Coated vesicle adaptors. (360G-Wellcome-053316_Z_98_C)

Adaptors, or APs, are heterotetrameric protein complexes involved in vesicle budding. The AP-1 and AP-2 adaptors are associated with clathrin-coated pits and vesicles at the trans-Golgi network (TGN) and plasma membrane respectively. We plan to investigate the mechanisms by which AP-1 and AP-2 are recruited onto the appropriate membrane, and how this leads to the production of a coated vesicle. We will identify adaptor-binding proteins which might act as membrane docking sites by cross-linking, affinity purification, and two-hybrid library screening, and we will clone and sequence their corresponding cDNAs. We also plan to clone cDNAs encoding some of the minor proteins of unknown function that co-purify with clathrin-coated vesicles. Characterisation of these proteins will include immunolocalisation studies at both the light and electron microscope levels, transfecting tissue culture cells with both wild-type and mutant constructs and then examining the phenotype, and adding antibodies and/or constructs to in vitro systems that we have developed to determine at what stage a particular protein acts. The second aim of the proposal is to identify and characterise novel adaptor-related complexes. We have recently discovered an adaptor-like complex, AP-3, which is not associated with clathrin, and we plan to establish its function by antibody microinjection, by expressing truncated subunits that might act as dominant mutants, and in particular by taking advantage of the finding that two of the subunits are encoded by genes for which mutations are already known. In addition, we have evidence for at least one other adaptor-related complex, and we plan to investigate both its subunit composition and its function.