Figure 6.

Interaction with TORC2 stabilizes Slm1 association with the plasma membrane. (A) The Slm1 C terminus can mediate interaction with TORC2 components. In vitro-transcribed and -translated ³⁵S-labeled Avo2, Bit61, and Ybr270c were incubated with resin alone or with resin containing GST fused inframe to the Slm1 C terminus (GST-Slm1C; containing amino acids 441–686 of Slm1). Bound ³⁵S-labeled proteins that remained after washing were analyzed by SDS-PAGE and autoradiography. The molecular masses of protein markers are indicated to the left. (B) Plasmid pJK702 (GAL1-HA-SLM1) was introduced into wild-type cells (W303a), and isogenic avo2Δ, bit61Δ, ybr270cΔ single mutant, or avo2Δ bit61Δ double mutant cells. Cells were grown to early exponential phase in raffinose medium at 25°C and HA-Slm1 expression was induced by addition of galactose and further incubation for 2 h. HA-Slm1 was visualized in fixed cells as described in Figure 3. (C) Heightfield visualizations were performed on single z-sections of representative cells (marked with arrows) by using Amira 3.0. Maximum pixel intensity is indicated in red; minimum pixel intensity in dark blue in the color scheme.