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Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+

Nature Structural & Molecular Biology volume 15, pages 827–835 (2008)Cite this article

Abstract

Neuronal communication is mediated by Ca2+-triggered fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. Synaptotagmin I functions as a Ca2+ sensor that regulates exocytosis, whereas soluble N-ethylmaleimide–sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in the vesicle and target membrane assemble into complexes that directly catalyze bilayer fusion. Here we report that, before the Ca2+ trigger, synaptotagmin interacts with SNARE proteins in the target membrane to halt SNARE complex assembly at a step after donor vesicles attach, or dock, to target membranes. This results in fusion complexes that, when subsequently triggered by Ca2+, drive rapid, highly efficient lipid mixing. Ca2+-independent interactions with SNAREs also predispose synaptotagmin to selectively penetrate the target membrane in response to Ca2+; we demonstrate that Ca2+–synaptotagmin must insert into the target membrane to accelerate SNARE-catalyzed fusion. These findings demonstrate that Ca2+ converts synaptotagmin from a clamp to a trigger for exocytosis.

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Figure 1: Reconstitution of rapid, efficient, Ca2+-triggered membrane fusion.
Figure 2: Functional trans-SNARE pairing is arrested by apo-syt.
Figure 3: Apo-syt enables docking of multiple vesicles by arresting fusion; Ca2+–syt then triggers synchronous fusion.
Figure 4: Syt and the Vc peptide synergize to increase the rate of SNARE-catalyzed fusion.
Figure 5: Dual function of syt in the regulation of SNARE-catalyzed fusion.
Figure 6: Syt must act on t-SNARE membranes to stimulate fusion.
Figure 7: t-SNAREs predispose syt to penetrate the target membrane in response to the Ca2+ signal.

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Acknowledgements

We thank members of the Chapman laboratory, W. Tucker, J.M. Edwardson and M. Jackson for helpful discussions. This work was supported by the Howard Hughes Medical Institute and by grants from the American Heart Association and US National Institutes of Health.

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Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Physiology, University of Wisconsin, Madison, 1300 University Avenue, SMI 129, Madison, 53706, Wisconsin, USA

    Michael C Chicka, Enfu Hui, Huisheng Liu & Edwin R Chapman

  2. Graduate Program in Cellular and Molecular Biology, University of Wisconsin, Madison, 1300 University Avenue, SMI 129, Madison, 53706, Wisconsin, USA

    Michael C Chicka

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Contributions

M.C.C. carried out all biochemical experiments and fusion assays, and, together with E.R.C., planned all experiments and wrote the manuscript; E.H. carried out the fluorescence experiments in Figure 7; and H.L. carried out the electrophysiology and neuronal imaging experiments shown in Figure 5d–g and Supplementary Figure 3.

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Correspondence to Edwin R Chapman.

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Chicka, M., Hui, E., Liu, H. et al. Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+. Nat Struct Mol Biol 15, 827–835 (2008). https://doi.org/10.1038/nsmb.1463

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