Figure 2.

Requirement of the N and C termini of Ste50p in the pheromone response and the HOG pathways. (A) Both N- and C-terminal domains of Ste50p are required for the pheromone-induced morphogenesis. Yeast strain YCW335 (MATa ste50Δ sst1) was transformed with wild-type Ste50p as a GST fusion construct (pVL57) or various Ste50p deletion mutants as GST fusion constructs (pCW205, encoding Ste50p^27–346; pCW207, encoding Ste50p^115–346; and pCW213, encoding Ste50p^1–218) or with the cloning vector expressing GST only. Transformants were grown to exponential phase in medium with 0.5 mM CuSO₄, and cultures were split and either treated with 1 μM α-factor or left untreated. The treated cultures were monitored every 0.5 h, and the time indicated was the time required for clear pheromone-mediated morphological changes to occur. Wild-type and Ste50p^27–346 looked to have the same response after 2 h. Ste50p^115–346 took 5 h to respond, and the Ste50p^1–218 was totally unresponsive and looked like untreated cells after 5 h. (B) The C-terminally truncated allele of Ste50p had dominant inhibitory effect in the HOG pathway. Yeast strain YCW362 (ssk2Δ ssk22Δ) was transformed with plasmids pVL57, pCW207, pCW213, and pCW215 (encoding Ste50p^Δ131–218) or the cloning vector, grown on selective medium, and challenged with high-osmolarity selective medium containing 1.5 M sorbitol at 30°C for 3 d.