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
Kinetic analysis of ATP-driven SNARE disassembly by NSF. Disassembly assays were set up and measured as described under “Experimental Procedures,” except that [ATP] was varied and MgCl2 was added to a concentration that was 1 mm above [ATP] in each case. A, Hill analysis with no ATP-regenerating system. Data were fit to a Hill equation yielding K0.5 = 0.14, Kcat = 9.4 SNAREs min−1, n = 1.12 with an R2 of 0.984. B and C, kinetics including high [ATP] conditions, showing inhibition at high [ATP]. B, no ATP-regenerating system (data from panel A are replotted along with higher [ATP] data). C, ATP-regenerating system added.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Kinetic analysis of ATP-driven SNARE disassembly by NSF. Disassembly assays were set up and measured as described under “Experimental Procedures,” except that [ATP] was varied and MgCl2 was added to a concentration that was 1 mm above [ATP] in each case. A, Hill analysis with no ATP-regenerating system. Data were fit to a Hill equation yielding K0.5 = 0.14, Kcat = 9.4 SNAREs min−1, n = 1.12 with an R2 of 0.984. B and C, kinetics including high [ATP] conditions, showing inhibition at high [ATP]. B, no ATP-regenerating system (data from panel A are replotted along with higher [ATP] data). C, ATP-regenerating system added.

Mentions: Most previous studies of ATP hydrolysis by NSF have been performed with the uncoupled basal ATPase (32). We exploited our observation of strong SNARE activation of ATPase to study the kinetics of SNARE-coupled ATP hydrolysis. We measured the initial SNARE disassembly rates as a function of ATP concentration (Fig. 5). At ATP concentrations under 2 mm, NSF is well fit by a Hill equation, with an apparent K0.5 of 0.14 mm, a Kcat of 9.4 SNARES min−1, and a Hill coefficient n = 1.1. At ATP concentrations above 2 mm, NSF showed substrate inhibition. Substrate inhibition has been seen for other ring ATPases (36) but is shown for NSF for the first time here. Substrate inhibition was seen with or without an ATP-regenerating system, but was much more pronounced in the presence of ATP regeneration (compare Fig. 5, B and C).


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)

Kinetic analysis of ATP-driven SNARE disassembly by NSF. Disassembly assays were set up and measured as described under “Experimental Procedures,” except that [ATP] was varied and MgCl2 was added to a concentration that was 1 mm above [ATP] in each case. A, Hill analysis with no ATP-regenerating system. Data were fit to a Hill equation yielding K0.5 = 0.14, Kcat = 9.4 SNAREs min−1, n = 1.12 with an R2 of 0.984. B and C, kinetics including high [ATP] conditions, showing inhibition at high [ATP]. B, no ATP-regenerating system (data from panel A are replotted along with higher [ATP] data). C, ATP-regenerating system added.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Kinetic analysis of ATP-driven SNARE disassembly by NSF. Disassembly assays were set up and measured as described under “Experimental Procedures,” except that [ATP] was varied and MgCl2 was added to a concentration that was 1 mm above [ATP] in each case. A, Hill analysis with no ATP-regenerating system. Data were fit to a Hill equation yielding K0.5 = 0.14, Kcat = 9.4 SNAREs min−1, n = 1.12 with an R2 of 0.984. B and C, kinetics including high [ATP] conditions, showing inhibition at high [ATP]. B, no ATP-regenerating system (data from panel A are replotted along with higher [ATP] data). C, ATP-regenerating system added.
Mentions: Most previous studies of ATP hydrolysis by NSF have been performed with the uncoupled basal ATPase (32). We exploited our observation of strong SNARE activation of ATPase to study the kinetics of SNARE-coupled ATP hydrolysis. We measured the initial SNARE disassembly rates as a function of ATP concentration (Fig. 5). At ATP concentrations under 2 mm, NSF is well fit by a Hill equation, with an apparent K0.5 of 0.14 mm, a Kcat of 9.4 SNARES min−1, and a Hill coefficient n = 1.1. At ATP concentrations above 2 mm, NSF showed substrate inhibition. Substrate inhibition has been seen for other ring ATPases (36) but is shown for NSF for the first time here. Substrate inhibition was seen with or without an ATP-regenerating system, but was much more pronounced in the presence of ATP regeneration (compare Fig. 5, B and C).

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