The clathrin-mediated endocytic pathway is vital to a variety of biological processes ranging from synaptic vesicle recycling to nutrient uptake. In addition to the major coat protein complexes, the GTPase dynamin has an essential role in clathrin-coated vesicle (CCV) formation. Dynamin is currently postulated to function as a molecular `pinchase' that facilitates the budding of CCVs from the plasma membrane, however recent evidence from the Schmid lab supports its role as a key regulator of endocytosis. Thus our goals are to establish dynamin's role as a regulator of endocytosis by: l) defining molecules that are required for dynamin recruitment to clathrin-coated pits and 2) identifying upstream and downstream effectors of dynamin function. To accomplish these goals, we will take a strict biochemical approach using functionally relevant constructs to screen cellular extracts by affinity chromatography. Factors found to specifically interact with dynamin will be immediately tested for their ability to support discrete steps in vesicle formation using cell-free assays for endocytosis that faithfully reconstitute the production of CCVs in vitro. The clathrin-mediated endocytic pathway is fundamental and highly conserved, and since abnormal expression or mutations of some of the endocytic machinery proteins have been reported in human disease and development, information obtained from this project will not only add to our understanding of the basic mechanisms of clathrin-mediated endocytosis, but may provide insight into the development of certain disease types.
