High affinity interaction of syntaxin and SNAP-25 on the plasma membrane is abolished by botulinum toxin E.
The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.
Rickman
Colin
C
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Meunier
Frederic A
FA
Binz
Thomas
T
Davletov
Bazbek
B
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism*; D001905:Botulinum Toxins / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D019439:Chromaffin Cells / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins; C096326:botulinum toxin type E