Limits...
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.

Show MeSH

Related in: MedlinePlus

ATP-powered disassembly of SNAREs by NSF.A, schematic representation of bulk fluorescence-dequenching-based SNARE disassembly assay. Stars represent the fluorescent dye, Oregon Green 488. B, structure of the minimal neuronal SNARE complex (Protein Data Bank (PDB) 1N7S) illustrating sites used for labeling with Oregon Green maleimide for the dequenching assay. Helices are colored using the scheme in panel A. Yellow spheres represent the cysteine residues used for labeling. C, SDS-PAGE gel of purified proteins used in this study. Proteins were suspended to 0.5 μg/μl in Laemmli sample buffer with DTT and either boiled (+) or not (−). 10 μl were loaded into each lane of an Any kD denaturing polyacrylamide gel (Bio-Rad). D, SDS-PAGE-based SNARE disassembly assay. E, fluorescence dequenching-based disassembly assay (colored lines, left axis). The black arrow indicates the addition of NSF at time 0. Red, typical disassembly assay. Yellow, same assay performed in the absence of α-SNAP. Green, disassembly assay performed in the presence of 10 mm EDTA. The blue curve shows a disassembly assay that was stopped at 40 s by the addition of 10 mm EDTA. For comparison, densitometry was performed on the gel in panel C and overlaid (black squares, right axis). F, SNARE-dependent activation of NSF ATPase activity. Data shown are the average of three independent assays ± S.E. collected at 37 °C. Numbers shown above each bar represent the average rates. G, effect of ionic strength on ATP-driven SNARE disassembly by NSF. Black squares and blue squares, the effect of added NaCl on the disassembly rate (black squares) and SNARE-activated ATPase rate (blue squares) of NSF. For comparison, the effects of 100 and 200 mm KCl (solid line) and NaCH3COO (dashed line) on the disassembly rates are shown. All data shown are the average of triplicate assays with standard errors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520572&req=5

Figure 2: ATP-powered disassembly of SNAREs by NSF.A, schematic representation of bulk fluorescence-dequenching-based SNARE disassembly assay. Stars represent the fluorescent dye, Oregon Green 488. B, structure of the minimal neuronal SNARE complex (Protein Data Bank (PDB) 1N7S) illustrating sites used for labeling with Oregon Green maleimide for the dequenching assay. Helices are colored using the scheme in panel A. Yellow spheres represent the cysteine residues used for labeling. C, SDS-PAGE gel of purified proteins used in this study. Proteins were suspended to 0.5 μg/μl in Laemmli sample buffer with DTT and either boiled (+) or not (−). 10 μl were loaded into each lane of an Any kD denaturing polyacrylamide gel (Bio-Rad). D, SDS-PAGE-based SNARE disassembly assay. E, fluorescence dequenching-based disassembly assay (colored lines, left axis). The black arrow indicates the addition of NSF at time 0. Red, typical disassembly assay. Yellow, same assay performed in the absence of α-SNAP. Green, disassembly assay performed in the presence of 10 mm EDTA. The blue curve shows a disassembly assay that was stopped at 40 s by the addition of 10 mm EDTA. For comparison, densitometry was performed on the gel in panel C and overlaid (black squares, right axis). F, SNARE-dependent activation of NSF ATPase activity. Data shown are the average of three independent assays ± S.E. collected at 37 °C. Numbers shown above each bar represent the average rates. G, effect of ionic strength on ATP-driven SNARE disassembly by NSF. Black squares and blue squares, the effect of added NaCl on the disassembly rate (black squares) and SNARE-activated ATPase rate (blue squares) of NSF. For comparison, the effects of 100 and 200 mm KCl (solid line) and NaCH3COO (dashed line) on the disassembly rates are shown. All data shown are the average of triplicate assays with standard errors.

Mentions: The purified SNARE and GCN4 proteins are shown in Fig. 2C. Immediately after purification, all proteins were aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. After thawing, aliquots were stored on ice and used the same day. All protein concentrations were estimated using the Bradford method (28).


