Far3 and Five Interacting Proteins Prevent Premature Recovery from Pheromone Arrest in the Budding Yeast Saccharomyces cerevisiae

Abstract

In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G₁ arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

ACKNOWLEDGMENTS

We gratefully acknowledge Becky Drees and Stan Fields for performing the comprehensive two-hybrid screen. We thank Kathy Chicas-Cruz for making purified bovine heart mitochondria, and we thank Douglass Forbes for performing preliminary sequence analysis of the Far protein sequences. We are grateful to members of the laboratories of Tom Stevens and Rod Capaldi—particularly Kate Bowers, Liz Connibear, Laurie Graham, and James Murray—for sharing antibodies and protocols. Paul Cullen, April Goehring, Joe Horecka, Megan Keniry, Dave Mitchell, David Rivers, and Greg Smith all provided invaluable discussion, reagents, and technical support.

This work was supported by a research grant (GM-30027) from the U.S. Public Health Service.