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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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NSF-driven disassembly of engineered SNARE substrates.A, schematic diagram illustrating the domain structure of the different SNARE substrates used in this study. B, SNARE-activated ATPase activity. C, NSF-driven disassembly. D, average time required for one NSF hexamer to disassemble a single SNARE complex. E, number of ATP molecules hydrolyzed for each SNARE molecule disassembled. Data shown are the average of three independent assays ± S.E. collected at 37 °C.
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Figure 3: NSF-driven disassembly of engineered SNARE substrates.A, schematic diagram illustrating the domain structure of the different SNARE substrates used in this study. B, SNARE-activated ATPase activity. C, NSF-driven disassembly. D, average time required for one NSF hexamer to disassemble a single SNARE complex. E, number of ATP molecules hydrolyzed for each SNARE molecule disassembled. Data shown are the average of three independent assays ± S.E. collected at 37 °C.

Mentions: To learn about the mechanism of NSF-driven SNARE disassembly, we assessed the structural features of the SNARE complex required for NSF and α-SNAP to recognize and disassemble it. In addition to the soluble SNARE complex, we also co-expressed a “minimal” soluble SNARE complex (1) consisting of only the SNARE core domains (i.e. missing the Habc domain of syntaxin and the connecting loop of SNAP-25). Separately, we purified an unrelated four-stranded coiled-coil: a tandem repeat of the GCN4-pLI tetramer (27) and an Ext-SNARE complex where the SNARE domains were duplicated (Fig. 3A). All constructs were SDS-stable, including the GCN4 tetramer, which was stable in SDS even after boiling the sample (Fig. 2C).


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)

NSF-driven disassembly of engineered SNARE substrates.A, schematic diagram illustrating the domain structure of the different SNARE substrates used in this study. B, SNARE-activated ATPase activity. C, NSF-driven disassembly. D, average time required for one NSF hexamer to disassemble a single SNARE complex. E, number of ATP molecules hydrolyzed for each SNARE molecule disassembled. Data shown are the average of three independent assays ± S.E. collected at 37 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: NSF-driven disassembly of engineered SNARE substrates.A, schematic diagram illustrating the domain structure of the different SNARE substrates used in this study. B, SNARE-activated ATPase activity. C, NSF-driven disassembly. D, average time required for one NSF hexamer to disassemble a single SNARE complex. E, number of ATP molecules hydrolyzed for each SNARE molecule disassembled. Data shown are the average of three independent assays ± S.E. collected at 37 °C.
Mentions: To learn about the mechanism of NSF-driven SNARE disassembly, we assessed the structural features of the SNARE complex required for NSF and α-SNAP to recognize and disassemble it. In addition to the soluble SNARE complex, we also co-expressed a “minimal” soluble SNARE complex (1) consisting of only the SNARE core domains (i.e. missing the Habc domain of syntaxin and the connecting loop of SNAP-25). Separately, we purified an unrelated four-stranded coiled-coil: a tandem repeat of the GCN4-pLI tetramer (27) and an Ext-SNARE complex where the SNARE domains were duplicated (Fig. 3A). All constructs were SDS-stable, including the GCN4 tetramer, which was stable in SDS even after boiling the sample (Fig. 2C).

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