Newly synthesized proteins are delivered to final destinations by way of vesicular transport. Assembly of the complex between target membrane (t-) SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and vesicle-associated (v-) SNARE is essential for docking and membrane fusion. The goal of this project is to elucidate the process of SNARE assembly and the mechanism of membrane fusion involved in yeast protein trafficking. SNAREs are amphipathic integral membrane proteins that offer quite a challenge to x-ray crystallography and NMR. The present project utilizes site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) that has proven powerful in determining structures and topologies of integral membrane proteins. In this method, native residues are substituted to cysteines and modified with a nitroxide spin label. Recent advances in EPR spectroscopy allow the measurement of membrane immersion depths as well as distances between pairs of positions in the range of 6-50 Angstroms. Data analysis and computer modeling on a sufficiently large set of distances give a three-dimensional model of the protein structure at backbone resolution. SNARE assembly proceeds in multiple steps. In this project, structures and membrane topologies of individual full-length SNARE proteins, their assembly intermediates, and the final complex are investigated with SDSL EPR. It is widely believed that SNAREs are the minimal fusion machinery. Thus, EPR data will provide insights into the mechanism of SNARE-induced membrane fusion. Remarkably, all SNAREs involved in vesicular transports are highly conserved. Thus, what is learned from yeast SNAREs will have implications for other systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067629-03
Application #
6915689
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Shapiro, Bert I
Project Start
2003-07-01
Project End
2007-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
3
Fiscal Year
2005
Total Cost
$216,265
Indirect Cost
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
Lai, Ying; Shin, Yeon-Kyun (2012) The importance of an asymmetric distribution of acidic lipids for synaptotagmin 1 function as a Ca2+ sensor. Biochem J 443:223-9
Diao, Jiajie; Su, Zengliu; Ishitsuka, Yuji et al. (2010) A single-vesicle content mixing assay for SNARE-mediated membrane fusion. Nat Commun 1:54
Yoon, T-Y; Shin, Y-K (2009) Progress in understanding the neuronal SNARE function and its regulation. Cell Mol Life Sci 66:460-9
Lu, Xiaobing; Zhang, Yinghui; Shin, Yeon-Kyun (2008) Supramolecular SNARE assembly precedes hemifusion in SNARE-mediated membrane fusion. Nat Struct Mol Biol 15:700-6
Su, Zengliu; Ishitsuka, Yuji; Ha, Taekjip et al. (2008) The SNARE complex from yeast is partially unstructured on the membrane. Structure 16:1138-46
Zhang, Yinghui; Shin, Yeon-Kyun (2006) Transmembrane organization of yeast syntaxin-analogue Sso1p. Biochemistry 45:4173-81
Yoon, Tae-Young; Okumus, Burak; Zhang, Fan et al. (2006) Multiple intermediates in SNARE-induced membrane fusion. Proc Natl Acad Sci U S A 103:19731-6