Dissecting the N-ethylmaleimide-sensitive factor: required elements of the N and D1 domains.
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.
Zhao
Chunxia
C
Department of Molecular and Cellular Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536-0509, USA.
Matveeva
Elena A
EA
Ren
Qiansheng
Q
Whiteheart
Sidney W
SW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000737:chemistry* / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050600:SNARE Proteins / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism
D000596:Amino Acids; D050505:Mutant Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins