FIG. 4.

Hbs1p interacts with Dom34p. (A) Gal4DBD-Hbs1p and Gal4ACT-Dom34p fusion proteins were assayed for interaction in two-hybrid reporter strain pJ69-4A on C-Leu-Trp-His + 1 mM 3-AT and on C-Leu-Trp-Ade plates. Gal4DBD-Snf1p and Gal4ACT-Snf4p serve as positive control for an interaction and as negative controls for the interaction with the Hbs1p and Dom34p fusions. (B) Twenty micrograms of protein extracts was prepared from yeast strain TKY514 expressing Dom34p-triple-HA-tag (HA₃) and either the full-length, aa-1-to-161, or aa-162-to-611 form of GST-tagged Hbs1p, incubated with 20 μl of 50% glutathione Sepharose 4B slurry, washed three times with 150 mM KCl buffer, and analyzed by Western blotting.