Defining the SNARE complex binding surface of alpha-SNAP: implications for SNARE complex disassembly.
N-Ethylmaleimide-sensitive factor (NSF) and its adaptor protein alpha-soluble NSF attachment protein (alpha-SNAP) sustain membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes that form during membrane fusion. To better understand the role of alpha-SNAP in this process, we used site-directed mutagenesis to identify residues in alpha-SNAP that interact with SNARE complexes. We find that mutations in charged residues distributed over a concave surface formed by the N-terminal nine alpha-helices of alpha-SNAP affect its ability to bind synaptic SNARE complex and promote its disassembly by NSF. Replacing basic residues on this surface with alanines reduced SNARE complex binding and disassembly, whereas replacing acidic residues with alanines enhanced alpha-SNAP efficacy in both assays. These findings show that the ability of NSF to take apart SNARE complexes depends upon electrostatic interactions between alpha-SNAP and the acidic surface of the SNARE complex and provide insight into how NSF and alpha-SNAP work together to drive disassembly.
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Karla E
KE
Marz
Joshua M
JM
Lauer
Phyllis I
PI
Hanson
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D052066:N-Ethylmaleimide-Sensitive Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D017386:Sequence Homology, Amino Acid; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D055672:Static Electricity; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins