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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM035370-27
Application #
8237216
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
1985-07-01
Project End
2015-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
27
Fiscal Year
2012
Total Cost
$415,580
Indirect Cost
$147,031
Name
University of California San Diego
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Novick, Peter (2016) Regulation of membrane traffic by Rab GEF and GAP cascades. Small GTPases 7:252-256
Stalder, Danièle; Novick, Peter J (2016) The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p. Mol Biol Cell 27:686-701
Casey, Amanda K; Chen, Shuliang; Novick, Peter et al. (2015) Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum. Mol Biol Cell 26:2833-44
Stalder, Danièle; Novick, Peter J (2015) Assaying the interaction of the Rab guanine nucleotide exchange protein Sec2 with the upstream Rab, a downstream effector, and a phosphoinositide. Methods Mol Biol 1298:85-98
Novick, Peter J (2014) A pathway of a hundred genes starts with a single mutant: isolation of sec1-1. Proc Natl Acad Sci U S A 111:9019-20
Liu, Dongmei; Novick, Peter (2014) Bem1p contributes to secretory pathway polarization through a direct interaction with Exo70p. J Cell Biol 207:59-72
Ling, Yading; Hayano, Scott; Novick, Peter (2014) Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature. Mol Biol Cell 25:3389-400
Riquelme, Meritxell; Bredeweg, Erin L; Callejas-Negrete, Olga et al. (2014) The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth. Mol Biol Cell 25:1312-26
Stalder, Danièle; Mizuno-Yamasaki, Emi; Ghassemian, Majid et al. (2013) Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners. Proc Natl Acad Sci U S A 110:19995-20002
Shen, David; Yuan, Hua; Hutagalung, Alex et al. (2013) The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p. J Cell Biol 202:509-26

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