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 JournalArticleAcademicpeer-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 languageEnglish
JournaleLife
Volume6
DOIs
Publication statusPublished - 23 Dec 2017

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Synaptotagmin I
SNARE Proteins
Calcium
Sensors
Synaptotagmins
Dyneins
Chromaffin Cells
Proteins
Cell membranes
Protein Binding
Aspartic Acid
Phospholipids
Binding Sites
Cell Membrane
Mutation

Keywords

  • Journal Article

Cite this

Houy, Sébastien ; Groffen, Alexander J ; Ziomkiewicz, Iwona ; Verhage, Matthijs ; Pinheiro, Paulo S ; Sørensen, Jakob Balslev. / Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs. In: eLife. 2017 ; Vol. 6.
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Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs. / Houy, Sébastien; Groffen, Alexander J; Ziomkiewicz, Iwona; Verhage, Matthijs; Pinheiro, Paulo S; Sørensen, Jakob Balslev.

In: eLife, Vol. 6, 23.12.2017.

Research output: Contribution to JournalArticleAcademicpeer-review

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AU - Houy, Sébastien

AU - Groffen, Alexander J

AU - Ziomkiewicz, Iwona

AU - Verhage, Matthijs

AU - Pinheiro, Paulo S

AU - Sørensen, Jakob Balslev

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AB - 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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