Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• SNARE proteins mediate membrane fusion by forming parallel helical bundles.
• N-ethylmaleimide-sensitive factor (NSF), an AAA+ ATPase, disassembles these complexes post-fusion.

Purpose of the Study:

• To investigate the mechanism by which NSF disassembles SNARE proteins.
• To correlate NSF's disassembly rate with its ATPase activity.

Main Methods:

• Real-time fluorescence dequenching assay to monitor SNARE disassembly.
• Measurement of NSF's ATPase activity in the presence of SNAREs.
• Substrate specificity studies using truncated and engineered SNARE complexes.

Main Results:

• NSF disassembles neuronal SNARE complexes at a rate approximately 26-fold higher than its basal ATPase rate.
• Disassembly of one SNARE complex takes ~5 seconds and consumes ~50 ATP.
• NSF disassembles truncated SNARE domains and engineered double-length complexes, but not unrelated coiled-coils.
• Single-turnover experiments indicate SNAREs can be unwound in a single NSF encounter.

Conclusions:

• NSF employs a processive, helicase-like unwinding mechanism to disassemble SNARE proteins.
• Approximately one residue is unwound per hydrolyzed ATP molecule during disassembly.