Ca 2+ /calmodulin transfers the membrane-proximal lipid-binding domain of the v-SNARE synaptobrevin from cis to trans bilayers

• DOI: 10.1073/pnas.0303274101
• Publication Date: 2004-02-10T18:32:51Z
• Authors: Luc de Haro; Géraldine Ferracci; Sandrine Opi; Cécile Iborra; Stéphanie Quetglas; Raymond Miquelis; Christian Lévêque; Michael Seagar
• Keywords: Synaptobrevin

Soluble N -ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) interactions at the synaptic vesicle/plasma membrane interface play an essential role in neurotransmitter release. The membrane-proximal region (amino acids 77–90) of v-SNARE vesicle-associated 2 (VAMP 2, synaptobrevin) binds acidic phospholipids or Ca 2+ /calmodulin a mutually exclusive manner, processes that are required for -dependent exocytosis. To address mechanisms involved, we asked whether this VAMP can interact with cis (outer vesicle leaflet) and/or trans (inner plasma lipids. evaluate lipid binding, recombinant was reconstituted into liposomes and accessibility to site-directed antibodies probed by surface plasmon resonance. Data indicated domain dips bilayer, sequestering epitopes between tetanus toxin cleavage site anchor. These were unmasked double mutation W89A, W90A, which abolishes interactions. liposomes, then immobilized on beads. ability capture protein-free 3 H-labeled measured. When eliminated omitting negatively charged lipids, binding revealed. In contrast, when both contained headgroups (i.e., approximating physiological conditions), totally occluded binding. these conditions displaced inhibition, transferring lipid-binding from bilayer. Our results suggest calmodulin acts as unidirectional -activated shuttle docks juxtamembrane portion target prepare fusion.