Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids

Abstract

Synaptotagmin-1 is the calcium sensor for neuronal exocytosis, but the mechanism by which it triggers membrane fusion is not fully understood. Here we show that synaptotagmin accelerates SNARE-dependent fusion of liposomes by interacting with neuronal Q-SNARES in a Ca 2+ -independent manner. Ca 2+ -dependent binding of synaptotagmin to its own membrane impedes the activation. Preventing this cis interaction allows Ca 2+ to trigger synaptotagmin binding in trans, accelerating fusion. However, when an activated SNARE acceptor complex is used, synaptotagmin has no effect on fusion kinetics, suggesting that synaptotagmin operates upstream of SNARE assembly in this system. Our results resolve major discrepancies concerning the effects of full-length synaptotagmin and its C2AB fragment on liposome fusion and shed new light on the interactions of synaptotagmin with SNAREs and membranes. However, our findings also show that the action of synaptotagmin on the fusionarrested state of docked vesicles in vivo is not fully reproduced in vitro.Neurotransmitters are stored in synaptic vesicles that undergo Ca 2+ -dependent exocytosis upon stimulation. Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins, including synaptobrevin-2 (also referred to as VAMP2) in the membrane of synaptic vesicles and syntaxin-1 and SNAP-25 in the plasma membrane. SNAREs are characterized by conserved stretches of 60-70 amino acid residues, referred to as SNARE motifs. Syntaxin-1 and synaptobrevin-2 each possess a single SNARE motif adjacent to the C-terminal transmembrane domain, whereas SNAP-25 contains two SNARE motifs that are separated by a palmitoylated linker 1,2 . The SNARE motifs are unstructured as monomers 3 but assemble into a tight bundle of four a-helices 4 . SNARE motifs are divided into four conserved subfamilies, referred to as Qa-, Qb-, Qc-and R-SNARE motifs. Each SNARE complex contains one member of each subfamily 5 . The assembly of SNARE complexes is currently believed to be the essential reaction in driving membrane fusion. According to this model, the formation of the SNARE complex is initiated in a trans configuration at the N-terminal ends of the SNARE motifs, forming a bridge between the membranes. Assembly then proceeds toward the C-terminal membrane anchor domains, clamping the membranes together and thus overcoming the energy barrier for fusion [6][7][8] .In contrast to several other SNARE-dependent fusion reactions, neuronal exocytosis is strongly upregulated by calcium 9 . The fast component of Ca 2+ -dependent release, which is essential for synchronous, action potential-coupled release, is mediated by the proteins synaptotagmin-1, synaptotagmin-2 and probably synaptotagmin-9, which reside in the membrane of synaptic vesicles 2 . Synaptotagmins constitute a family of type I membrane proteins with widespread tissue distribution 10 . The cytoplasmic part of the synaptotagmins contains t...

Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

“…To dissect the effect of 5-IP 7 in the regulation of synaptic vesicle exocytosis, we used an in vitro vesicle fusion system that allowed monitoring of SNARE-and Syt1-mediated membrane fusion down to the single-vesicle level (34)(35)(36)(37)(38)(39)(40)(41) (Fig. 2).…”

The Ca2+ Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate

“…Expression constructs of the isolated C2A domain (aa 97-273), the C2B domain (aa 262-421), the soluble domain of synaptotagmin (aa 97-421), and of the full-length protein (aa 1-421) have been described before (24). Also the following calcium mutants of the full-length protein and of the soluble domain have been described earlier (24): C2a*B (D178A, D230A, and D232A), C2Ab* (D309A, D363A, and D365A), and C2a*b* (D178A, D230A, D232A, D309A, D363A, and D365A). The constructs for the neuronal SNAREs were the SNARE motif of syntaxin 1A with its transmembrane domain (aa 183-288), a cysteine-free variant of SNAP-25A (aa 1-206), and fulllength synaptobrevin 2 (aa 1-116).…”

SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes

“…Fusion was monitored over a time of B60 min, showing consistency with earlier kinetic studies of SNARE-mediated fusion (Fig. 1d) 14 .…”