Author(s):
Houy, Sébastien ; Groffen, Alexander J ; Ziomkiewicz, Iwona ; Verhage, Matthijs ; Pinheiro, Paulo S. ; Sørensen, Jakob Balslev
Date: 2017
Persistent ID: https://hdl.handle.net/10316/108139
Origin: Estudo Geral - Universidade de Coimbra
Subject(s): adrenaline; capacitance measurements; chromaffin cell; exocytosis; membrane fusion; mouse; neuroscience; Animals; Calcium; Calcium-Binding Proteins; Cells, Cultured; Chromaffin Cells; Gene Expression; Gene Knockout Techniques; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Nerve Tissue Proteins; SNARE Proteins; Secretory Vesicles; Synaptotagmin I
Description
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.
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).
