Supplementary Figure 5: The CAAX box rescues TORC2 function in the mss4-103 mutant.

(a) Representative equatorial sections and maximum projections for 0.5µM-spaced Z-planes of mss4-103 cells expressing Avo3-GFP at the permissive (30 °C) and non-permissive (37 °C) temperatures. Scale bar, 5μm. This experiment was repeated three times with similar results. (b) Evolution of TORC2 activity, monitored by Ypk1 T⁶⁶² phosphorylation, upon shifting WT or mss4-103 cells to the non-permissive temperature. This experiment was repeated twice with similar results. (c) Quantification of TORC2 basal activity, monitored by Ypk1 T⁶⁶² phosphorylation, in WT and mss4-103 cells at the permissive (30 °C) and non-permissive (37 °C) temperatures. Represented are the mean values +/- SD from three independent experiments and normalized to the WT at 30 °C (*** p<0.001, two-tailed unpaired t-test, see Supplementary Table 3 for the exact p values). Source data are included in Supplementary Table 3. (d) Transient growth at 37 °C does not affect survival of either WT or mss4-103 TORC2^CAAX cells. Cells were grown at 37 °C for the indicated times. Serial dilutions of cells were then spotted onto YPD plates and growth was assessed 24h later. This experiment was repeated twice with similar results. All unprocessed scans of blots are shown in Supplementary Figure 7.