CWI requirements for thermal induction of FKS2 and PRM5. (A) Induction of FKS2 expression is dependent on either Mpk1 or its pseudokinase paralog, Mlp1. An FKS2-lacZ reporter plasmid (p2052) was transformed into the wild-type (DL3193), bck1Δ (DL3529), mkk1Δ mkk2Δ (DL3541), mpk1Δ (DL3195), mlp1Δ (DL3194), or mpk1Δ mlp1Δ (DL3196) strain. Transformants were cultured as described in the legend of Fig. 1 in the presence of YEPD medium containing 10% sorbitol for osmotic support. β-Galactosidase (β-Gal) activity was measured in crude extracts. (B) Mpk1 does not require catalytic activity for thermal induction of FKS2, but Mpk1 and Mlp1 must be phosphorylated. An FKS2-lacZ reporter plasmid (p2052) was cotransformed with centromeric plasmids bearing MPK1 (p2188), mpk1-TA/YF (p2190), mpk1-K54R (p2193), MLP1 (p2346), mlp1-Y192F (p2347), or vector (pRS315) into an mpk1Δ mlp1Δ strain (DL3196). Transformants were treated as above. (C) Mpk1 catalytic activity is required for thermal induction of PRM5. A PRM5-lacZ reporter plasmid (p1366) was cotransformed with the indicated plasmids from panel B into an mpk1Δ mlp1Δ strain (DL3196). Transformants were treated as above, except that culture time was 5 h rather than 15 h. Each value represents the mean and standard deviation from three independent transformants. Sp. Act., specific activity; U, unit.