Mapping of a Yeast G Protein ß Signaling Interaction

Corresponding author: Simon J. Dowell, Gene Function Unit, Cellular Sciences, Glaxo Wellcome Medicines Research Centre, Gunnels Wood Rd., Stevenage, Hertfordshire, SG1 2NY, United Kingdom., sd14041{at}glaxowellcome.co.uk (E-mail).

Communicating editor: F. WINSTON

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein ß subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gß (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gß coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (G) and Ste18p (G) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gß coiled-coil in Ste5p binding may set a precedent for Gß-effector interactions in more complex organisms.

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