Single cell analysis of membrane recruitment dynamics of the mating MAPK pathway scaffold protein

VICTORIA REPETTO; ALAN BUSH; MATTHEW WINTERS; PETER PRYCIAK; ALEJANDRO COLMAN LERNER

Cold Spring Harbor

Congreso; CELL BIOLOGY OF YEASTS; 2013

Cold Spring Harbor Laboratory

In S. cerevisiae, pheromone signaling arrests cell cycle progression in G1. But in cells that have already committed to a new round of cell division, Cdk activity associated with Cln1/2 cyclins blocks pheromone response. Key aspects of this mutually inhibitory interaction have been elucidated previously. Pheromone-mediated arrest involves activation of the MAPK Fus3, which then activates the Cdk inhibitor Far1. Activation of this MAPK occurs when the pheromone-activated Gbetagamma dimer (Ste4/Ste18) recruits the MAPK cascade scaffold protein, Ste5, to the plasma membrane. In addition to binding Gbetagamma, membrane recruitment of Ste5 also requires a separate interaction with membrane phospholipids through its plasma membrane (PM) domain. It is this membrane interaction that is regulated by Cdk activity: Cln1/2-Cdk inhibits Ste5 recruitment by phosphorylating multiple residues flanking the Ste5 PM domain. Recently we developed a quantitative and semi-automated method to measure protein relocalization over time using microscope cytometry. Using this method, we are now able to study the early dynamics of Ste5 recruitment, immediately after pheromone exposure, in single cells at various different stages of the division cycle. Unexpectedly, we found that the initial recruitment of Ste5 is similar in G1 and S phase cells, but then it rapidly declines in S phase cells. Remarkably, this decline in S phase is strictly dependent on the activity of Fus3, since it does not happen when Fus3 is deleted or chemically inhibited, and hence it is a product of negative feedback. This feedback explains the delayed effect of cell cycle position, since there is a requirement for initial pathway activation to acquire Fus3 activity. Nevertheless, Fus3 activity alone cannot displace Ste5 from the membrane, since Ste5 recruitment persists over time in G1 cells or when it lacks the Cdk sites flanking its PM domain. These findings reveal an unanticipated collaboration between the pheromone-activated MAPK pathway and Cdk activity. They indicate that Fus3, in addition to its known roles in promoting mating, cooperates with Cdk phosphorylation to generate a complex negative feedback system that results in distinct patterns of temporal recruitment dynamics at different cell cycle stages.