The proposal is divided into four sections, all of which share a common focus on brain tissue vesicles. These vesicles transport materials to different parts of the neuron cytoplasm to sustain cell functions. Among these vesicles, the clathrincoated vesicles play the dual role of bringing molecules into the cell by the process of receptor-mediated endocytosis and of transporting materials through the cytoplasm from the perinuclear area to the plasma membrane. The proposed studies will focus on new structural proteins of the clathrin coat and on molecules present in the membrane of the various subtypes of coated vesicles we recently identified. One set of studies will consist of identifying and separating subpopulations of coated vesicles based on a panel of monoclonal antibodies recognizing different constituent """"""""cargo"""""""" molecules in these vesicles. The vesicle subtypes will be immunoprecipitated, enzyme and protein content established biochemically, the cargo molecules visualized immunologically and receptor sites characterized by their binding affinity for specific ligands. In the second set of studies we will characterize a group of surface antigens present in one form in synaptosomal membranes, in a modified state in synaptic vesicles and in another for coated vesicles. The properties of these antigens will be ascertained using a combination of protein solubilization, separation by immunoprecipitation and by determination of post-translational modifications. The third and fourth series of studies will consist of the biochemical and immunological characterization respectively of a novel protein identified in clathrincoated vesicles named NP185. This polypeptide seems specific for brain clathrincoated vesicles, differs from clathrin, and has binding affinity for other coat proteins, for protein kinases and for tubulin. For these studies we produced a pair of monoclonal antibodies to NP185 and will determine the nature of the binding sites for other proteins, characterize structurally the molecule and determine its localiza- tion in brain cells. A better understanding of neuronal cell functions may be obtained by elucidation of the properties of these various proteins and determination of their interactions in these most conspicuous brain organelles.
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