The long-term objectives of these studies are to elucidate the mechanism of clathrin-coated vesicle assembly and budding, and the role of this phenomenon in the dynamic behavior of lysosomes. We recently discovered that clathrin coats assemble on lysosomes providing a model system for studying this process. Clathrin-coat formation on lysosomes is strictly dependent on ATP, and a biochemical approach will be taken to identify and characterize the cytosolic ATP-requiring component(s). Once identified, I plan to analyze how this protein primes lysosomes to initiate the coordinated protein-protein interactions that result in the assembly of the coated vesicle. This will be achieved by developing in vitro assays that follow sequential steps in the assembly process. Utilizing these sub-step assays, additional studies will be aimed at defining the precise role that AP180, a neuronal clathrin-associated protein, which is shown here to be also present in non-neuronal tissues, plays in coat formation. Studies will then be aimed at perturbing the clathrin-coated vesicle-dependent trafficking pathway out of the lysosome in vivo. This will center on the identification of a pathway- specific rab protein and the use of site-directed mutagenesis to create dominant-inhibitory mutants with opposing effects on the nucleotide- binding properties of the protein. The study is designed to provide a detailed understanding of the nature and function of the proteins responsible for coated vesicle formation on lysosomes and the role that these vesicles play in lysosome homeostasis.
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