The primary goal of this project is to identify proteins that are involved in synaptic vesicle endocytosis that specifically interact with cytoskeletal components. Work with yeast endocytic mutants has suggested a functional role of the cell cytoskeleton in endocytosis that is speculated to also play a physiological role in mammalian system endocytosis. Several of these yeast END (endocytosis) genes have homologues in mammalian systems and in synaptic vesicles. For example the protein products of the END4, END5, END6, and END7 gene mutants are homologues of talin, verprolin, amphiphysin, and actin, respectively. The strategy to be pursued in this proposal is to isolate cytoskeletal- interacting proteins in a mammalian system that are involved in the process of endocytosis and determine their functional significance. The initial approach will be to identify a synaptic vesicle homologue of the yeast END3 gene that has been shown in yeast to affect the cell cytoskeleton. Alternatively, using the methodology of degenerate PCR, hybridization, and complementation cloning, a protein related to the END3 gene may be identified, or other novel cytoskeletal-interacting protein. For instance, the esp15 gene, which shares some homology with END3 and has been isolated from mammalian cells, may show enrichment in the nerve terminal. Ultimately, the gene(s) isolated will be expressed and the role of the encoded protein(s) will be biochemically characterized for interactions with other synaptic vesicle proteins and its cellular localization identified. In this way, it will be possible to study the function of the END3-like gene product and contribute to the growing body of knowledge revealing the mechanisms involved in synaptic vesicle endocytosis.
