Bem1p-binding–deficient mutations in Exo70p do not impair its interactions with Rho3p-GTP. (A) GST-tagged WT Exo70p or mutant proteins (M26, M30, and ΔdC) were immobilized on glutathione beads and then incubated with yeast lysate expressing His6-Rho3Q74Lp (GTP-locked form) or His6-Rho3T30Np (GDP-locked form). Bound proteins were detected as described in the Materials and methods. (B) Bem1p does not compete with Rho3p for Exo70p binding. GST-tagged WT Exo70p or M26 mutant proteins were immobilized on glutathione beads and then incubated with yeast lysate expressing His6-Rho3Q74Lp (GTP-locked form) in the presence or absence of purified His6-Bem1p. Bound proteins were detected as described in the Materials and methods. Two different exposures (labeled long and short) of the Rho3p blot are shown. (C) Exo70p single domain deletion mutants with an N-terminal GST-tag were pre-immobilized on glutathione beads and incubated with yeast lysate expressing His6-Rho3Q74Lp or His6-Rho3T30Np. Bound proteins were detected as described in the Materials and methods. Two different exposures of the Rho3p blots are shown. (D) The percentage of His6-Rho3Q74Lp or His6-Rho3T30Np bound to Exo70p WT or mutants was quantified. Error bars represent SD; n = 3.
