Mapping of a yeast G protein by signaling interaction

The mating pathway of Saccharomyces cerevisiae is widely used as a model sy stem for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein py subu nits transmit the signal to a MAP kinase cascade, which involves interactio n of GP (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identi fy residues in Ste4p required for the interaction with Ste5p. These residue s define a new signaling interface close to the Ste20p binding site within the G beta gamma coiled-coil. Ste4p mutants defective in the Ste5p interact ion interact efficiently with Gpa1p (G alpha) and Ste18p (G gamma) but cann ot function in signal transduction because cells expressing these mutants a re sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste 5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong ev idence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of t he Ste4p-Ste5p interaction and sterility confirms the importance of this in teraction in signal transduction. Identification of the G beta gamma coiled -coil in Ste5p binding may set a precedent for G beta gamma-effector intera ctions in more complex organisms.