Figure 6.

Vps21 stimulates TORC2 activity on Ypk1. (A) Equivalent amounts of TORC2 immunopurified from WT (BY4741) or yMLT85 (vps21Δ ypt52Δ) strains (see Fig. S4 A) were added in increasing amounts and incubated with [γ-³²P]ATP and MBP-Ypk1^CT. The reaction products were resolved by SDS-PAGE and analyzed by autoradiography and staining with SYPRO Ruby, as described in Materials and methods. Results of one representative experiment are shown. (B) The results of multiple experiments (n = 3) as in A are plotted as the mean ³²P incorporation into MBP-Ypk1^CT versus the amount of TORC2 complex present. Error bars, SEM. ³²P incorporation was measured by quantifying the corresponding autoradiograms using ImageJ and estimating the amount of TORC2 present from the staining intensity of the SYPRO Ruby-stained gel and accompanying standards (not depicted). The values were then normalized to the mean ³²P incorporation into MBP-Ypk1^CT catalyzed by the lowest amount of TORC2 from the vps21Δ ypt52Δ cells, which was set at [1]. (C) TORC2 immunopurified from yMLT85 (vps21Δ ypt52), denoted TORC2^ΔΔ, was incubated with [γ-³²P]ATP and MBP-Ypk1^CT in the absence or presence of Vps21-GTPγS (0.2 µg and 0.4 µg). (D) Equivalent amounts of TORC2 immunopurified from yMLT85 (vps21Δ ypt52Δ) as in A, denoted TORC2^ΔΔ, were incubated with [γ-³²P]ATP, MBP-Ypk1^CT, and either Vps21-GTPγS (2 µg) or Ypt7-GTPγS (4 µg), as indicated. Reactions were terminated at the indicated time points.