Pathway-Specific Inhibitors of Post-Golgi Transport

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DESCRIPTION (provided by applicant): Intracellular transport of cell surface and secreted proteins is a fundamental process in all eukaryotic cells. The yeast Saccharomyces cerevisiae has been an invaluable tool in identifying and characterizing many of the components in the protein transport machinery and is a well-established model in the study of secretory mechanisms. However, the branching of transport routes in both yeast and mammalian cells has complicated studies of the late (post-Golgi) secretory pathway, and the molecular machinery required for exocytic cargo sorting and exit from the Golgi and endosomes is largely unknown. Our long-term goal is to define the processes by which cargo is transported from the Golgi to the cell surface. The identification of the components that mediate transport at this step is critical for reaching this goal. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit an easily screened secretory phenotype and are therefore difficult to isolate. As an efficient alternative to a mutant screen, we devised a chemical genetic screen strategy that has succeeded in identifying structurally related compounds that cause a rapid (within 15 min) accumulation of secretory cargo and Golgi membranes at low (as little as 500 nM) concentration, without causing ER-to-Golgi or Golgi-to-lysosome defects, indicating that they likely target the transport machinery at the Golgi. We propose to use a compound identified by our current strategy as a tool in a new high-throughput screen aimed at identifying compounds that are highly pathway-specific. Such compounds, especially when used in various combinations, will be useful in future studies aimed at delineating how the numerous transport routes interact, with or without the presence of mutations. Many compounds will likely have specific targets, and identifying these targets will identify and determine the roles of novel components of the secretory machinery. Furthermore, pathway-specific inhibitors have potential as leads for therapeutic drugs, since they do not have a general effect on secretion and have low toxicity, a requirement in our screen. The knowledge and tools gained from our work will result in an improved understanding of late secretory transport mechanisms and of diseases that involve perturbations of these mechanisms.