title: SNAP-25 with mutations in the zero layer supports normal membrane fusion kinetics.
abstract: Considerable data support the idea that intracellular membrane fusion involves a conserved machinery containing the SNARE proteins. SNAREs assembled in vitro form a stable 4-helix bundle and it has been suggested that formation of this complex provides the driving force for bilayer fusion. We have tested this possibility in assays of exocytosis in cells expressing a botulinum neurotoxin E (BoNT/E)-resistant mutant of SNAP-25 in which additional disruptive mutations have been introduced. Single or double mutations of glutamine to glutamate or to arginine in the central zero layer residues of SNAP-25 did not impair the extent, time course or Ca2+-dependency of exocytosis in PC12 cells. Using adrenal chromaffin cells, we found that exocytosis could be reconstituted in cells transfected to express BoNT/E. A double Q-->E mutation did not prevent reconstitution and the kinetics of single granule release events were indistinguishable from control cells. This shows a high level of tolerance of changes in the zero layer indicating that the conservation of these residues is not due to an essential requirement in vesicle docking or fusion and suggests that formation of a fully stable SNARE complex may not be required to drive membrane fusion.
authors.affiliation: The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK.
authors.forename: M E
authors.initials: ME
authors.lastname: Graham
authors.forename: P
authors.initials: P
authors.lastname: Washbourne
authors.forename: M C
authors.initials: MC
authors.lastname: Wilson
authors.forename: R D
authors.initials: RD
authors.lastname: Burgoyne
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D001905:Botulinum Toxins / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology; D005089:Exocytosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D005973:Glutamine / Q000235:genetics; D006868:Hydrolysis / Q000187:drug effects; D007425:Intracellular Membranes / Q000502:physiology*; D007700:Kinetics; D008561:Membrane Fusion / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008839:Microelectrodes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D016716:PC12 Cells; D051381:Rats; D050825:Synaptosomal-Associated Protein 25; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D005973:Glutamine; D001120:Arginine; D001905:Botulinum Toxins; C096326:botulinum toxin type E