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

Ucheor B Choi,K Ian White,Minglei Zhao,Luis Esquivies,Richard A Pfuetzner,Qiangjun Zhou,Axel T Brunger

doi:10.7554/elife.36497

Ucheor B Choi, K Ian White + Show 5 more

Open Access

https://doi.org/10.7554/elife.36497

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Journal: eLife	Publication Date: Jul 9, 2018
Citations: 39	License type: CC BY 4.0

Affiliation: Stanford University, University of Chicago

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 s) and long-lived (≥0.32 s) disassembled states. The long-lived states represent fully disassembled SNARE complex, while the short-lived states correspond to failed disassembly or immediate reassembly. 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, implicating it 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, possibly differentially regulating cis and trans SNARE complexes disassembly.

Highlights

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential for cellular processes including membrane trafficking, neurotransmitter release, and hormone secretion (Rothman, 2014; Sudhof, 2013; Wickner and Schekman, 2008)
In addition to N-ethylmaleimide-sensitive factor (NSF) and SNAPs, several proteins interact with SNAREs, including complexin (Cpx – we primarily focus on the complexin-1 isoform), synaptotagmin-1 (Zhou et al, 2017), Munc18 (Misura et al, 2000), and Munc13 (Lai et al, 2017)
We expressed synthetically-linked SNARE complex (L-SNARE) as a single polypeptide with all endogenous cysteines mutated to serines (Gao et al, 2012; Zorman et al, 2014) (Figure 1A) and introduced specific cysteine mutations for labeling purposes (Materials and methods)

Summary

Introduction

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential for cellular processes including membrane trafficking, neurotransmitter release, and hormone secretion (Rothman, 2014; Sudhof, 2013; Wickner and Schekman, 2008). The cis SNARE complex is disassembled by the ATPase N-ethylmaleimide-sensitive factor (NSF) (Block et al, 1988; Malhotra et al, 1988) in combination with SNAP (soluble NSF attachment protein) adapter proteins (Weidman et al, 1989). Prior to ATP hydrolysis and together with SNAPs and the SNARE complex, NSF forms the so-called 20S complex; the structural details of the starting point for this process have been described previously (Zhao et al, 2015; White et al, 2018).

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