Figure 4.

Hypophosphorylated Rod1 does not require Rsp5 binding to squelch mating pheromone-evoked growth arrest. (A) The adaptation-promoting capacity of the indicated 2A, 6A, and 8A alleles of Rod1 was assessed, as in Figure 1A, with or without mutation of all three Rsp5-binding motifs (^V/_PPxY-less). (B) Expression of the Rod1 variants shown in A was confirmed as in Figure 2C. (C) Cultures of a GEV-carrying derivative of the protease-deficient strain BJ5459 expressing the indicated Rod1 mutant were grown to midexponential phase. Protein expression was induced with β-estradiol and, after 3 hr, the cells were harvested by centrifugation and ruptured by vigorous vortex mixed with glass beads, and the GST-fusion proteins in the resulting extracts were captured by binding to glutathione-agarose beads. After washing, bead-bound proteins were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (D) The adaptation-promoting capacity of the indicated Rod1 alleles was assessed, as in Figure 1A, in mutant cells lacking endogenous Rod1, Rog3 and Ldb19. (E) Expression of the Rod1 variants shown in A was confirmed as in Figure 2C.