Figure 4.
Mpk1 activity is stimulated by tunicamycin treatment independently of Ire1 and Ca2+. (A) Immune complex kinase assays were performed using Mpk1-HA isolated from wild-type cells (strain DL1101 transformed with YEp352 MPK1-HA) that had been grown at 23°C and either treated with 250 μM chlorpromazine for 2 h (lane 1), mock-shifted to 23°C (lane 2), heat shocked at 39°C for 30 min (lane 3), or treated with 2.5 μg/ml tunicamycin for the indicated times (lanes 5–10). Immunoprecipitates were also analyzed by SDS-PAGE Western blotting for immunodetection of Mpk1-HA (bottom). (B) Wild-type cells, cch1 mutants, mpk1 mutants, rlm1 mutants, and ire1 mutants (strains K1257, RG04847, RG00993, RG02739, and K1259) bearing the MPK1-lacZ reporter gene were grown to log phase in SC medium lacking uracil and treated with tunicamycin with or without FK506. After 4 h, the cells were harvested and assayed for β-galactosidase activity. The mean of three independent transformants is shown (± SD).