Our studies have defined three systems that work together to mediate the polarized delivery of secretor vesicles, their localized tethering to exocytic sites and their fusion with the plasma membrane. Delivery requires actin, a type V myosin, the GTPase Sec4 and its exchange protein Sec2. Tethering requires a complex containing Sec3, Sec5, Sec6, Sec8, SeclO, Secl5, Exo 70 and Exo84, termed the exocyst.It interacts with Sec4-GTP on the vesicles and Rhol on the plasma membrane. Fusion requires the SNAREs, Snc, Sso and Sec9 as well as Sec 1, a protein that interacts with the assembled SNARE complex. This proposal focuses on the exocyst complex, its assembly and its interactions with the secretory vesicles and plasma membrane. In specific: 1) We will use a biochemical approach to identify the component of the secretory vescie that is recognized by Secl 5 and may provide specificity to the tethering process. 2) We will determine if activation by Sec4 mediates the binding of Sec 15 to Sec 10 or the interaction of a Seci 5/Sec 10 subcomplex with Sec5 and we will reconstitute this assembly step in vitro. 3) We will analyze the process of exocyst assembly using a battery of approaches. We will identify the subcomplexes present in a sec4 mutant and test the effects of Rhol and Rho3 on assembly. 4) We will reconstitute vesicle tethering in vitro and test the requirements for Sec4-GTP and each of the subunits of the exocyst in the tethering reaction. 5) We will use a biochemical approach to identify the component of the plasma membrane that binds to the exocyst. 6) We will determine if Seci or Sso overexpression can bypass the deletion of any of the exocyst structural genes and determine the effects on the spatial regulation of surface growth. 7) We will test the role of the exocyst in catalyzing the assembly of the SNARE complex and look for interactions of specific subunits of the exocyst with component SNAREs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM035370-20
Application #
6769348
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
1985-07-01
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
20
Fiscal Year
2004
Total Cost
$367,272
Indirect Cost
Name
Yale University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Liu, Dongmei; Li, Xia; Shen, David et al. (2018) Two subunits of the exocyst, Sec3p and Exo70p, can function exclusively on the plasma membrane. Mol Biol Cell 29:736-750
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
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
Liu, Dongmei; Novick, Peter (2014) Bem1p contributes to secretory pathway polarization through a direct interaction with Exo70p. J Cell Biol 207:59-72
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
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
Mizuno-Yamasaki, Emi; Rivera-Molina, Felix; Novick, Peter (2012) GTPase networks in membrane traffic. Annu Rev Biochem 81:637-59
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

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