NSF-mediated disassembly of on and off-pathway SNARE complexes and inhibition by complexin

Abstract
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Abstract

SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter release and other membrane trafficking processes. We developed a single molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly and reassembly of individual SNARE complexes. For ternary neuronal SNARE complexes, disassembly proceeds in a single step within 100 msec. We observed short- (< 0.32 sec) and long-lived ({greater than or equal to} 0.32 sec) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate re-assembly. Either high ionic strength or decreased αSNAP concentration reduces the disassembly rate while increasing the frequency of short-lived states. NSF is also capable of disassembling anti-parallel ternary SNARE complexes, suggesting a role in quality control. Finally, complexin-1 competes with αSNAP binding to the SNARE complex; addition of complexin-1 has an effect similar to that of decreasing the αSNAP concentration, suggesting a possible regulatory role in disassembly.

Data availability

The EM map associated with this paper has been deposited in the wwPDB under the accession number EMD-8944.

The following data sets were generated

1. Axel Brunger
2. Minglei Zhao
(2018) 20S supercomplex consisting of linked neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)
EMD-8944.

http://www.wwpdb.org/

Article and author information

Author details

Ucheor B Choi

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Minglei Zhao

Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, United States

Competing interests
No competing interests declared.
K Ian White

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Richard A Pfuetzner

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Luis Esquivies

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Qiangjun Zhou

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-1789-9588
Axel T Brunger

Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States

For correspondence
brunger@stanford.edu

Competing interests
Axel T Brunger, Reviewing editor, eLife.

"This ORCID iD identifies the author of this article:" 0000-0001-5121-2036

Funding

National Institutes of Health (R37MH63105)

Axel T Brunger

Howard Hughes Medical Institute

Axel T Brunger

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

Vivek Malhotra, The Barcelona Institute of Science and Technology, Spain

Version history

Received: March 8, 2018
Accepted: July 6, 2018
Accepted Manuscript published: July 9, 2018 (version 1)
Version of Record published: September 10, 2018 (version 2)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.