The overall objective of this proposal is to understand the molecular mechanisms of neuronal membrane turnover pathways. In brain nerve synaptic endings the transport of membrane, with its intrinsic and associated protein components, is mediated by endocytic vesicles. An important type of endocytic organelles is represented by a vesicle coated with a lattice of clathrin molecules. Clathrin is one of the most abundant proteins in mammalian brain and highly concentrated in nerve endings. Thus, in studying specific neuronal processes where clathrin-coated vesicles are involved, we will also study their relationship with exocytic events like synaptic vesicle-mediated neurotransmitter release. From brain cortex we will obtain the endocytic vesicles, identify and characterize their specific functions mediated by their constituent polypeptides and enzymes. The studies described are based on our recent finding of a novel protein specific for brain clathrin-coated vesicles. This protein caries a protein kinase that phosphorylates one of the clathrin light chain molecules. Upon phosphorylation, phospholipase A2 activation ensues, followed by vesicle membrane release from the coat lattice and fusion with other vesicle membranes to form large cisternae. We will address: a) the functional and biochemical significance of the clathrin light chain phosphorylation; b) properties of the protein molecule that acts as a carrier of the kinase; c) separation of subpopulations of coated vesicles that possess one or more protein kinases and phospholipase A2 activity; and d) the sequence of changes in the denuded vesicles leading to fused cisternae formation. We will determine membrane events by light scattering and observe them by electron microscopy, measure phosphorylation by quantitative gel electrophoresis and aut oradiography, phospholipase A2 activation by thin-layer chromatography and HPLC and separate the vesicle subpopulations containing specific marker antigens by immunoprecipitation with a panel of monoclonal antibodies that we produced and characterized.
