MUTATIONAL ANALYSIS OF STE5 IN THE YEAST SACCHAROMYCES-CEREVISIAE - APPLICATION OF A DIFFERENTIAL INTERACTION TRAP ASSAY FOR EXAMINING PROTEIN-PROTEIN INTERACTIONS

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially af fect the ability of Ste5 to interact with either of two MAPK cascade c onstituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affec t association with Ste7 or with Ste11 delineate discrete regions of St e5 that are critical for each interaction. Co-immunoprecipitation anal ysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G p rotein beta subunit), and Fus3 (MAPK), confirmed that each mutation sp ecifically affects the interaction of Ste5 with only one protein. When expressed in a ste5 Delta cell, mutant Ste5 proteins that are defecti ve in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakene d (but did not eliminate) interaction of Ste5 with Ste7 permitted mati ng at wild-type efficiency, indicating that an efficacious signal is g enerated even when Ste5 associates with only a small fraction of (or o nly transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expr essed, suggesting that the functional form of Ste5 in vivo is an oligo mer.