SNARE disassembly requires Sec18/NSF side-loading.

Area of Science:

• Molecular biology
• Cellular biology
• Protein structure and function

Background:

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

Purpose of the Study:

• To elucidate the mechanism of Sec18/NSF in disassembling SNARE complexes.
• To resolve the topological constraints of SNARE substrate threading through Sec18/NSF.
• To visualize the structural states of Sec18/NSF during the disassembly cycle.

Main Methods:

• In vivo mass-spectrometry to identify protein interactions.
• Cryo-electron microscopy (Cryo-EM) to determine high-resolution structures.
• Structural analysis of Sec18/NSF-Sec17/α-SNAP-SNARE complexes in different functional states.

Main Results:

• N-terminal SNARE domains interact with Sec18/NSF, complicating threading.
• Cryo-EM structures show SNARE Sso1 threaded through both D1 and D2 ATPase rings of Sec18/NSF.
• The N-terminal Habc domain of Sso1 remains folded and interacts with the D2 ring.
• Structures under hydrolyzing conditions reveal substrate release via coordinated ring opening.

Conclusions:

• Sec18/NSF employs a side-loading mechanism to engage and disassemble topologically constrained SNARE substrates.
• The SNARE substrate does not need to unfold completely during disassembly.
• This mechanism allows for efficient recycling of SNARE proteins for subsequent membrane fusion events.