SNARE disassembly requires Sec18/NSF side loading.

Area of Science:

• Cell biology
• Molecular machinery
• Membrane fusion

Background:

• SNARE proteins mediate membrane fusion by forming four-helix bundles.
• Sec18/NSF and Sec17/α-SNAP disassemble these bundles for recycling.
• Previous models faced topological challenges with SNARE transmembrane domains.

Purpose of the Study:

• To elucidate the mechanism of SNARE complex disassembly by Sec18/NSF.
• To resolve the topological constraints of SNARE threading through Sec18/NSF.
• To visualize Sec18/NSF and Sec17/α-SNAP interactions with SNAREs.

Main Methods:

• Cryo-electron microscopy (cryo-EM) of yeast SNARE complexes.
• Mass spectrometry to identify protein interactions.
• Structural analysis under non-hydrolyzing and hydrolyzing conditions.

Main Results:

• Cryo-EM structures show SNARE Sso1 threaded through Sec18/NSF's D1 and D2 ATPase rings.
• The N-terminal Habc domain of Sso1 interacts with the D2 ring and remains folded.
• Structures under hydrolyzing conditions reveal substrate release and ring opening.
• Sec18/NSF utilizes side loading and unloading for topologically constrained SNAREs.

Conclusions:

• Sec18/NSF disassembles SNAREs via a side-loading mechanism.
• The ATPase rings of Sec18/NSF open to accommodate and release SNARE substrates.
• This mechanism overcomes topological constraints posed by SNARE domains.