Abstract
The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein βγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gβγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.
Highlights
The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction
On the basis of these findings, we propose that vacuolar targeting represents a third and distinct branch of the mating response pathway, one that is uniquely mediated by the G-protein ␣ subunit, the PI 3-kinase Vps34, and the F-box protein Dia2
Given that Vps34 is required for autophagy and cytoplasm–to–vacuole transport, we hypothesized that Vps34 might promote these processes in response to G-protein activation
Summary
Mating pheromone ␣-factor promotes vacuolar targeting of a cytoplasmic reporter protein. Dia and Vps promote pheromone-induced vacuolar targeting of cytoplasmic proteins and limit the abundance of organelle markers, at least under basal (unstimulated) conditions. Our findings reveal a novel form of vacuolar targeting that is mediated by mating pheromone, the G-protein ␣ subunit Gpa, the PI 3-kinase Complex II, the MAPK Fus, and the F-box protein Dia. Cells were treated with pheromone, resolved by gel electrophoresis and immunoblotting, and probed with antibodies that detect the dual-phosphorylated form of the protein In this case, the dia2⌬ mutant sustained full Fus activation, despite the marked decrease in Fus3-mediated transcription and vacuolar targeting (110). In light of the pheromone-dependent phenotype and its documented role in ubiquitination (41), we named the gene PFU1 (pheromone F-box ubiquitination)
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