NEW YEAST MUTANTS IN POSTGOLGI TRANSPORT

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Previous genetic screens have identified yeast secretory mutants have block protein traffic at various points along the secretory pathway. While these mutants most likely block the transport of all cargo, two classes of post-Golgi vesicles transporting unique cargo to the plasma membrane have been recently characterized. This proposal aims to isolate new mutants blocked in the transport of only one of these vesicle species. One strategy involves a selection scheme in which a secretory block would allow cells to grow. While this method will most likely identify only non- essential genes, the use of a selection as an initial step should greatly facilitate mutant isolation. A second screen utilizes K/28 killer toxin as a secretory marker for identifying conditional mutations in essential genes required for the secretion of K/28 and other cargo in the pathway transporting this toxin. A better understanding of the mechanisms of vesicle-mediated transport from the Golgi to the plasma membrane will require the identification of the key components involved in this process, and characterization of the mutants isolated in the proposed screens should greatly contribute to this end. These mutants, as well as a new in vitro budding assay in yeast for the biochemical analysis of the formation of late secretory vesicles, will provide powerful tools for subsequent biochemical and genetic studies.