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bioRxiv · 10.1101/2024.08.30.610324

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts

Khan, Y. A.·White, K. I.·Pfuetzner, R. A.·Singal, B.·Esquivies, L.·Mckenzie, G.·Liu, F.·DeLong, K.·Choi, U.·Montabana, E.·Mclaughlin, T.·Wickner, W.·Brunger, A. T.
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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/-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--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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Khan, Y. A., White, K. I., Pfuetzner, R. A., Singal, B., Esquivies, L., Mckenzie, G., Liu, F., DeLong, K., Choi, U., Montabana, E., Mclaughlin, T., Wickner, W., Brunger, A. T.. 2024-09-01. Sec18 side-loading is essential for universal SNARE recycling across cellular contexts. https://doi.org/10.1101/2024.08.30.610324

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