Figure 2. Myriocin-induced TORC2 phosphorylation of Ypk1 requires Slm1/2. (A) Model for sphingolipid regulation of TORC2 activity. Scheme 1 refers to the model described in¹³ describes a regulation of TORC2 activity by level of sphingolipids, while Scheme 2 refers to the model from¹² describing sphingolipid regulation of Slm1/2 localization as a major influence on the ability of TORC2 to phosphorylate Ypk1. See text for details. (B) WT (SEY6210) and slm1Δslm2Δsac7Δ (PLY1447) strains expressing empty vector (pPL420), Ypk1-HA (pPL433) or SlmPH-Ypk1-HA (pPL495) were grown at 30°C as described in reference 1. Protein extracts were prepared using the NaOH cell lysis method³⁰ and loaded onto SDS-PAGE gels and transferred to nitrocellulose membrane. Membranes were probed with α-HA (Covance; 12CA5, 1:5000), α-phospho-Ypk1 (T662) (1:20,000; described in ref. 1) and α-G6PDH (Sigma-Aldrich; 1:100,000) primary antibodies, and visualized using the appropriate secondary antibodies conjugated to IRDye (LI-COR Biosciences; 1:5000) on the Odyssey Infrared Imaging System (LI-COR Biosciences). Quantification below the blot describes the difference relative to WT after normalizing to the α-HA signal.