Sequential SNARE assembly underlies priming and triggering of exocytosis.
Changes in SNARE conformations during MgATP-dependent priming of cracked PC12 cells were probed by their altered accessibility to various inhibitors. Dominant negative soluble syntaxin and, to a much lesser extent, VAMP coil domains inhibited exocytosis more efficiently after priming. Neurotoxins and an anti-SNAP25 antibody inhibited exocytosis less effectively after priming. We propose that SNAREs partially and reversibly assemble during priming, and that the syntaxin H3 domain is prevented from fully joining the complex until the arrival of the Ca2+ trigger. Furthermore, we find that mutation of hydrophobic residues of the SNAP25 C-terminal coil that contribute to SNARE core interactions affects the maximal rate of exocytosis, while mutation of charged residues on the surface of the complex affects the apparent affinity of the coil domain for the partially assembled complex.
Chen
Y A
YA
Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Scales
S J
SJ
Scheller
R H
RH
D000818:Animals; D000906:Antibodies / Q000187:drug effects / Q000378:metabolism; D001905:Botulinum Toxins / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D050600:SNARE Proteins; D013570:Synaptic Membranes / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D000906:Antibodies; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins; D009638:Norepinephrine