Figure 2. Mpk1 phosphorylates Rom2.

A. Yeast cells expressing Myc tagged Rom2, including those of wild type (Y1243), mpk1Δ (Y1272)^*, wsc1Δ mid2Δ (Y1273)^*, pkc1Δ (Y1274)^*, bck1Δ (Y1275)^* and rlm1Δ (Y1276) were transformed with GAL1p-MKK1 ^S386P or a control vector. Transformed cells were grown overnight in medium containing 2% raffinose to exponential phase, followed by addition of galactose to a final concentration of 1%. An aliquot of cells was collected before or 3 hr after the addition of galactose. Cells were lysed and lysates were analyzed by western blotting with anti-Myc antibody. B. Cells expressing HA₃-MPK1 (Y1054) or HA₃-mpk1 ^K54R (Y1052) were grown to exponential phase at 23°C and shifted to 38°C. Cells were collected at the indicated time points after the shift. HA tagged Mpk1 proteins were immunopurified and incubated with Rom2-myc₁₃ in the presence of ATPγ³²P. The phosphorylation levels of Rom2-myc₁₃ by HA₃-Mpk1 were monitored by the incorporation of ³²P into the protein (lower panel). The levels of the Mpk1 protein (upper panel) and phospho-Mpk1 (middle panel) were determined by western blotting. Note: * these strains are not isogenic to the wild type strain (Y1243).