Characterizing the Conformation of the Yeast Endocytic Scaffold Protein Pan1

Endocytosis is a sequential process that involves the coordination of numerous proteins to internalize membrane bound cargo and extracellular material. The steps of endocytosis include cargo selection, assembly of the endocytic machinery, and the activation of factors that enable vesicle internalization. Transitions between these stages are carefully regulated to ensure successful endocytosis. The essential Saccharomyces cerevisiae protein Pan1 acts as a “scaffold”, mediating endocytosis by binding factors that act in both early and late stages of endocytosis. The Pan1 N-terminus interacts with adaptor proteins involved in the early process of cargo selection. At its C-terminus, Pan1 binds late-acting factors such as the type I myosins and the Arp2/3 complex, which stimulate actin polymerization to promote vesicle scission. In vitro data suggest that phosphorylation of Pan1 plays a role in its regulation. The Pan1 N-terminus is phosphorylated by the kinase Prk1, which inhibits Pan1 stimulation of Arp2/3 dependent actin polymerization in vitro. These and other data suggest a model whereby Pan1 adopts a conformation that prevents its interaction with late-acting factors in endocytosis. The inhibited state mediated by self-association may be an intra-molecular interaction, where Pan1 folds upon itself, or an inter-molecular interaction, through Pan1 oligomers formed by the central coiled-coil domain. Consistent with this model, initial electron microscopy and biophysical data indicate that Pan1 undergoes Prk1-dependent conformation changes. To characterize the conformations Pan1 can assume, I am defining the domains involved in Pan1 self-interactions and quantifying their affinities and dependence on phosphorylation. I am also analyzing the tryptophan fluorescence and anisotropy values of Pan1 mutants containing only one of four tryptophans. A greater understanding of Pan1 structural changes and how these conformations regulate endocytosis is necessary to develop a complete model of the endocytic process.