Determinants of liposome fusion mediated by synaptic SNARE proteins.
Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobrevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome fusion mediated by synaptic SNAREs is inhibited by botulinum neurotoxin E (BoNT/E) but can be rescued by supplementing the C-terminal portion of SNAP-25. Furthermore, fusion is prevented by a SNAP-25-specific antibody known to block exocytosis in chromaffin cells, and it is competed for by soluble fragments of the R-SNAREs synaptobrevin 2, endobrevin/VAMP-8, and tomosyn. No accumulation of clustered vesicles is observed during the reaction. Rapid artificial clustering of SNARE-containing proteoliposomes enhances the fusion rate at low but not at saturating liposome concentrations. We conclude that the rate of liposome fusion is dominated by the intrinsic properties of the SNAREs rather than by the preceding docking step.
Schuette
Christina G
CG
Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany.
Hatsuzawa
Kiyotaka
K
Margittai
Martin
M
Stein
Alexander
A
Riedel
Dietmar
D
Küster
Petra
P
König
Marcelle
M
Seidel
Claus
C
Jahn
Reinhard
R
D000818:Animals; D003001:Cloning, Molecular; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011510:Proteolipids / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D050600:SNARE Proteins; D013569:Synapses / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D008081:Liposomes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; C020485:proteoliposomes