Identification and Functional Characterization of Pheromone Induced Fus3 Specific Phosphorylation Sites of the Yeast 26S Proteasome by Quantitative Mass Spectrometry

摘要

Transitions between different phases of the cell cycle are tightly regulated to maintain genome integrity and prevent uncontrolled cell proliferation. Unidirectional progression through the cell cycle is driven by the degradation of cyclins and regulatory proteins by the ubiquitin-proteasome system. In order to understand the molecular mechanisms underlying cell cycle regulated proteasomal degradation, we recently characterized cell cycle dependent protein interaction networks of the yeast 26S proteasome. We discovered that pheromone induced G1 phase arrest led to increased proteasome phosphorylation and interaction with multiple key players of the pheromone signaling pathway including MAP kinase Fus3 and several Fus3 substrates/interacting proteins. Fus3 regulates several key signaling events including the phosphorylation and activation of the transcription activator Ste12 and the Far1 protein which mediates polarized bud emergence as well as cell cycle arrest in G1 phase. We hypothesize that the proteasome may regulate the cell cycle in response to pheromone signaling. To test this, we have used quantitative mass spectrometry to investigate 26S proteasome phosphorylation dynamics in response to pheromone signaling as well as identified protein interactions with the proteasome which are enhanced or induced by pheromone treatment.