Coat protein assembly on membranes

At the trans-Golgi network (TGN), diversion of newly synthesized lysosomal enzymes to late endosomes occurs via adaptor protein complex 1 (AP-1) containing clathrin-coated vesicles. Although AP-I recruitment is dependent upon prior translocation of the GTP binding protein ADP-ribosylation factor (ARF), ARF alone is insufficient to confer selective binding to the TGN. Our laboratory hypothesized that ARF-GTP activates an AP-1-specific docking protein complex on the TGN. I have biochemically characterized a high molecular weight complex containing AP-1 in an effort to isolate the putative AP-1 docking protein(s).; To biochemically define the minimal molecular requirements for AP-1 coated vesicle formation, we developed a liposomal recruitment assay. I found that AP-1 recruitment onto synthetic membranes was ARF-GTP dependent and was enhanced when the membranes contained acidic phospholipids. When viewed by electron microscopy, the synthetic membranes were found to support clathrin-coated vesicle formation. I also found that AP-3 recruitment onto liposomes requires ARF-GTP, and occurs efficiently with ARF5 as well as ARF1. A central question within the field is whether AP-3 interacts with clathrin. I showed that AP-3 recruits clathrin to membranes to form clathrin-coated vesicles. Further, clathrin binding is dependent upon the membrane concentration of AP-3.; Soluble NSF attachment protein receptors (SNAREs) play central roles in vesicular targeting and fusion events. Little is known, however, about mechanisms for their selective inclusion into coated vesicles. We have identified a di-leucine motif within the cytoplasmic domain of a TGN localized SNARE, vesicle-associated membrane protein 4 (VAMP 4), that mediates the selective interaction of VAMP 4 with the AP-1 complex.; Adaptor protein complex 2 (AP-2) containing CCVs formed at the plasma membrane function in receptor-mediated endocytosis. Recent studies suggest that the cooperative activity of multiple proteins underlies the membrane assembly. Epsin is a recently identified protein believed to play an important role in this process due to its ability to serve as a molecular link between proteins involved in the endocytic event. I extended the network of proteins with which epsin interacts by demonstrating a direct association between epsin and the clathrin terminal domain.