Figure 7.
Hog1 acts subsequent to Sld2 phosphorylation and before Dpb2 phosphorylation. (a) Sld2 phosphorylation kinetics are identical in nonstress and osmostress conditions. Sic1 cells with endogenously tagged Sld2-HA were synchronized with α-fac for 3 h and released into fresh medium (control). Half of the culture was exposed to 0.4 M NaCl 20 min after release from α-factor (NaCl). Protein extracts were prepared, separated on 7% SDS-PAGE, and probed with α-HA antibodies to detect Sld2-HA. The slower migrating phosphorylated form of Sld2 (P-Sld2) and the nonphosphorylated form (Sld2) are indicated. (b) Dpb2 phosphorylation is delayed in osmostressed cells. sic1 mutant cells with endogenously tagged Dpb2-HA were grown and assayed as described in a. (c) The delayed Dpb2 phosphorylation kinetics depends on Hog1. Dpb2-HA tagged sln1ts4 sic1 with wild-type HOG1 (right) or a hog1 deletion (left) were grown, synchronized with α-fac and released into fresh medium as described in a. The cultures were shifted to 37°C 20 min after their release from α-fac. Dpb2 phosphorylation was followed as described above. Experiments were performed at least twice, and representative results are shown.