Mechanism and function of synaptotagmin-mediated membrane apposition (original) (raw)
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Reconstitution of Ca 2+ -Regulated Membrane Fusion by Synaptotagmin and SNAREs
Science, 2004
We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca 2+ , the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca 2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca 2+ -binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca 2+ -triggered exocytosis.
Nature Structural & Molecular Biology, 2007
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.
Structural Basis for the Clamping and Ca2+Activation of SNARE-mediated Fusion by Synaptotagmin
2019
Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to Ca2+-influx. To understand the underlying molecular mechanism, we determined the structure of the Syt1-SNARE complex on lipid membranes using cryo-electron microscopy. Under resting conditions, the Syt1 C2 domains adopt a novel membrane orientation with a Mg2+-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently binds the SNARE bundle via the ‘primary’ interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, allowing the attached SNAREpins to complete zippering and drive fusion. Overall, we find cation (Mg2+/Ca2+) dependent...
Molecular mechanism of the synaptotagmin–SNARE interaction in Ca2+-triggered vesicle fusion
Nature Structural & Molecular Biology, 2010
In neurons, SNAREs, synaptotagmin and other factors catalyze Ca2+-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process, especially the interaction between synaptotagmin and SNAREs, remains an enigma. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca2+-binding domains of synaptotagmin 3 (Syt3) undergo large relative motions in solution. Interaction with
Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion
The Journal of cell biology, 2011
The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca(2+) and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca(2+). In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3-9 min) that was required for subsequent Ca(2+)-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. T...
eLife
The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a s...
Analysis of the Synaptotagmin Family during Reconstituted Membrane Fusion
Journal of Biological Chemistry, 2008
Ca 2؉-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca 2؉-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca 2؉ , while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca 2؉. Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca 2؉-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.
Molecular biology of the cell, 2007
Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding...