INTERACTIONS BETWEEN THE ANKYRIN REPEAT-CONTAINING PROTEIN AKR1P AND THE PHEROMONE RESPONSE PATHWAY IN SACCHAROMYCES-CEREVISIAE

Akr1p, which contains six ankyrin repeats, was identified during a scr een for mutations that displayed synthetic lethality with a mutant all ele of the bud emergence gene BEM1. Cells from which AKR1 had been del eted were alive but misshapen at 30 degrees C and inviable at 37 degre es C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G(alpha) subunit of the pheromone receptor-cou pled G protein. (The active subunit of this G protein is G(beta gamma) , and G(alpha) plays an inhibitory role in G(beta gamma)-mediated sign al transduction.) AKR1 could serve as a multicopy suppressor of the le thality caused by either loss of GPA1 or overexpression of STE4, which encodes the G(beta) subunit of this G protein, suggesting that pherom one signaling is inhibited by overexpression of Akr1p. Mutations in AK R1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just, when overexpressed) inhibits signa ling. In contrast, deletion of BEM1 resulted in decreased expression o f FUS1, suggesting that Bem1p normally facilitates pheromone signaling . During a screen for proteins that displayed two-hy brid interactions with Akr1p, we identified Ste4p, raising the possibility that an inte raction between Akr1p and Ste4p contributes to proper regulation of th e pheromone response pathway.