
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.30.610324
preprint
1
Article
Sec18 side-loading is essential for universal SNARE recycling across cellular contexts
Khan Yousuf A. http://orcid.org/0000-0003-0201-2796

White K. Ian
Pfuetzner Richard A.
Singal Bharti
Esquivies Luis
Mckenzie Garvey
Liu Fang
DeLong Katherine
Choi Uchoer B.
Montabana Elizabeth
Mclaughlin Theresa
Wickner William T.
Brunger Axel T. http://orcid.org/0000-0001-5121-2036

01 9 2024
2024.08.30.610324https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.30.610324
nihpp-2024.08.30.610324.pdf
Summary

SNARE proteins drive membrane fusion as their core domains zipper into a parallel four-helix bundle 1,2 . After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/ α-SNAP 3,4 to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains 5 . Previous structures of the NSF–α-SNAP–SNARE complex revealed SNARE domain threaded through the D1 ATPase ring 6 , posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems 7 . Here, in vivo mass-spectrometry reveals N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. Cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition shows SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.
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pmc
