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Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

View ORCID ProfileManindra Bera, View ORCID ProfileSathish Ramakrishnan, Jeff Coleman, View ORCID ProfileShyam S. Krishnakumar, James E. Rothman
doi: https://doi.org/10.1101/2021.07.05.451112
Manindra Bera
1Departments of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
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Sathish Ramakrishnan
1Departments of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
2Department of Pathology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
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Jeff Coleman
1Departments of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
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Shyam S. Krishnakumar
1Departments of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
3Department of Neurology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
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  • For correspondence: shyam.krishnakumar@yale.edu james.rothman@yale.edu
James E. Rothman
1Departments of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
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  • For correspondence: shyam.krishnakumar@yale.edu james.rothman@yale.edu
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ABSTRACT

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 05, 2021.
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Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion
Manindra Bera, Sathish Ramakrishnan, Jeff Coleman, Shyam S. Krishnakumar, James E. Rothman
bioRxiv 2021.07.05.451112; doi: https://doi.org/10.1101/2021.07.05.451112
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Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion
Manindra Bera, Sathish Ramakrishnan, Jeff Coleman, Shyam S. Krishnakumar, James E. Rothman
bioRxiv 2021.07.05.451112; doi: https://doi.org/10.1101/2021.07.05.451112

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