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Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs

  1. Sébastien Houy
  2. Alexander J Groffen
  3. Iwona Ziomkiewicz
  4. Matthijs Verhage
  5. Paulo S Pinheiro
  6. Jakob Balslev Sørensen  Is a corresponding author
  1. University of Copenhagen, Denmark
  2. Vrije Universiteit Amsterdam, Netherlands
Research Article
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Cite this article as: eLife 2017;6:e27000 doi: 10.7554/eLife.27000

Abstract

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.

Article and author information

Author details

  1. Sébastien Houy

    Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
  2. Alexander J Groffen

    Department of Functional Genomics, Vrije Universiteit Amsterdam, Amsterdam, Netherlands
    Competing interests
    No competing interests declared.
  3. Iwona Ziomkiewicz

    Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    Iwona Ziomkiewicz, Performed experiments as an employee of University of Copenhagen and is now an employee of AstraZeneca. Has no financial investments in AstraZeneca.
  4. Matthijs Verhage

    Department of Functional Genomics, Vrije Universiteit Amsterdam, Amsterdam, Netherlands
    Competing interests
    No competing interests declared.
  5. Paulo S Pinheiro

    Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
  6. Jakob Balslev Sørensen

    Department of Neuroscience, University of Copenhagen, Copenhagen, Denmark
    For correspondence
    jakobbs@sund.ku.dk
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5465-3769

Funding

Lundbeckfonden

  • Jakob Balslev Sørensen

Novo Nordisk Foundation

  • Jakob Balslev Sørensen

Danish Medical Research Council

  • Sébastien Houy

European Research Council (ERC-ADG-322966-DCVfusion)

  • Matthijs Verhage

Danish Medical Research Council

  • Jakob Balslev Sørensen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Permission to keep and breed knockout mice for this study was obtained fromThe Danish Animal Experiments Inspectorate (2006/562−43, 2012−15−2935−00001). The animals were maintained in an AAALAC-accredited stable in accordance with institutional guidelines as overseenby the Institutional Animal Care and Use Committee (IACUC).

Reviewing Editor

  1. Christian Rosenmund, Charité-Universitätsmedizin Berlin, Germany

Publication history

  1. Received: March 20, 2017
  2. Accepted: December 21, 2017
  3. Accepted Manuscript published: December 23, 2017 (version 1)
  4. Version of Record published: January 8, 2018 (version 2)

Copyright

© 2017, Houy 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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