Polarized exocytosis is fundamental to a wide range of physiological processes such as the release of neurotransmitters and the establishment of epithelial asymmetry. The budding yeast Saccharomyces cerevisiae grows asymmetrically, which requires polarized exocytosis for the incorporation of proteins and lipids to the daughter cell membrane for surface expansion and cell growth. This property, combined with its powerful genetics, makes the budding yeast an excellent model organism to study the molecular basis of polarized exocytosis. The """"""""exocyst"""""""" is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles to specific domains of the plasma membrane for exocytosis. Our goal is to understand the molecular mechanism of polarized exocytosis mediated by the exocyst complex. Central to this question are how the exocyst is targeted to the plasma membrane, and how its function is spatially and kinetically regulated there. We hypothesize that the exocyst directly binds to the membrane through its interaction with phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2), and the assembly of the exocyst complex mediates the tethering of post- Golgi secretory vesicles to the plasma membrane for exocytosis. In particular, the dual interactions of the exocyst component Sec3 with PI(4,5)P2 and Cdc42, a member of the Rho family of small GTPases, play important roles in exocyst targeting and function at the plasma membrane. Here we propose to analyze the molecular interactions of Cdc42 and phospholipids with Sec3 using biochemical methods. In addition we will determine the roles of Cdc42 and PI(4,5)P2 in the polarized localization of the exocyst, exocytosis, and cell morphogenesis. Finally, using a combination of biochemical, genetic, and cell biological approaches, we plan to characterize the novel """"""""EXR"""""""" (""""""""Exocytosis Regulators"""""""") genes recently identified from our genetic screen for proteins that spatially and kinetically regulate the exocyst function in cells. Overall these studies will help us elucidate the molecular basis and regulatory mechanisms of exocytosis, and contribute to our understanding of diseases such as neurological degeneration, polycystic kidney diseases, and cancer. PUBLIC HEALTH REVELANCE: Polarized exocytosis is fundamental to many physiological processes such as neurotransmission, embryogenesis, and the establishment of epithelial cell asymmetry in kidney. Studying the molecular basis of polarized exocytosis will help us understand these physiological processes and shed light on the pathogenesis of diseases such as diabetes, neurological disorders, and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064690-09
Application #
8212457
Study Section
Cell Structure and Function (CSF)
Program Officer
Ainsztein, Alexandra M
Project Start
2002-09-05
Project End
2013-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
9
Fiscal Year
2012
Total Cost
$305,222
Indirect Cost
$109,202
Name
University of Pennsylvania
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Feng, Shanshan; Knödler, Andreas; Ren, Jinqi et al. (2012) A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis. J Biol Chem 287:15602-9
Sakamori, Ryotaro; Das, Soumyashree; Yu, Shiyan et al. (2012) Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice. J Clin Invest 122:1052-65
Orlando, Kelly; Sun, Xiaoli; Zhang, Jian et al. (2011) Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth. Mol Biol Cell 22:624-33
Das, Amlan; Guo, Wei (2011) Rabs and the exocyst in ciliogenesis, tubulogenesis and beyond. Trends Cell Biol 21:383-6
Baek, Kyuwon; Knödler, Andreas; Lee, Sung Haeng et al. (2010) Structure-function study of the N-terminal domain of exocyst subunit Sec3. J Biol Chem 285:10424-33
He, Bing; Guo, Wei (2009) The exocyst complex in polarized exocytosis. Curr Opin Cell Biol 21:537-42
Orlando, Kelly; Guo, Wei (2009) Membrane organization and dynamics in cell polarity. Cold Spring Harb Perspect Biol 1:a001321
Zuo, Xiaofeng; Guo, Wei; Lipschutz, Joshua H (2009) The exocyst protein Sec10 is necessary for primary ciliogenesis and cystogenesis in vitro. Mol Biol Cell 20:2522-9
Orlando, Kelly; Zhang, Jian; Zhang, Xiaoyu et al. (2008) Regulation of Gic2 localization and function by phosphatidylinositol 4,5-bisphosphate during the establishment of cell polarity in budding yeast. J Biol Chem 283:14205-12

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