Figure 4.

Win1 is required for stress-induced activation of the Sty1 MAPK. (A) Tyrosine phosphorylation of StyI in cells lacking win1. Log phase cultures of wild-type (WT) (JM1520) or win1–1 (win1–1) (MW 1539) cells bearing an integrated and epitope-tagged version of Sty1 were incubated in medium containing 0.6 M KCl for the times indicated. Approximately 2 × 10⁸ cells were harvested and lysed at each time point, and the Sty1 protein was precipitated using Ni⁺⁺-NTA agarose. Precipitates were probed by Western blot for the presence of phosphotyrosine (α-pTyr) or the HA epitope tag (α-HA). (B) Activation of Sty1 in cells lacking win1. Log phase cultures of either wild-type (WT) (PR109) or win1–1 (win1–1) (JM1413) cells growing in YEPD at 30°C were shifted to 42°C for the times indicated. Cell extracts were prepared as above, and Sty1 was precipitated with GST-Atf1 prebound to glutathione beads. Precipitates were washed extensively and incubated in the presence of [γ-³²P]-ATP for 20 min at 30°C. Phosphorylation of Atf1 was visualized after SDS-PAGE and autoradiography.