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Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids

Nature Structural & Molecular Biology volume 13, pages 209–217 (2006)Cite this article

Abstract

Synaptotagmin acts as a Ca2+ sensor in neurotransmitter release through its two C2 domains. Ca2+-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca2+ binding to the C2B domain is more crucial for release than Ca2+ binding to the C2A domain. Here we show that Ca2+ induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C2B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C2B domain.

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Figure 1: The synaptotagmin-1 C2 domains are flexibly linked.
Figure 2: Analysis of Ca2+-dependent synaptotagmin-1–phospholipid interactions by NBD fluorescence.
Figure 3: The synaptotagmin-1 C2AB fragment does not oligomerize on phospholipid vesicles.
Figure 4: Synaptotagmin-1 clusters phospholipid vesicles.
Figure 5: The C2AB fragment brings two membranes into close proximity.
Figure 6: Proposed model of Ca2+-triggered neurotransmitter release.

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Acknowledgements

We thank T. Kodadek and L. Burdine for assistance with the cross-linking experiments, M. Schmid for assistance on the three-dimensional tomographic reconstruction and N. Mizuno and Z. Metlagel for help with the negative-staining experiments. This work was supported by grants from the Welch Foundation (to A.E.J. and J.R.) and by US National Institutes of Health grants P41RR02250 (to W.C.), GM26494 (to A.E.J.) and NS40944 (to J.R.).

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Authors and Affiliations

  1. Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Demet Araç, Xiaocheng Chen, Josep Ubach & Josep Rizo

  2. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    Htet A Khant, Steven J Ludtke & Wah Chiu

  3. Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Masahide Kikkawa

  4. Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, 77843, Texas, USA

    Arthur E Johnson

  5. Department of Molecular Genetics and Howard Hughes Medical Institute, Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Thomas C Südhof

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Supplementary information

Supplementary Fig. 1

Problems with labeling C277 of synaptotagmin 1 (PDF 690 kb)

Supplementary Fig. 2

Reconstruction of the oligomeric model of synaptotagmin 1 (PDF 618 kb)

Supplementary Fig. 3

Negative stain em analysis of synpatotagmin/calcium/lipid samples (PDF 1135 kb)

Supplementary Fig. 4

Synaptotagmin does not oligomerize (PDF 226 kb)

Supplementary Fig. 5

Calcium dependence of synaptotagmin induced vesicle clustering (PDF 77 kb)

Supplementary Fig. 6

Diverse considerations supporting the model of Figure 6 (PDF 256 kb)

Supplementary Video 1

Tomographic 3D reconstruction of a vesicle cluster (MPG 2310 kb)

Supplementary Methods (PDF 16 kb)

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Araç, D., Chen, X., Khant, H. et al. Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nat Struct Mol Biol 13, 209–217 (2006). https://doi.org/10.1038/nsmb1056

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