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SNARE disassembly requires Sec18/NSF side-loading.

Yousuf A Khan1,2,3,4, K Ian White1,2,3,4,5, Richard A Pfuetzner1,2,3,4,5

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Sec18/NSF (Sec18/N-ethylmaleimide-sensitive factor) disassembles SNARE protein complexes for membrane fusion. New structures reveal Sec18/NSF uses side-loading to handle topologically constrained SNAREs without unfolding their domains.

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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.