Figure 4.

Mak2p and Mak3p are required for Sty1p activation in response to peroxide stress. (A) Mak2p and Mak3p are required for Sty1p phosphorylation. Log-phase cultures of wild-type (JM1521), Δmak1 (VB1828), Δmak2 (VB1829), or Δmak3 (VB1934) cells bearing a hemagglutinin- and six-histidine–tagged version of Sty1p growing in YEPD medium at 30°C were incubated in the same medium containing 1 mM hydrogen peroxide for the times indicated. Approximately 2 × 10⁸ cells were harvested at each time point and phosphorylation of Sty1p was measured as described in MATERIALS AND METHODS. (B) Mak2p and Mak3p are required for Sty1p-dependent Atf1p phosphorylation. Log-phase cultures of wild-type (KS1479), Δmak1 (TS2029), Δmak2 (TS2030), or Δmak3 (TS2031) cells bearing a six-histidine– and hemagglutinin-tagged version of Atf1p growing in YEPD medium at 30°C were incubated in the same medium containing 1 mM hydrogen peroxide for 5 min. Atf1p was precipitated from each sample using Ni²⁺-nitriloacetic acid agarose and analyzed by probing Western blots with the 12CA5 anti-hemagglutinin antibody.