In virtually all tissues of every eukaryotic organism, polarized export of cell surface components is critical for a range of important cellular functions including protein secretion, assembly of the plasma membrane, cell signaling, cell polarization, cell migration, ciliogenesis, and cytokinesis. Defects in polarized export underlie or exacerbate a number of human diseases including cancer. We pioneered the genetic analysis of polarized export in yeast, identifying and characterizing a system of components that are all structurally and functionally conserved in higher eukaryotic organisms. Sec4p, the founding member of the rab GTPase family, acts as a master regulator by promoting the function of three different effectors: Myo2p a type V myosin that actively transports vesicles along polarized actin cables, the yeast homolog of the Lgl tumor suppressor Sro7p that physically links Sec4-GTP to the t-SNARE Sec9p, and the exocyst a complex of eight different gene products that interacts with components on the vesicle surface as well as polarity determinants on the cell cortex and thus tethers incoming secretory vesicles to these specialized sites. This proposal seeks to probe in greater detail the mechanism by which the exocyst acts to promote polarized export. 1. We will test the hypothesis that the exocyst is recruited to the vesicle surface by recognizing two coincident signals, Sec4-GTP and the v-SNARE Snc2p. 2. We will determine if the exocyst can bind the vSNARE Snc2p and the t-SNARE Sec9p at the same time, forming a Snc2p-exocyst-Sec9p ternary complex. Mutagenesis of the interaction sites will establish their function in vivo. 3. SNAREs will self-assemble in vitro, but at rates far too slow to account for their function in vivo. We will determine if the exocyst, either alone or in combination with its binding partners, can catalyze assembly of the exocytic SNARE complex. 4. The exocyst subunit Exo70p can localize to exocytic sites independent of the actin used for delivery of secretory vesicles, implying an interaction with a polarity determinant at the cell cortex. We will determine the identity of this polarity determinant. 5. Bacillus anthraxis secretes two toxins that impair the function of the exocyst in both drosophila and mammalian cells. We will determine if they also act on the yeast exocyst. This would allow us to exploit the facile genetics of yeast to identify the direct targets of these toxins and define their mechanism of action.
In virtually all tissues of every eukaryotic organism, polarized export of cell surface components is critical for a range of important cellular functions including protein secretion, assembly of the plasma membrane, cell signaling, cell polarization, cell migration, ciliogenesis, and cytokinesis. Defects in polarized export underlie or exacerbate a number of human diseases including cancer. We will determine how the key components of the export system work together to direct efficient delivery of material to the cell surface.
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