The proteins Boi1/2p link cell polarity establishment with exocytosis and actin organization in budding yeast Saccharomyces cerevisiae

Abstract

The establishment of cell polarity in yeast requires a tight coordination of diverse biological processes, involving the enrichment of Cdc42GTP at the incipient bud site, followed by the recruitment of the actin cytoskeleton, the septin cytoskeleton and the machinery for polarized exocytosis and endocytosis. Boi1p and Boi2p are functionally redundant proteins that were reported to bind to Cdc42GTP, Bem1p, and phospholipids. However, their precise cellular functions in polarity establishment and maintenance were unknown.

A large scale Split-Ubiquitin screen for interaction partners of the Boi-proteins defined a novel and complex network of protein interactions between the Boi-proteins and proteins that regulate the GTPase Cdc42p, proteins that stimulate the formations of actin filaments, proteins that catalyse the fusion of vesicles with the plasma membrane and proteins involved in cytokinesis. Starting from this network I could show that the Boi-proteins bind to the actin nucleation factors Bud6p and Bni1p. Cells that only express Boi-proteins lacking the regions interacting with Bud6p and Bni1p display a severely altered actin cytoskeleton.

The interactions with the exocyst complex and the SM-like protein Sec1p correlated with the accumulation of secretory vesicles in cells depleted for Boi1/2p. Further genetic analysis suggested that the Boi1/2 proteins might connect the vesicle-tethering exocyst complex with the vesicle fusion machinery, thereby helping to form the active fusion complex at sites of polarized growth. My work thus revealed the Boi-proteins as down-stream effectors of Cdc42GTP that coordinate actin filament formation with secretory vesicle tethering and fusion.