The general objective is to study clathrin and its associated proteins (AP's) in order to understand their role in the formation and function of coated pits and coated vesicles. We have obtained a relatively detailed knowledge of the organization and interaction of clathrin heavy and light chains, through combined use of structural approaches and molecular cloning. We now turn to analyze the functional role of clathrin and its associated proteins in the process of vesicular membrane traffic. Several parts of the long range project are presented in this proposal. The first part is the identification, characterization and assignment of cDNA clones to the protein components of the AP-1 and AP-2 complexes. Complete sequences will be determined. In the second part we will use the sequence information to study the molecular organization and multiple forms of AP complexes at four levels: 1) Determine the domain structure of the AP polypeptide chains. 2) Analyze the molecular contacts within AP complexes, using chemical crosslinking and 2-D peptide maps. 3) Determine the subunit composition and cellular distribution of different complexes, using antibodies raised against selected synthetic peptides of the three classes of AP proteins. 4) Establish the extent and significance of the multiple species and classes of AP complexes within a cell type and between tissues. The third part of this proposal describes our long-range plans that will capitalize on our current knowledge of clathrin and on the new structural information that we expect to gain about the AP's. We will study the effects of selective modifications of clathrin and AP's on various aspects of coat formation and function. We will modify DNA sequences and transfect cells in an effort to influence aspects of coat formation and function.
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