FIG 5.

Ric8 interacts with Gpa1 and Gpa2 in vivo and in vitro. (A) Yeast two-hybrid screen showing host cell growth in SD-Leu-Trp medium (DD) and SD-Leu-Trp-His-Ade medium plus 3 mM 3-amino-1,2,4-triazole (3-AT) [QD (3mM 3-AT)], suggesting that Ric8 interacts with Gpa2 (top) and Gpa1 and Gpa1^Q284L (bottom). (B) Heterologous expression of Gpa1, Gpa1^Q284L, Gpa2, Gpa2^Q203L, Gpa3, Gpa3^Q206L, and Ric8 proteins revealed by Coomassie blue staining following SDS-PAGE. The expected molecular masses of these proteins are 78.4, 78.3, 72.5, 72.5, 73.1, 73.1, and 80.2 kDa, respectively. (C) Protein pulldown assay indicating that Ric8 physically interacts with Gpa1 and Gpa2, but not Gpa3. The GST-tagged Gpa1, Gpa1^Q284L, Gpa2, Gpa2^Q203L, Gpa3, and Gpa3^Q206L and Xpress-tagged Ric8 proteins were prepared as previously described (14). The GST fusion Gα proteins were adsorbed to glutathione Sepharose beads and washed, and Ric8 was then added. Bound proteins were separated and analyzed by blotting with either the anti-GST antibody for Gαs (top) or the anti-Xpress antibody for Ric8 (bottom).