A modulatory role for clathrin light chain phosphorylation in Golgi membrane protein localization during vegetative growth and during the mating response of Saccharomyces cerevisiae

The role of clathrin light chain phosphorylation in regulating clathrin fun ction has been examined in Saccharomyces cerevisiae. The phosphorylation st ate of yeast clathrin light chain (Clc1p) in vivo was monitored by [P-32]ph osphate labeling and immunoprecipitation. Clc1p was phosphorylated in growi ng cells and also hyperphosphorylated upon activation of the mating respons e signal transduction pathway. Mating pheromone-stimulated hyperphosphoryla tion of Clc1p was dependent on the mating response signal transduction path way MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred ex clusively on serines. Mutagenesis of Clc1p was used to map major phosphoryl ation sites to serines 52 and 112, but conversion of all 14 serines in Clc1 p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells e xpressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacu olar protein, or receptor-mediated endocytosis of mating pheromone. However , the trans-Golgi network membrane protein Kex2p was not optimally localize d in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p l ocalization defect and caused a corresponding defect in Kex2p-mediated matu ration of the alpha-factor precursor. The results reveal a novel requiremen t for clathrin during the mating response and suggest that phosphorylation of the light chain subwnit modulates the activity of clathrin at the trans- Golgi network.