FIG 4.

Ahk1 is necessary for activation of Hog1 by constitutively active Opy2 and Ste50 mutants. (A) Hog1 phosphorylation in AHK1⁺ and ahk1Δ cells in response to osmostress. KT034 (ssk2/22Δ msb2Δ) and AN10 (ssk2/22Δ msb2Δ ahk1Δ) were grown exponentially. Cells were collected at the indicated times after addition of 0.4 M NaCl to the cultures, and the amounts of phosphorylated Hog1 (p-Hog1) and total Hog1 (Hog1) were determined by immunoblotting of the whole-cell lysate (20 μg protein per lane). Intensities of the p-Hog1 bands were quantified using the Image Lab program (Bio-Rad) and were normalized with the intensities of the corresponding Hog1 bands. The strongest band was set to 100%, and the relative intensity of each band is shown below the p-Hog1 blot. (B) Hog1-specific reporter expression in AHK1⁺ and ahk1Δ cells in response to osmostress. KT034 and AN10 were transformed with a reporter plasmid and stimulated with 0.4 M NaCl for 30 min, and expression of 8xCRE-lacZ was determined. (C) Hog1 phosphorylation in AHK1⁺ and ahk1Δ cells in response to expression of constitutively active Opy2-F96I A104V. KT034 and AN10 were transformed with a plasmid that expresses Opy2-F96I A104V from P_GAL1. Cells were collected at the indicated times after addition of 2% galactose to the cultures, and the amounts of p-Hog1 and total Hog1 were determined by immunoblotting of the whole-cell lysate (30 μg protein per lane). Quantification of the band intensities was done as described for panel A. (D) Induction of a Hog1-specific reporter gene in AHK1⁺ and ahk1Δ cells in response to expression of Opy2-F96I A104V. KT034 and AN10 were cotransformed with a reporter plasmid and a plasmid that expresses Opy2-F96I A104V from P_GAL1. Cells were stimulated with 2% galactose for 2 h, and expression of 8xCRE-lacZ was determined. (E and F) Induction of a Hog1-specific reporter gene in response to expression of the hyperactive Ste50-D146F (E) or Ssk2ΔN (F). The yeast strains shown at the bottom of the panels were cotransformed with a reporter plasmid and a plasmid that expresses Ste50-D146F (E) or Ssk2ΔN (F) from P_GAL1. KT018 and AN25 used in the experiments in panel E carry the constitutively active ste11-Q301P mutation in the chromosome. The cells were stimulated with 2% galactose for 2 h, and expression of 8xCRE-lacZ was determined. (B and D to F) β-Galactosidase activity is expressed in Miller units. Error bars represent SD (n ≥ 3). (G) A simplified model of the HOG signal pathway. Shading indicates proteins that were shown to interact with Ahk1.