Many biologically important macromolecules such as nutrients, peptide hormones, growth factors and immunoglobulins bind to specific receptors on the cell surface and are internalized into cells by the process of receptor-mediated endocytosis. The early steps in receptor-mediated endocytosis are: 1, formation of clathrin coated pits; 2, sorting of cell surface receptors for inclusion into a coated pit; 3, invagination and membrane fission to form a primary endocytic """"""""coated"""""""" vesicle; and 4, fusion of the newly formed primary endocytic vesicle to deliver its contents to an 'early' endosomal compartment. The overall goal of this project is to identify protein components which mediate the earliest steps of receptor-mediated endocytosis and eventually to elucidate the mechanisms involved in vesicular membrane transport along the endocytic pathway. This goal will be accomplished through the development and use of novel cell-free assays which faithfully reproduced these early events. An 'endocytosis' assay will be developed which follows coated pit formation and the internalization of receptor-bound ligands into """"""""semi-intact"""""""" cells (a preparation of cells which have had a portion of their plasma membrane removed so as to deplete them of cytoplasm but otherwise maintain their intracellular organelles intact an accessible to exogenously added reagents). This 'endocytosis' assay will be used to study the mechanisms of clathrin-coated pit formation and of coated vesicle budding. In addition, the role of clathrin and other coated proteins in the sorting of cell surface receptors into coated pits will be examined. A second assay which measures the specific membrane fusion event between highly enriched newly formed endocytic vesicles and early endosomes will also be developed and employed. This 'endosome-fusion' assay will be used to identify integral and/or peripheral endosome and endocytic vesicle-specific membrane components which mediate specific organelle recognition and membrane fusion. These studies will be facilitated by the availability of highly enriched endocytic vesicles and endosomal fractions which will be obtained through the continued development of free flow electrophoresis as a novel method for the subcellular fractionation of endocytic organelles. It is expected that this basic approach of developing functional cell-free assays for well- defined cellular events during receptor-mediated endocytosis will facilitate the biochemical dissection of these events. The biochemical approach described should eventually lead to an understanding of some of the molecular mechanisms of membrane sorting and transport in mammalian cells.
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