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

• Main Authors: Sébastien Houy, Alexander J Groffen, Iwona Ziomkiewicz, Matthijs Verhage, Paulo S Pinheiro, Jakob Balslev Sørensen
• Format: Article
• Language: English
• Published: eLife Sciences Publications Ltd 2017-12-01
• Series: eLife
• Subjects: chromaffin cell; capacitance measurements; exocytosis; adrenaline; membrane fusion
• Online Access: https://elifesciences.org/articles/27000

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.