Loss of Cyc8 SUMOylation suppresses the osmosensitivity of hog1<i>Δ</i> cells

(A) Spot titer assay comparing osmosensitivity of various cells. Parent, cyc8Δ, hog1Δ, or cyc8Δhog1Δ yeast cells were transformed with the indicated constructs and spotted in ten-fold serial dilutions on YPD or YPD+0.8MKCl plates and grown at 30°C for two days. (B) Quantitative measure of growth rates generated by Bioscreen C. Indicated cells were grown in triplicate at 30°C in YPD or YPD+1M sorbitol for 24 hours with continuous shaking. Absorbance at 600nm was measured every 30 minutes and average absorbance was plotted versus time. (C) Average doubling times in specific media as measured by Bioscreen C. Doubling times for indicated cells were calculated using the yeast outgrowth data analyzer (YODA, [22]). Error bars show SD for triplicate samples. (D) Confirmation of reduced Cyc8 SUMOylation in suppressor cells. Indicated cells bearing 6His-FLAG-Smc3 and 3HSV-tagged Cyc8 were treated with 1.2M sorbitol and collected at the indicated time points. SUMOylated proteins were collected and analyzed as described in Fig 1A. SUMOylated-Cyc8 was identified by Western analysis using anti-HSV antibodies. Total Cyc8 in the input fraction was used as a loading control, while activated p38 was used as a control for Hog1 presence and activation.