Point mutations identify a conserved region of the Saccharomyces cerevisiae AER1 gene that is essential for both the pheromone signaling and morphogenesis functions

Mating pheromone receptors activate a G protein signal pathway that leads t o the conjugation of the yeast Saccharomyces cerevisiae. This pathway also induces the production of Afr1p, a protein that negatively regulates pherom one receptor signaling and is required to form pointed projections of new g rowth that become the site of cell fusion during mating. Afr1p lacks strong similarity to any well-characterized proteins to help predict how it acts. Therefore, we investigated the relationship between the different function s of Afr1p by isolating and characterizing seven mutants that were defectiv e in regulating pheromone signaling. The AFR1 mutants were also defective w hen expressed as fusions to STE2, the alpha-factor receptor, indicating tha t the mutant Afr1 proteins are defective in function and not in co-localizi ng with receptors. The mutant genes contained four distinct point mutations that all occurred between codons 254 and 263, identifying a region that is critical for AFR1 function. Consistent with this, we found that the corres ponding region is very highly conserved in the Afr1p homologs from the yeas ts S. uvarum and S. douglasii. In contrast, there were no detectable effect s on pheromone signaling caused by deletion or overexpression of YER158c, a n open reading Game with overall sequence similarity to Afr1p that lacks th is essential region. interestingly, all of the AFR1 mutants showed a defect in their ability to form mating projections that was proportional to their defect in regulating pheromone signaling. This suggests that both function s may be due to the same action of Afr1p. Thus, these studies identify a sp ecific region of;Afr1p that is critical for its function in both signaling and morphogenesis.