Three αSNAP and 10 ATP Molecules Are Used in SNARE Complex Disassembly by N-ethylmaleimide-sensitive Factor (NSF)

• Published in: The Journal of biological chemistry Vol. 290; no. 4; pp. 2175 - 2188
• Main Authors: Shah, Niket, Colbert, Karen N., Enos, Michael D., Herschlag, Daniel, Weis, William I.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.01.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - chemistry; alpha-SNAP; Animals; Anisotropy; ATPase; ATPases Associated with Diverse Cellular Activities (AAA); Base Sequence; Cell Biology; Cell Membrane - metabolism; Cricetulus; Escherichia coli - metabolism; Hydrolysis; Intracellular Trafficking; Membrane Fusion; Membrane Trafficking; Microscopy, Fluorescence; Molecular Sequence Data; NSF; Nucleotides - chemistry; Protein Binding; Protein Engineering; Protein Transport; SNARE Proteins - chemistry; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins - chemistry; Soluble NSF Attachment Protein Receptor (SNARE); Membrane Trafficking; NSF; ATPase; Soluble NSF Attachment Protein Receptor (SNARE); Intracellular Trafficking; ATPases Associated with Diverse Cellular Activities (AAA); alpha-SNAP
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.620849

The fusion of intracellular membranes is driven by the formation of a highly stable four-helix bundle of SNARE proteins embedded in the vesicle and target membranes. N-Ethylmaleimide sensitive factor recycles SNAREs after fusion by binding to the SNARE complex through an adaptor protein, αSNAP, and using the energy of ATP hydrolysis to disassemble the complex. Although only a single molecule of αSNAP binds to a soluble form of the SNARE complex, we find that three molecules of αSNAP are used for SNARE complex disassembly. We describe an engineered αSNAP trimer that supports more efficient SNARE complex disassembly than monomeric αSNAP. Using the trimerized αSNAP, we find that N-ethylmaleimide-sensitive factor hydrolyzes 10 ATP molecules on average to disassemble a single SNARE complex.The ATPase NSF works with the adaptor protein αSNAP to disassemble SNARE protein complexes involved in membrane fusion. Kinetic assays demonstrate that three αSNAP and 10 ATP molecules are required to disassemble a SNARE complex. The energy requirements and functional stoichiometry of αSNAP in SNARE complex disassembly are established. The results suggest models of NSF action.