Effects of the mutations and osmotic stresses on the <i>in vivo</i> binding between Sho1 and Opy2.

(A-C) In vivo binding between Sho1 and Opy2 was assayed by co-precipitation of Sho1 and Opy2. The yeast strain TM257 (ssk2/22Δ) (A), FP75 (ssk2/22Δ ste11Δ) (B), or KY590-1(ssk2Δ ssk22Δ sho1Δ opy2Δ) (C) was co-transfected with a plasmid expresses the indicated derivatives of Opy2Δ SR1-GFP and another plasmid that expresses either GST-Sho1 (A and B) or HA-Sho1 (C) all under the GAL1 promoter. vec, empty vector. Transformed cells were grown in CARaf, and expression of the tagged proteins was induced by 2% galactose for 2 hr. Sorbitol (0.8M, final conc.) was added (+) for 3 min (B), or not added (-). Cell extracts were prepared using Buffer A containing the following detergents: (A) 0.3% Brij-L23, (B) 0.1% Brij-97, (C) 1% digitonin. (A) HA-Sho1 was immunoprecipitated (IP) from cell extracts using anti-HA antibody and Protein G Sepharose beads, whereas (B and C) GST proteins were captured by the Gluthatione-Sepharose beads. In all cases, beads were washed three times with the same buffer used for cell lysate preparation, and co-precipitated proteins were detected by immunoblotting (IB) using the indicated antibodies.