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

Sébastien Houy; Alexander J Groffen; Iwona Ziomkiewicz; Matthijs Verhage; Paulo S Pinheiro; Jakob Balslev Sørensen

doi:10.7554/eLife.27000

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

Sébastien Houy, Alexander J Groffen, Iwona Ziomkiewicz, Matthijs Verhage, Paulo S Pinheiro, Jakob Balslev Sørensen

Research output: Contribution to Journal › Article › Academic › peer-review

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.

Original language	English
Article number	e27000
Journal	eLife
Volume	6
DOIs	https://doi.org/10.7554/eLife.27000
Publication status	Published - 23 Dec 2017

Funding

We would like to thank Anne-Marie Nordvig Petersen for expert technical assistance. This investigation was supported by the The Lundbeck Foundation (to JBS), the Novo Nordic Foundation (JBS), the Danish Medical Research Council (JBS and SH), and the European Research Council (ERC-ADG-322966-DCVfusion, to MV). We would like to thank Anne-Marie Nordvig Petersen for expert technical assistance. This investiga-tion was supported by the The Lundbeck Foundation (to JBS), the Novo Nordic Foundation (JBS), the Danish Medical Research Council (JBS and SH), and the European Research Council (ERC-ADG-322966-DCVfusion, to MV). Danish Medical Research Council Sébastien Houy Jakob Balslev Sørensen European Research Council ERC-ADG-322966-DCVfusion Matthijs Verhage Lundbeckfonden Jakob Balslev Sørensen Novo Nordisk Foundation Jakob Balslev Sørensen University of Copenhagen 2016 (KU2016) excellence program Jakob Balslev Sørensen The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funders	Funder number
Sundhed og Sygdom, Det Frie Forskningsråd
Seventh Framework Programme	322966
European Research Council
Københavns Universitet	2016 (KU2016)
Lundbeckfonden
Novo Nordisk
Novo Nordisk Fonden

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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.",

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AU - Verhage, Matthijs

AU - Pinheiro, Paulo S

AU - Sørensen, Jakob Balslev

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

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