Understanding how polarized cells are generated and maintained has emerged as a central problem in cell biology. Although this process is controlled by the presence of cytoplasmic domain sorting signals, nothing is known about components that decode these signals. My objective is to identify and characterize likely candidates involved in these functions. Drawing on expertise available in the laboratory as well as my experience as a biochemist, three interrelated approaches will be attempted. First, I will apply a recently developed in vitro assay that reconstitutes vesicle budding and sorting from isolated endosomes to determine whether any known proteins play important roles in this process. Second, I will make a novel application of a conventional biochemical approach, to assay the inherently weak interactions between fusion peptides bearing active sorting signals with specific cytosolic proteins. Such an approach will be used as an assay to purify potential sorting molecules. Finally, in collaboration with others in the group, I will analyze the products of newly identified genes that bear significant relationship to clathrin adaptor subunits, these being the type of proteins predicted to interact with basolateral targeting determinants.
