A Stimulation Function of Synaptotagmin-1 in Ternary SNARE Complex Formation Dependent on Munc18 and Munc13.

Yun Li,Yuanyuan Xu,Shen Wang,Tianzhi Li,Le Zhu,Cong Ma

doi:10.3389/fnmol.2017.00256

Yun Li, Yuanyuan Xu + Show 4 more

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https://doi.org/10.3389/fnmol.2017.00256

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Abstract

The Ca2+ sensor synaptotagmin-1 (Syt1) plays an essential function in synaptic exocytosis. Recently, Syt1 has been implicated in synaptic vesicle priming, a maturation step prior to Ca2+-triggered membrane fusion that is believed to involve formation of the ternary SNARE complex and require priming proteins Munc18-1 and Munc13-1. However, the mechanisms of Syt1 in synaptic vesicle priming are still unclear. In this study, we found that Syt1 stimulates the transition from the Munc18-1/syntaxin-1 complex to the ternary SNARE complex catalyzed by Munc13-1. This stimulation can be further enhanced in a membrane-containing environment. Further, we showed that Syt1, together with Munc18-1 and Munc13-1, stimulates trans ternary SNARE complex formation on membranes in a manner resistant to disassembly factors NSF and α-SNAP. Disruption of a proposed Syt1/SNARE binding interface strongly abrogated the stimulation function of Syt1. Our results suggest that binding of Syt1 to an intermediate SNARE assembly with Munc18-1 and Munc13-1 is critical for the stimulation function of Syt1 in ternary SNARE complex formation, and this stimulation may underlie the priming function of Syt1 in synaptic exocytosis.

Highlights

Neurotransmitter release by synaptic exocytosis is accomplished by the fusion of synaptic vesicles to the plasma membrane upon Ca2+ influx into the nerve terminal (Südhof, 2004; Rizo and Rosenmund, 2008)
Fluorescence donor and acceptor were separately labeled to synaptobrevin-2 and SNAP-25 (S187C), respectively, and ternary SNARE complex formation was measured by a decrease of donor fluorescence based on fluorescence resonance energy transfer (FRET) (Figure 1A)
C2AB did not affect formation of synaptobrevin-2 with preassembled syntaxin-1/SNAP-25 heterodimers in the absence and presence of Ca2+ (Figures 1B,C). These results are consistent with previous observations (Stein et al, 2007; Chicka et al, 2008), suggesting that Syt1 has no obvious effect on ternary SNARE complex formation when beginning merely with the three SNAREs

Summary

Introduction

Neurotransmitter release by synaptic exocytosis is accomplished by the fusion of synaptic vesicles to the plasma membrane upon Ca2+ influx into the nerve terminal (Südhof, 2004; Rizo and Rosenmund, 2008). In contrast to most intracellular membrane fusion processes, synaptic vesicle fusion occurs in a sub-millisecond timescale in response to Ca2+ (Augustine et al, 1987; Südhof, 2013) To achieve this goal, most of synaptic vesicles undergo a series of maturation steps before Ca2+-triggered fast fusion, which include: (i) ‘‘tethering’’: recruitment of synaptic vesicles to specialized sites at the presynaptic membrane called active zones (Pfeffer, 1999); (ii) ‘‘docking’’: close attachment of synaptic vesicles to the fusion sites (Schimmöller et al, 1998); and (iii) ‘‘priming’’ that renders the docked vesicles in a semi-stable state ready for fast membrane fusion (Klenchin and Martin, 2000). Full assembly of the ternary SNARE complex towards the C-terminal membrane anchors coincides with Ca2+-triggered membrane fusion and this step is highly regulated by neuronal specific proteins such as synaptotagmin-1 (Syt1) and complexins (Südhof and Rothman, 2009; Südhof, 2013; Rizo and Xu, 2015)

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