NSF-mediated disassembly of on and off-pathway SNARE complexes and inhibition by complexin
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.
Article and author information
Author details
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.
Copyright
© 2018, Choi et al.
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.
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