Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion

  1. Ewa Sitarska
  2. Junjie Xu
  3. Seungmee Park
  4. Xiaoxia Liu
  5. Bradley Quade
  6. Karolina Stepien
  7. Kyoko Sugita
  8. Chad A Brautigam
  9. Shuzo Sugita
  10. Josep Rizo  Is a corresponding author
  1. European Molecular Biology Laboratory, Germany
  2. University of Texas Southwestern Medical Center, United States
  3. 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

  1. Ewa Sitarska

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Junjie Xu

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Seungmee Park

    Department of Physiology, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  4. Xiaoxia Liu

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Bradley Quade

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Karolina Stepien

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Kyoko Sugita

    Department of Physiology, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  8. Chad A Brautigam

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Shuzo Sugita

    Department of Physiology, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9182-873X
  10. Josep Rizo

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    Jose.Rizo-Rey@UTSouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1773-8311

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

  1. Axel T Brunger, Stanford University Medical Center, United States

Version history

  1. Received: December 15, 2016
  2. Accepted: May 4, 2017
  3. Accepted Manuscript published: May 6, 2017 (version 1)
  4. 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.

Metrics

  • 1,912
    Page views
  • 520
    Downloads
  • 60
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Ewa Sitarska
  2. Junjie Xu
  3. Seungmee Park
  4. Xiaoxia Liu
  5. Bradley Quade
  6. Karolina Stepien
  7. Kyoko Sugita
  8. Chad A Brautigam
  9. Shuzo Sugita
  10. Josep Rizo
(2017)
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

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics
    Bronwyn A Lucas, Benjamin A Himes, Nikolaus Grigorieff
    Research Advance

    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.

    1. Plant Biology
    2. Structural Biology and Molecular Biophysics
    Jinping Lu, Ingo Dreyer ... Rainer Hedrich
    Research Article

    To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca2+. In our search for species-dependent functional TPC1 channel variants with different luminal Ca2+ sensitivity, we found in total three acidic residues present in Ca2+ sensor sites 2 and 3 of the Ca2+-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca2+. When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca2+ sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca2+ sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.