Internalization of integral membrane proteins during receptor- mediated endocytosis (RME) is accompanied by the polymerization of clathrin on the cytoplasmic side of invaginated membrane. This results in the formation of coated pits and coated vesicles, composed of internalized membrane and receptors, covered with a polyhedral lattice of clathrin. It has been proposed that self-assembly of clathrin into this polyhedral structure is fundamental to the process of selective receptor internalization and membrane translocation which occurs during RME. To understand the mechanism and ultimately the control of this selective membrane movement, we propose to investigate the molecular basis of clathrin polymerization both in vitro and in situ. In preliminary studies, we have identifed molecular sites involved in clathrin-clathrin interactions during assembly by using anti-clathrin monoclonal antibodies (MAbs) to inhibit clathrin assembly. Based on the aborted clathrin structures produced, we have proposed a model for clathrin assembly which involves sequential molecular interactions. The first specific aim of the studies outlined in this proposal is to focus on molecular localization of clathrin interaction sites involved in assembly and to test the proposed model. The second specific aim is to address the role of accessory proteins in clathrin polymerization to assess additional fators which influence clathrin assembly in situ and affect coated vesicle movement. The third specific aim is to investigae in situ, clathrin assembly by following biosynthesis and assembly of the clathrin subunits. As a whole, these studies should lead to a clearer understanding of the process of clathrin assembly at the molecular and cellular level and establish directions for investigating additional processes contributing to receptor-mediated endocytosis.
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