The long term goal of this project is to understand the mechanism of intracellular membrane fusion events. In the last project period, utilizing a cell-free system that reconstitutes intercisternal transport in the Golgi stack as an assay system, we discovered and purified an N- ethylmaleimide-sensitive fusion ATPase (NSF) that is required for fusion processes involving multiple compartments in yeast and animal cells. We also found and purified three NSF attachment proteins (SNAPs) that assemble with NSF and an integral membrane SNAP receptor to form a 20S particle that seems likely to be involved in fusion processes. We propose to build upon this foundation by better characterizing the structure and function of NSF, SNAPs and snap receptor, to definitively establish their roles in fusion in living cells, and to isolate additional fusion components. We will test our hypothesis that the 20S NSF-containing particle is the """"""""core"""""""" of a general intracellular membrane fusion machinery that assembles from dispersed subunits upon demand when a fusion event is required, and disassembles in the course of catalyzing fusion.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK027044-21
Application #
2733996
Study Section
Special Emphasis Panel (NSS)
Program Officer
Haft, Carol R
Project Start
1991-09-30
Project End
2000-06-30
Budget Start
1998-07-10
Budget End
1999-06-30
Support Year
21
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Bello, Oscar D; Jouannot, Ouardane; Chaudhuri, Arunima et al. (2018) Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis. Proc Natl Acad Sci U S A 115:E7624-E7631
Rothman, James E; Krishnakumar, Shyam S; Grushin, Kirill et al. (2017) Hypothesis - buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission. FEBS Lett 591:3459-3480
Wang, Jing; Li, Feng; Bello, Oscar D et al. (2017) Circular oligomerization is an intrinsic property of synaptotagmin. Elife 6:
Li, Feng; Tiwari, Neeraj; Rothman, James E et al. (2016) Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion. Proc Natl Acad Sci U S A 113:10536-41
Xu, Weiming; Nathwani, Bhavik; Lin, Chenxiang et al. (2016) A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion. J Am Chem Soc 138:4439-47
Bello, Oscar D; Auclair, Sarah M; Rothman, James E et al. (2016) Using ApoE Nanolipoprotein Particles To Analyze SNARE-Induced Fusion Pores. Langmuir 32:3015-23
Xu, Weiming; Wang, Jing; Rothman, James E et al. (2015) Accelerating SNARE-Mediated Membrane Fusion by DNA-Lipid Tethers. Angew Chem Int Ed Engl 54:14388-92
Zorman, Sylvain; Rebane, Aleksander A; Ma, Lu et al. (2014) Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins. Elife 3:e03348
Li, Feng; K├╝mmel, Daniel; Coleman, Jeff et al. (2014) A half-zippered SNARE complex represents a functional intermediate in membrane fusion. J Am Chem Soc 136:3456-64
Shi, Lei; Howan, Kevin; Shen, Qing-Tao et al. (2013) Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores. Nat Protoc 8:935-48

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