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)

ATP-powered disassembly of SNAREs by NSF.A, schematic representation of bulk fluorescence-dequenching-based SNARE disassembly assay. Stars represent the fluorescent dye, Oregon Green 488. B, structure of the minimal neuronal SNARE complex (Protein Data Bank (PDB) 1N7S) illustrating sites used for labeling with Oregon Green maleimide for the dequenching assay. Helices are colored using the scheme in panel A. Yellow spheres represent the cysteine residues used for labeling. C, SDS-PAGE gel of purified proteins used in this study. Proteins were suspended to 0.5 μg/μl in Laemmli sample buffer with DTT and either boiled (+) or not (−). 10 μl were loaded into each lane of an Any kD denaturing polyacrylamide gel (Bio-Rad). D, SDS-PAGE-based SNARE disassembly assay. E, fluorescence dequenching-based disassembly assay (colored lines, left axis). The black arrow indicates the addition of NSF at time 0. Red, typical disassembly assay. Yellow, same assay performed in the absence of α-SNAP. Green, disassembly assay performed in the presence of 10 mm EDTA. The blue curve shows a disassembly assay that was stopped at 40 s by the addition of 10 mm EDTA. For comparison, densitometry was performed on the gel in panel C and overlaid (black squares, right axis). F, SNARE-dependent activation of NSF ATPase activity. Data shown are the average of three independent assays ± S.E. collected at 37 °C. Numbers shown above each bar represent the average rates. G, effect of ionic strength on ATP-driven SNARE disassembly by NSF. Black squares and blue squares, the effect of added NaCl on the disassembly rate (black squares) and SNARE-activated ATPase rate (blue squares) of NSF. For comparison, the effects of 100 and 200 mm KCl (solid line) and NaCH3COO (dashed line) on the disassembly rates are shown. All data shown are the average of triplicate assays with standard errors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: ATP-powered disassembly of SNAREs by NSF.A, schematic representation of bulk fluorescence-dequenching-based SNARE disassembly assay. Stars represent the fluorescent dye, Oregon Green 488. B, structure of the minimal neuronal SNARE complex (Protein Data Bank (PDB) 1N7S) illustrating sites used for labeling with Oregon Green maleimide for the dequenching assay. Helices are colored using the scheme in panel A. Yellow spheres represent the cysteine residues used for labeling. C, SDS-PAGE gel of purified proteins used in this study. Proteins were suspended to 0.5 μg/μl in Laemmli sample buffer with DTT and either boiled (+) or not (−). 10 μl were loaded into each lane of an Any kD denaturing polyacrylamide gel (Bio-Rad). D, SDS-PAGE-based SNARE disassembly assay. E, fluorescence dequenching-based disassembly assay (colored lines, left axis). The black arrow indicates the addition of NSF at time 0. Red, typical disassembly assay. Yellow, same assay performed in the absence of α-SNAP. Green, disassembly assay performed in the presence of 10 mm EDTA. The blue curve shows a disassembly assay that was stopped at 40 s by the addition of 10 mm EDTA. For comparison, densitometry was performed on the gel in panel C and overlaid (black squares, right axis). F, SNARE-dependent activation of NSF ATPase activity. Data shown are the average of three independent assays ± S.E. collected at 37 °C. Numbers shown above each bar represent the average rates. G, effect of ionic strength on ATP-driven SNARE disassembly by NSF. Black squares and blue squares, the effect of added NaCl on the disassembly rate (black squares) and SNARE-activated ATPase rate (blue squares) of NSF. For comparison, the effects of 100 and 200 mm KCl (solid line) and NaCH3COO (dashed line) on the disassembly rates are shown. All data shown are the average of triplicate assays with standard errors.
Mentions: The purified SNARE and GCN4 proteins are shown in Fig. 2C. Immediately after purification, all proteins were aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C. After thawing, aliquots were stored on ice and used the same day. All protein concentrations were estimated using the Bradford method (28).

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