A time resolved interaction analysis of Bem1 reconstructs the flow of Cdc42 during polar growth

Cdc42 organizes cellular polarity and directs the formation of cellular structures in many organisms. By locating Cdc24, the source of active Cdc42, to the growing edge of the yeast cell, the scaffold protein Bem1 is instrumental in shaping the cellular gradient of Cdc42. This gradient instructs bud formation, bud growth, or cytokinesis through the actions of a diverse set of effector proteins. To address how Bem1 participates in this transformation we systematically mapped its protein interactions in time and space. SPLIFF analysis defined a unique ensemble of Bem1 interaction-states for each cell cycle stage. The characterization of mutants of Bem1 that interact with a discrete subset of the interaction partners allowed to assign specific functions to different interaction states and identified the determinants for their cellular distributions. The analysis characterizes Bem1 as a cell cycle specific shuttle that distributes active Cdc42 from its source to its effectors and helps to convert the PAKs Cla4 and Ste20 into their active conformation.