Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion
- European Molecular Biology Laboratory, Germany
- University of Texas Southwestern Medical Center, United States
- University of Toronto, Canada
Abstract
Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the 'furled conformation' of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in C. elegans unc-18 nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.
Article and author information
Author details
Funding
National Institutes of Health (R35 NS097333)
- Josep Rizo
Welch Foundation (I-1304)
- Josep Rizo
Canadian Institute of Health Research (MOP-130573)
- Shuzo Sugita
National Institutes of Health (S10OD018027)
- Josep Rizo
National Institutes of Health (S10RR026461)
- Josep Rizo
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Axel T Brunger, Stanford University Medical Center, United States
Version history
- Received: December 15, 2016
- Accepted: May 4, 2017
- Accepted Manuscript published: May 6, 2017 (version 1)
- Version of Record published: June 8, 2017 (version 2)
Copyright
© 2017, Sitarska 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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Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion
eLife 6:e24278.
https://doi.org/10.7554/eLife.24278
Further reading
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Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.
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