Figure 2.

Hog1 induces an S phase delay independently of the Sic1 and Swe1 Hog1 targets. (a) Osmostressed cells are delayed in S phase progression. Wild-type cells were grown exponentially (async.) and then incubated with α-factor for 3 h (α-fac), washed free of the α-factor, and incubated with 200 mM HU for 1 h (left-hand side). The cells were then washed and liberated of HU and allowed to progress through the cell cycle at 25°C in YPD medium (control) or YPD supplemented with 0.4 M NaCl (NaCl). Samples were analyzed by FACS. (b) HU treated cells are synchronized in S phase. Protein and RNA samples were extracted from α-fac and HU-synchronized cells. C-Terminally tagged Sic1-HA and Clb5-TAP levels were measured by Western blot. The asterisk indicates a nonspecific band. Northern blot analysis was performed with a probe directed at RNR1 mRNA. (c) S phase delay is independent of SIC1 but depends on HOG1. Exponential cultures of sln1^ts4 and the isogenic hog1 or sic1 mutants were grown at the permissive temperature of 25°C in which they were synchronized first with α-factor and then with HU as described in a. The cells were then washed free of HU at 37°C. Samples were analyzed by FACS. (d) S phase delay is independent of Swe1. sln1^ts4, sln1^ts4 hog1, and sln1^ts4 Swe1 cells analyzed as described in c. Experiments were performed at least twice, and representative results are shown.