Ca2+-dependent release of Synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes

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Abstract

The Ca²⁺ sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving polybasic, primary and tripartite interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca²⁺-free synaptotagmin-1 binds to SNARE complexes anchored on PIP₂-containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca²⁺ induces tight binding of synaptotagmin-1 to PIP₂-containing nanodiscs, disrupting synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca²⁺-dependent synaptotagmin-1-PIP₂-membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca²⁺ releases this interaction, inducing PIP₂/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release.

Data availability

The NMR data corresponding to Figs. 1-4 and corresponding figure supplements are publicly available at https://doi.org/10.5061/dryad.0zpc866vt. Source data are provided for all the FRET experiments shown in Figs. 5-9 and corresponding figure supplements.

The following data sets were generated

(2020) NMR data in 'Ca2+-dependent release of Synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphatecontaining membranes' by Voleti, Jaczynska and Rizo, eLife 2020
Dryad Digital Repository, doi:10.5061/dryad.0zpc866vt.

https://doi.org/10.5061/dryad.0zpc866vt

Article and author information

Author details

Rashmi Voleti

Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

Competing interests
The authors declare that no competing interests exist.
Klaudia Jaczynska

Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

Competing interests
The authors declare that no competing interests exist.
Josep Rizo

Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

For correspondence
Jose.Rizo-Rey@UTSouthwestern.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-1773-8311

Funding

National Institute of Neurological Disorders and Stroke (R35 NS097333)

Josep Rizo

Welch Foundation (I-1304)

Josep Rizo

Howard Hughes Medical Institute

Rashmi Voleti

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.