MAP kinase dynamics in response to pheromones in budding yeast

Although scaffolding is a major regulator of mitogen-activated protein kina se (MAPK) pathways, scaffolding proteins are poorly understood. During yeas t mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated b y MAPKKK Ste11p. This MAPK module interacts with the scaffold molecule Ste5 p. Here we show that Ste11p and Ste7p were predominantly cytoplasmic protei ns, while Ste5p and Fus3p were found in the nucleus and the cytoplasm. Ste5 p, Ste7p and Fus3p also localized to tips of mating projections in pheromon e-treated cells. Using fluorescence recovery after photobleaching (FRAP), w e demonstrate that Fus3p rapidly shuttles between the nucleus and the cytop lasm independently of pheromones, Fus3p phosphorylation and Ste5p. Membrane -bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex. S te5p remains stably bound at the plasma membrane, unlike activated Fus3p, w hich dissociates from Ste5p and translocates to the nucleus.