Vesicular traffic in eukaryotic cells involves the formation, transport and fusion of carrier vesicles with their target membrane. We are studying the post-Golgi export of secretory proteins in yeast as a well defined example of this general mechanism of intracellular membrane transport. These events are genetically defined by the 10 late-acting SEC genes. The goal of this proposal is to understand, at a molecular level, the events controlled by the 10 SEC gene products and their possible interaction with the cytoskeleton. Five specific projects are proposed: 1) The role of the ras-like protein product of SEC4 will be examined. Genetic and biochemical approaches will be used to determine the mechanism by which this protein is attached to the cytoplasmic surface of the plasma membrane and secretory vesicles. The putative downstream effectors controlled by Sec4p will be identified by reversion of a dominant-lethal allele of SEC4 and by a simple test for suppression of a sec4 deletion. the role of second messenger molecules will be probed biochemically. 2) The sequence of Sec2p predicts that it may be a cytoskeletal protein. The association of Sec2p with cytoskeletal elements will be tested. In addition, association of purified secretory vesicles with actin fibers and movement of vesicles along actin fibers will be studied in vitro. 3) Sec15p exhibits divalent cation dependent membrane association. A membrane re-attachment assay will be developed and Sec15p interacting proteins will be identified. 4) The remaining late SEC genes will be cloned and sequenced. Antibody directed against the gene products will be generated and the intracellular location of the Sec proteins will be determined. Interactions among the Sec proteins at a functional and physical level will be assessed. 5) An in vitro exocytosis assay will be optimized utilizing reversible, vesicle accumulating sec mutants and a yeast cell permeabilization protocol. This assay will be used to address the biochemical function of the late acting SEC gene products. In total, these studies will begin to fill in the genetic framework of the secretory pathway with molecular and biochemical details concerning the components of the machinery essential for vesicular transport. We believe that these results will be broadly relevant to transport events in all eukaryotes.
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