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 #
5R01GM035370-29
Application #
8595311
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
2014-01-01
Budget End
2014-12-31
Support Year
29
Fiscal Year
2014
Total Cost
$374,627
Indirect Cost
$132,932
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
Liu, Dongmei; Novick, Peter (2014) Bem1p contributes to secretory pathway polarization through a direct interaction with Exo70p. J Cell Biol 207:59-72
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
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
Li, Xia; Ferro-Novick, Susan; Novick, Peter (2013) Different polarisome components play distinct roles in Slt2p-regulated cortical ER inheritance in Saccharomyces cerevisiae. Mol Biol Cell 24:3145-54
Stalder, Daniele; 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
Hutagalung, Alex H; Novick, Peter J (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119-49
Mizuno-Yamasaki, Emi; Medkova, Martina; Coleman, Jeff et al. (2010) Phosphatidylinositol 4-phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF Sec2p. Dev Cell 18:828-40
Vasan, Neil; Hutagalung, Alex; Novick, Peter et al. (2010) Structure of a C-terminal fragment of its Vps53 subunit suggests similarity of Golgi-associated retrograde protein (GARP) complex to a family of tethering complexes. Proc Natl Acad Sci U S A 107:14176-81
Williams, Daniel C; Novick, Peter J (2009) Analysis of SEC9 suppression reveals a relationship of SNARE function to cell physiology. PLoS One 4:e5449

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