Figure 1. TORC2/Ypk1 signaling regulates ROS.

(A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), and Ypk1- AS yap1Δ (PLY1527), were grown in SCD-Ura media, either with or without 20 mM NAC, then treated for 1 hr with 0.5 μM 2,3-DMB-PPI or with 1 mM H₂O₂. All strains were incubated with 10 μM 2,7-Dichlorofluorescin diacetate (DCF) for the last 30 min prior to imaging by fluorescence microscopy. Quantification represents percentage of 200–300 cells labelled with DCF, from at least three experiments. (B) WT (PLY062), ypk1Δ (PLY521), and ypk2Δ (PLY1360) cells were grown in SCD media, and ROS was determined and quantified as in (A). (C) WT (INA17-4D), pkh1/2-ts (INA106-3B), torc2-ts (PLY1134), and PLY1134 transformed with Ypk1^D242A (pPL240) were grown overnight at 25°C, then shifted to 37°C or 30°C as noted for 1 hour. ROS was determined and quantified as in (A). (D) Ypk1-AS cells were transformed with either Ypk1-WT (pPL433), Ypk1^T504A (pPL530), or Ypk1^S644A/T662A (pPL491). Cells were grown in SCD-Ura/-Leu media, and ROS was determined and quantified as in (A). Protein extracts were prepared and resolved by SDS/PAGE and immunoblotted with α-HA antibody. (E) Ypk1-WT, Ypk1-AS, and Ypk1-AS yap1Δ (PLY1527) were grown on SCD-Ura plates containing 0.5 μM 2,3-DMBPPI with and without 0.5 mM H₂O₂ for approximately 2 days.