Characterization of Fus3 localization: Active Fus3 localizes in complexes of varying size and specific activity

The MAP kinase Fus3 regulates many different signal transduction outputs th at govern the ability of Saccharomyces cerevisiae haploid cells to mate. He re we characterize Fus3 localization and association with other proteins. B y indirect immunofluorescence, Fus3 localizes in punctate spots throughout the cytoplasm and nucleus, with slightly enhanced nuclear localization afte r pheromone stimulation. This broad distribution is consistent with the cri tical role Fus3 plays in mating and contrasts that of Kss1, which concentra tes in the nucleus and is not required for mating. The majority of Fus3 is soluble and not bound to any one protein; however, a fraction is stably bou nd to two proteins of similar to 60 and similar to 70 kDa. Based on fractio nation and gradient density centrifugation properties, Fus3 exists in a num ber of complexes, with its activity critically dependent upon association w ith other proteins. In the presence of alpha factor, nearly all of the acti ve Fus3 localizes in complexes of varying size and specific activity, where as monomeric Fus3 has little activity. Fus3 has highest specific activity w ithin a 350- to 500-kDa complex previously shown to contain Ste5, Ste11, an d Ste7. Ste5 is required for Fus3 to exist in this complex. Upon ct factor withdrawal, a pool of Fus3 retains activity for more than one cell cycle. C ollectively, these results support Ste5's role as a tether and suggest that association of Fus3 in complexes in the presence of pheromone may prevent inactivation in addition to enhancing activation.