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Mechanism and function of synaptotagmin-mediated membrane apposition

Nature Structural & Molecular Biology volume 18, pages 813–821 (2011)Cite this article

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Abstract

Synaptotagmin-1 is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of this process were poorly understood. Here we show that synaptotagmin-1–mediated vesicle aggregation is driven by trans interactions between synaptotagmin-1 molecules bound to different membranes. We found a strong correlation between the ability of Ca2+-bound synaptotagmin-1 to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes—using non-synaptotagmin proteins—also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that synaptotagmin-1 drove the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion-competent heterodimers, independently of aggregation. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of fusion reactions that are regulated by Ca2+-bound synaptotagmin-1 and catalyzed by SNAREs.

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Figure 1: The cytoplasmic domain of synaptotagmin-1 aggregates phosphatidylserine-harboring vesicles in a Ca2+-dependent manner.
Figure 2: C2AB aggregates liposomes through a mechanism distinct from those of avidin-biotin and poly-D-lysine.
Figure 3: Mutational analysis of synaptotagmin-1–mediated membrane aggregation.
Figure 4: Mapping the membrane-binding interface of C2B using NBD fluorescence reporters.
Figure 5: FRET between NBD-labeled C2AB and rhodamine-labeled liposomes.
Figure 6: Synaptotagmin-1–mediated vesicle aggregation occurs through trans interactions.
Figure 7: The ability of synaptotagmin-1 to aggregate vesicles is correlated with its ability to stimulate fusion of v-SNARE vesicles with t-SNARE heterodimer vesicles.
Figure 8: Stimulation of SNARE-catalyzed fusion by other vesicle-aggregating proteins.
Figure 9: Ca2+-C2AB, but not other aggregating agents, accelerates fusion between syntaxin-bearing vesicles (SYXr) and Vr in the presence of soluble SNAP-25.

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Acknowledgements

We thank members of the Chapman laboratory for their discussions and comments. This work was supported by the Howard Hughes Medical Institute (E.R.C.), the US National Institutes of Health (NIH) National Institute of Mental Health grant MH061876 (to E.R.C.), and the NIH National Institutes on Deafness and Other Communication Disorders grant 1K99DC011267-01 (to C.P.J.).

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Author notes

  1. Enfu Hui

    Present address: Present address: Department of Molecular and Cellular Pharmacology, University of California, San Francisco, San Francisco, California, USA.,

  2. Enfu Hui and Jon D Gaffaney: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Neuroscience, University of Wisconsin, Madison, Wisconsin, USA

    Enfu Hui, Jon D Gaffaney, Zhao Wang, Colin P Johnson, Chantell S Evans & Edwin R Chapman

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Contributions

E.R.C. conceived of and supervised the project; E.H., J.D.G. and E.R.C. designed the experiments and wrote the manuscript; E.H. conducted all the aggregation assays on synaptotagmin-1 proteins and cPLA2-C2, membrane penetration assays and FRET-based membrane binding assays; J.D.G. conducted all the fusion assays on synaptotagmin-1 proteins and cPLA2-C2 and performed experiments for Figure 6 with Z.W.; Z.W. carried out experiments for Figure 9; C.P.J. conducted avidin-biotin–mediated aggregation and fusion assays in Figure 2d,i and Figure 8e–h; C.S.E. carried out experiments in Supplementary Figure 8.

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Correspondence to Edwin R Chapman.

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The authors declare no competing financial interests.

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Hui, E., Gaffaney, J., Wang, Z. et al. Mechanism and function of synaptotagmin-mediated membrane apposition. Nat Struct Mol Biol 18, 813–821 (2011). https://doi.org/10.1038/nsmb.2075

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