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PreprintCited by 9Cellular transport and secretionATP Synthase and ATPases ResearchMicrotubule and mitosis dynamics

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SNARE disassembly requires Sec18/NSF side-loading

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Khan, Yousuf A., White, K. Ian, Pfuetzner, Richard A., Singal, Bharti, Esquivies, Luis, Mckenzie, Garvey, Liu, Fang, DeLong, Katherine, Choi, Ucheor B., Montabana, Elizabeth, Mclaughlin, Theresa, Wickner, William T., Brunger, Axel T.·2024·Nature Structural & Molecular Biology
DOI 10.1101/2024.08.30.610324PMC PMC12440825

Preprint — published as 10.1038/s41594-025-01590-w

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Abstract

SNARE proteins drive membrane fusion at different cell compartments as their core domains zipper into a parallel four-helix bundle. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/α-SNAP to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains. Previous structures of the NSF–α-SNAP–SNARE complex revealed binding to the D1 ATPase pore, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems. Using mass-spectrometry in yeast cells, we show N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. We present cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition, which show 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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Metadata

Authors
Khan, Y. A., White, K. I., Pfuetzner, R. A., Singal, B., Esquivies, L., Mckenzie, G., Liu, F., DeLong, K., Choi, U. B., Montabana, E., Mclaughlin, T., Wickner, W. T., & Brunger, A. T.
Affiliations
  • Stanford University (US)
  • Howard Hughes Medical Institute (US)
  • Stanford Medicine (US)
  • Dartmouth College (US)
Corresponding author
Khan, Yousuf A.
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
openRxiv
Published
2024-09-01
Volume
32
Issue
9
Pages
1708-1720
ISSN
1545-9985 (ISSN-L)
DOI
10.1101/2024.08.30.610324
PMC
PMC12440825
OpenAlex
W4411924662
Cited by
9
References
1
Citations by year
2026: 8 · 2025: 1
FWCI
4.49
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Structural & Molecular Biology (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • National Science Foundation
  • Howard Hughes Medical Institute
  • National Institutes of Health (1s10od030473-01, NIH P30 CA124435, RO1-MH63105)
  • National Institute of Mental Health (RO1MH63105, R01MH063105, F31MH134477)
  • National Cancer Institute (P30CA124435)
  • National Institute of General Medical Sciences (R35GM118037)
  • Stanford Cancer Institute (P30CA124435)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
Keywords
SNARE proteins, membrane fusion, AAA+ ATPase, lateral loading, yeast, cryo-EM structure, ATP hydrolysis

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