The mechanisms by which endocytic vesicles form and by which vesicle formation is regulated are not understood in any biological system. The proposed SGER is directed at understanding these phenomena by taking advantage of the temperature sensitive recessive mutation, shibire, in Drosophila. At 30 degrees C coated visicle formation and endocytosis are stopped within 1-2 minutes in flies homozygous for the mutation. Resumption of uptake occurs within 1-2 minutes after return to 22 degrees C. Wild type Drosophila exhibit normal uptake and vesicle formation at both 22 and 30 degrees C. Such rapid onset and reversal in the mutants suggests that a protein intrinsic to vesicle formation is the target of the shibire mutation. P element transposon mutagenesis will be used to identify and mark the wild type shibire gene. Portions of the wild type shibire gene will then be used to identify and isolate both genomic and cDNA clones of the wild type shibire gene. Receptor-mediated endocytosis is an important step in the traffic of cellular membranes and a fundamental cellular function that is closely associated with the ability of cells to receive and respond to chemical signals. If this project is successful it will be the first isolation of any gene involved in endocytic vesicle formation during receptor-mediated endocytosis. This should then permit the identification, characterization, and localization of one or more of the cellular constituents which control this critical step in cellular membrane traffic.
