dependent phosphorylation of T737 on Sch9. (D) Ypk1 is phosphorylated directly by TORC2, as well as other protein kinases. A c-myc tagged YPK1 phosphosite mutant (S51A, S71A, T504A, S644A, T662A) retains only TORC2 specific phosphosites (YPK15A-myc). Cultures exogenously expressing this construct were treated with VPA (V; 1 mM), myriocin (M; 1.25 µM), or vehicles (C) for 0.5–4 h. Cell lysates were assayed by Mn2+ Phos-tag SDS-PAGE and WB analysis and probed with anti-myc antibodies. Phosphorylated proteins migrate slower on Phos-tag gels in a stoichiometric manner compared to their unphosphorylated counterpart. Data shown in A, C, and D is representative of 2 biological replicates which are shown in Supplementary figure S5. See supplementary figure S6 for uncropped Western blot images.
