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

Cipriano DJ, Jung J, Vivona S, Fenn TD, Brunger AT, Bryant Z - J. Biol. Chem. (2013)

Bottom Line: NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism.We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF.We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.

ABSTRACT
SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

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Single-turnover burst kinetics of NSF-driven SNARE disassembly. 20S complex was formed by premixing Oregon Green 488-labeled SNARE complex with a 1.5-fold excess of NSF hexamer and 5-fold excess of α-SNAP in the presence of ATP and 1 mm EDTA. A 50-fold excess of unlabeled SNARE was added as a trap, either before or after the initial 20S complex formation as indicated. At the time indicated by the arrow, the disassembly reaction was started by the addition of MgCl2 to 5 mm. Black, no trap added; red, trap added before NSF; green, purple, and blue, trap added after 20S complex formation. 20S complex was formed by incubation at 37 °C for 5 min with 188 nm labeled SNARE, 281 nm NSF hexamer, and 938 nm α-SNAP (green); 375 nm labeled SNARE, 563 nm NSF hexamer, and 1875 nm α-SNAP (purple); or 750 nm labeled SNARE, 1.1 μm NSF hexamer, and 3.8 μm α-SNAP (blue).
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Figure 4: Single-turnover burst kinetics of NSF-driven SNARE disassembly. 20S complex was formed by premixing Oregon Green 488-labeled SNARE complex with a 1.5-fold excess of NSF hexamer and 5-fold excess of α-SNAP in the presence of ATP and 1 mm EDTA. A 50-fold excess of unlabeled SNARE was added as a trap, either before or after the initial 20S complex formation as indicated. At the time indicated by the arrow, the disassembly reaction was started by the addition of MgCl2 to 5 mm. Black, no trap added; red, trap added before NSF; green, purple, and blue, trap added after 20S complex formation. 20S complex was formed by incubation at 37 °C for 5 min with 188 nm labeled SNARE, 281 nm NSF hexamer, and 938 nm α-SNAP (green); 375 nm labeled SNARE, 563 nm NSF hexamer, and 1875 nm α-SNAP (purple); or 750 nm labeled SNARE, 1.1 μm NSF hexamer, and 3.8 μm α-SNAP (blue).

Mentions: For single-turnover NSF assays, we preincubated labeled SNARE complex with a 1.5-fold molar access of NSF and a 5-fold molar excess of α-SNAP in the presence of ATP and EDTA. Under these conditions, we expect all of the labeled SNAREs should form the 20S complex with NSF and α-SNAP.4 The samples were then diluted into buffer containing a 50-fold excess of unlabeled SNAREs (dark trap), and the reaction was started by the addition of excess Mg2+. If a single NSF can disassemble a SNARE complex without dissociation and rebinding to another SNARE complex, we expect to see a rapid burst in the fluorescent signal. If NSF is infinitely processive, then we expect this burst to go to completion as all the SNARES should be disassembled during the burst phase. If NSF is moderately processive, then only a population of the 20S complexes will be disassembled during the burst. In this case, the burst magnitude will be proportional to the fraction of molecules disassembled and tells us the probability that a single NSF binding event results in disassembly. The NSF that dissociates is 50 times more likely to bind an unlabeled SNARE and contributes only to an overall upward creep that is easily distinguishable from the burst magnitude (Fig. 4, red line).


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

Cipriano DJ, Jung J, Vivona S, Fenn TD, Brunger AT, Bryant Z - J. Biol. Chem. (2013)

Single-turnover burst kinetics of NSF-driven SNARE disassembly. 20S complex was formed by premixing Oregon Green 488-labeled SNARE complex with a 1.5-fold excess of NSF hexamer and 5-fold excess of α-SNAP in the presence of ATP and 1 mm EDTA. A 50-fold excess of unlabeled SNARE was added as a trap, either before or after the initial 20S complex formation as indicated. At the time indicated by the arrow, the disassembly reaction was started by the addition of MgCl2 to 5 mm. Black, no trap added; red, trap added before NSF; green, purple, and blue, trap added after 20S complex formation. 20S complex was formed by incubation at 37 °C for 5 min with 188 nm labeled SNARE, 281 nm NSF hexamer, and 938 nm α-SNAP (green); 375 nm labeled SNARE, 563 nm NSF hexamer, and 1875 nm α-SNAP (purple); or 750 nm labeled SNARE, 1.1 μm NSF hexamer, and 3.8 μm α-SNAP (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520572&req=5

Figure 4: Single-turnover burst kinetics of NSF-driven SNARE disassembly. 20S complex was formed by premixing Oregon Green 488-labeled SNARE complex with a 1.5-fold excess of NSF hexamer and 5-fold excess of α-SNAP in the presence of ATP and 1 mm EDTA. A 50-fold excess of unlabeled SNARE was added as a trap, either before or after the initial 20S complex formation as indicated. At the time indicated by the arrow, the disassembly reaction was started by the addition of MgCl2 to 5 mm. Black, no trap added; red, trap added before NSF; green, purple, and blue, trap added after 20S complex formation. 20S complex was formed by incubation at 37 °C for 5 min with 188 nm labeled SNARE, 281 nm NSF hexamer, and 938 nm α-SNAP (green); 375 nm labeled SNARE, 563 nm NSF hexamer, and 1875 nm α-SNAP (purple); or 750 nm labeled SNARE, 1.1 μm NSF hexamer, and 3.8 μm α-SNAP (blue).
Mentions: For single-turnover NSF assays, we preincubated labeled SNARE complex with a 1.5-fold molar access of NSF and a 5-fold molar excess of α-SNAP in the presence of ATP and EDTA. Under these conditions, we expect all of the labeled SNAREs should form the 20S complex with NSF and α-SNAP.4 The samples were then diluted into buffer containing a 50-fold excess of unlabeled SNAREs (dark trap), and the reaction was started by the addition of excess Mg2+. If a single NSF can disassemble a SNARE complex without dissociation and rebinding to another SNARE complex, we expect to see a rapid burst in the fluorescent signal. If NSF is infinitely processive, then we expect this burst to go to completion as all the SNARES should be disassembled during the burst phase. If NSF is moderately processive, then only a population of the 20S complexes will be disassembled during the burst. In this case, the burst magnitude will be proportional to the fraction of molecules disassembled and tells us the probability that a single NSF binding event results in disassembly. The NSF that dissociates is 50 times more likely to bind an unlabeled SNARE and contributes only to an overall upward creep that is easily distinguishable from the burst magnitude (Fig. 4, red line).

Bottom Line: NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism.We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF.We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305, USA.

ABSTRACT
SNARE proteins promote membrane fusion by forming a four-stranded parallel helical bundle that brings the membranes into close proximity. Post-fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide-sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ∼26-fold. One SNARE complex takes an average of ∼5 s to disassemble in a process that consumes ∼50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled-coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single-turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ∼1 residue is unwound for every hydrolyzed ATP molecule.

Show MeSH
Related in: MedlinePlus