The long-range goal of this project is to understand the molecular mechanisms governing coated vesicle membrane traffic. The focus of the present proposal is the set of heterotetrameric complexes known as clathrin associated proteins or adaptors (""""""""APs"""""""": AP-1, associated with the TGN and AP-2 associated with the plasma membrane). Our recent important success in reconstituting functional AP complexes from expressed recombinant proteins now allows us to analyze AP activities hitherto not possible. In particular, we will pursue the following five set of aims. (A) We-will exchange AP-I and AP-2 chains to determine the combinations that can assemble successfully into functional complexes. (B)We will analyze AP/clathrin interactions in detail and use the results to purify and study cytosolic clathrin and to dissect the regulation of coat assembly. (C) We will determine which of the AP chains are responsible for initial membrane binding, and we will use this information to identify the proteins that mediate specific AP targeting. (D)We will work out which AP chains interact with the cytoplasmic domains of receptors and whether different receptors with different fates bind to different AP chains. (E)We will study receptor/AP association using cells expressing receptors of different types and in some cases expressing altered AP chains. By accomplishing these five set of aims, we will have established the identity of the specific molecular partners at various stages in coated vesicle traffic.
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