The ionic layer is required for efficient dissociation of the SNARE complex by alpha-SNAP and NSF.
The four-helical bundle soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex that mediates intracellular membrane fusion events contains a highly conserved ionic layer at the center of an otherwise hydrophobic core. This layer has an undetermined function; it consists of glutamine (Q) residues in syntaxin and the two synaptosomal-associated protein of 25 kDa (SNAP-25) family helices, and an arginine (R) in vesicle-associated membrane protein (a 3Q:1R ratio). Here, we show that the ionic-layer glutamine of syntaxin is required for efficient alpha-SNAP and NSF-mediated dissociation of the complex. When this residue is mutated, the SNARE complex still binds to alpha-SNAP and NSF and is released through ATP hydrolysis by NSF, but the complex no longer dissociates into SNARE monomers. Thus, one function of the ionic layer--in particular, the glutamine of syntaxin--is to couple ATP hydrolysis by NSF to the dissociation of the fusion complex. We propose that alpha-SNAP and NSF drive conformational changes at the ionic layer through specific interactions with the syntaxin glutamine, resulting in the dissociation of the SNARE complex.
Scales
S J
SJ
Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305-5345, USA.
Yoo
B Y
BY
Scheller
R H
RH
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D005973:Glutamine / Q000737:chemistry; D066298:In Vitro Techniques; D007477:Ions; D046911:Macromolecular Substances; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050827:Syntaxin 1; D014162:Transfection; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D002352:Carrier Proteins; D007477:Ions; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D005973:Glutamine; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat