Figure 3. Overactive Fpk1 results in defects in vacuolar acidification and ROS.

(A) Ypk1-WT (PLY1083), Ypk1-AS (PLY1098), Ypk1-AS fpk1Δ (PLY1533), Ypk1-AS dnf1Δdnf2Δdnf3Δ (PLY1534), Ypk1-AS fpk1Δ rho⁰ (PLY1536), Ypk1-AS fpk1Δtpk3Δ (PLY1535), were grown in SCD-Ura media, then treated for 1 hr with 0.5 μM 2,3-DMB-PPI. ROS was determined and quantified as in Figure 1A. (B) Ypk1-WT, Ypk1-AS, Ypk1-AS fpk1Δ, Ypk1-AS rho⁰ (PLY1528) were grown in either SCD-Ura or SCD-Ura + 50 mM MES pH 6.2, treated with 0.5 μM 2,3-DMB-PPI for 1 hr, and incubated for the last 30 min with 5 μM 5(6)-Carboxyfluorescein diacetate (5(6)-CFDA). Quantification corresponds to the percent of labeled cells with cytoplasmic labeling, including cells with both vacuolar and extra-vacuolar labeling, for three separate experiments. Scale bar, 5 μm. (C) Ypk1-WT, Ypk1-AS, Ypk1-AS rho⁰, Ypk1-AS fpk1Δ, and Ypk1-AS fpk1Δ rho⁰ were grown in either SCD-Ura or SCD-Ura + 50 mM MES pH 6.2, and treated with 0.5 μM 2,3-DMB-PPI for 1 hr. ROS was determined and quantified as in Figure 1A.