Uncoupling the ATPase Activity of the N-Ethylmaleimide Sensitive Factor (NSF) from 20S Complex Disassembly
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- 作者：Elena A. Matveeva ; Andrew P. May ; Ping He ; and Sidney W. Whiteheart
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：January 15, 2002
- 年：2002
- 卷：41
- 期：2
- 页码：530 - 536
- 全文大小：326K
- 年卷期：v.41,no.2(January 15, 2002)
- ISSN：1520-4995
文摘
The N-ethylmaleimide sensitive factor (NSF) plays a critical role in intracellular traffickingby disassembling soluble NSF attachment protein receptor (SNARE ) complexes. The NSF protomerconsists of three domains (NSF-N, NSF-D1, and NSF-D2). Two domains (NSF-D1 and NSF-D2) containa conserved ~230 amino acid cassette, which includes a distinctive motif termed the second region ofhomology (SRH) common to all ATPases associated with various cellular activities (AAA). In hexamericNSF, several SRH residues become trans elements of the ATP binding pocket. Mutation of two conservedarginine residues in the NSF-D1 SRH (R385A and R388A) did not effect basal or soluble NSF attachmentprotein (SNAP)-stimulated ATPase activity; however, neither mutant underwent ATP-dependent releasefrom SNAP-SNARE complexes. A trans element of the NSF-D2 ATP binding site (K631) has beenproposed to limit the ATPase activity of NSF-D2, but a K631D mutant retained wild-type activity. Amutation of the equivalent residue in NSF-D1 (D359K) also did not affect nucleotide hydrolysis activitybut did limit NSF release from SNAP-SNARE complexes. These trans elements of the NSF-D1 ATPbinding site (R385, R388, and D359) are not required for nucleotide hydrolysis but are important asnucleotide-state sensors. NSF-N mediates binding to the SNAP-SNARE complex. To identify the structuralfeatures required for binding, three conserved residues (R67, S73, and Q76) on the surface of NSF-Nwere mutated. R67E completely eliminated binding, while S73R and Q76E showed limited effect. Thissuggests that the surface important for SNAP binding site lies in the cleft between the NSF-N subdomainsadjacent to a conserved, positively charged surface.