Abstract
N-Ethylmaleimide-sensitive factor (NSF) is a homo-hexameric member of the AAA(+) (ATPases associated with various cellular activities plus) family. It plays an essential role in most intracellular membrane trafficking through its binding to and disassembly of soluble NSF attachment protein (SNAP) receptor (SNARE) complexes. Each NSF protomer contains an N-terminal domain (NSF-N) and two AAA domains, a catalytic NSF-D1 and a structural NSF-D2. This study presents detailed mutagenesis analyses of NSF-N and NSF-D1, dissecting their roles in ATP hydrolysis, SNAP.SNARE binding, and complex disassembly. Our results show that a positively charged surface on NSF-N, bounded by Arg(67) and Lys(105), and the conserved residues in the central pore of NSF-D1 (Tyr(296) and Gly(298)) are involved in SNAP.SNARE binding but not basal ATP hydrolysis. Mutagenesis of Sensor 1 (Thr(373)-Arg(375)), Sensor 2 (Glu(440)-Glu(442)), and Arginine Fingers (Arg(385) and Arg(388)) in NSF-D1 shows that each region plays a discrete role. Sensor 1 is important for basal ATPase activity and nucleotide binding. Sensor 2 plays a role in ATP- and SNAP-dependent SNARE complex binding and disassembly but does so in cis and not through inter-protomer interactions. Arginine Fingers are important for SNAP.SNARE complex-stimulated ATPase activity and complex disassembly. Mutants at these residues have a dominant-negative phenotype in cells, suggesting that Arginine Fingers function in trans via inter-protomer interactions. Taken together, these data establish functional roles for many of the structural elements of the N domain and of the D1 ATP-binding site of NSF.
Highlights
NSF and mutants formed hexamers analyzed by gel filtration chromatography and mixed hexamers in HeLa cells
Our studies show that the hydrophobic patch (Tyr296 to Gly298) in one of the loops that protrudes into the central pore of NSF-D1 might mediate an interaction with the SNAP1⁄7SNARE complexes
Summary—Using mutagenesis and a series of functional assays, we dissected the roles of specific elements of NSF
Summary
Plasmids—Plasmids encoding His6-NSF and His6-␣-SNAP in the pQE9 expression vector were described previously [17, 29]. Constructs containing wild-type NSF or mutants with C-terminal GFP fusion were transiently transfected into HeLa cells with FuGENE 6 (Roche Applied Science) according to the manufacturer’s recommendations. SNARE complexes (GST-VAMP-81⁄7His6-SNAP-231⁄7syntaxin 2), His6-NSF or mutants, and His6-␣-SNAP (1:1:3 molar ratio) were incubated for 30 min at 4 °C in binding buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 2 mM -mercaptoethanol, 1% (v/v) Triton X-100, and 5% glycerol with 0.5 mM AMP-PNP (Roche Applied Science) and 2 mM EDTA). The bound protein complexes were recovered by brief centrifugation, washed, eluted with SDS-PAGE sample buffer, and analyzed by Western blotting for His tags with India-His-HRP (Pierce). The bound complexes were incubated in 0.5 ml of binding buffer containing 5 mM ATP and 10 mM MgCl2 for 30 min at 4 °C. The correlation between NSF-GFP expression or transfection efficiency and cell death was analyzed using the correlation function (Pearson product-moment correlation coefficient) of GraphPad Prism 4.0 software (GraphPad Software Inc., La Jolla, CA)
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