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

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.