FIG 2.

Role of the cytoplasmic domain of Hkr1 in the HOG pathway (A) Schematic models of the Hkr1 and Msb2 constructs used for the experiments shown in this figure. The top bar shows the full-length Hkr1 WT molecule. Dotted lines represent deleted segments. Gray bars represent the Msb2-derived segments. Numbers indicate amino acid positions. TM, transmembrane domain; Cyto, cytoplasmic region. (B to F) Expression of the Hog1-specific reporter gene 8xCRE-lacZ. β-Galactosidase activity is expressed in Miller units. Error bars represent standard deviations (SD) (n ≥ 3). (B) The yeast strain KT063 (ssk2/22Δ hkr1Δ msb2Δ) was transformed with single-copy plasmids that expressed the indicated Hkr1 constructs from the HKR1 promoter (P_HKR1), together with a reporter plasmid. Cells were stimulated with 0.4 M NaCl for 30 min or not stimulated, and expression of the 8xCRE-lacZ gene was determined. (C and D) KT063 was transformed with single-copy plasmids that expressed the indicated Hkr1 and Msb2 constructs from their native promoter (P_OWN is either P_HKR1 or P_MSB2, which corresponds to the 5′ end of the cloned gene), another single-copy plasmid that expressed Opy2-F96I A104V from the inducible GAL1 promoter (P_GAL1), and a reporter plasmid. Expression of Opy2-F96I A104V was induced by 2% galactose for 2 h, and expression of 8xCRE-lacZ was determined. (E and F) The yeast strain AN01 (ssk2/22Δ hkr1Δ msb2Δ ste11-Q301P) was transformed with single-copy plasmids that expressed the indicated Hkr1 and Msb2 constructs from their native promoter, another single-copy plasmid that expressed Ste50-D146F from the inducible GAL1 promoter (P_GAL1), and a reporter plasmid. Expression of Ste50-D146F was induced by 2% galactose for 2 h, and expression of the 8xCRE-lacZ gene was determined.