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. 2015 Apr 21;6:6975. doi: 10.1038/ncomms7975

Figure 10. The roles of the Sho1–Opy2–Hkr1 interactions in osmotic activation of Hog1.

Figure 10

(a) Schematic model of Hkr1. Structures of the HMH domain deletion mutants are enlarged. Numbers are amino-acid positions. (b) Opy2–Hkr1-binding assay. The indicated Hkr1 and Opy2 constructs were expressed in TM257 (left) or QG158 (right) strains. Cell lysates were prepared with Buffer A containing 1% digitonin. Hkr1ΔSTR-HA was immunoprecipitated, and co-precipitated Opy2-GFP was detected by immunoblotting. (c) Hog1-specific reporter assays of KY585 and KY599-2 strains co-transfected with the indicated Hkr1 and Opy2 expression plasmids. Cells were stimulated with or without 0.4 M NaCl for 30 min, and the expression of the 8xCRE-lacZ reporter gene was determined. Error bars are s.d. (n⩾3). (d) Schematic model of sequential interaction among Hkr1, Opy2 and Sho1. (e) Summary of the interactions among Sho1, Opy2 and Hkr1. Only a portion of the Sho1 oligomer is shown. (f) Mutations that disrupt the interactions between Sho1, Opy2 and Hkr1. (g–h) Hog1-specific reporter assays of the yeast strains AN004 (g) and KY602-12 (h) transformed with expression plasmids for the indicated mutants of Opy2 and Hkr1 (native promoters). Cells were stimulated with or without 0.4 M NaCl for 30 min and expression of the 8xCRE-lacZ reporter gene was determined. Error bars are s.d. (n=4). (i) Summary of the functional effects of mutations that disrupt interactions between Sho1 (S), Opy2 (O) and Hkr1 (H).