Multiple forms of SNARE complexes in exocytosis from chromaffin cells: effects of Ca(2+), MgATP and botulinum toxin type A.
The changes that SNAREs undergo during exocytosis were studied in permeabilised chromaffin cells treated with Ca(2+), MgATP or botulinum neurotoxin A. High-resolution 2D SDS-PAGE revealed multiple SDS-resistant SNARE complexes having a wide range of sizes and in which SNAP-25 and syntaxin predominate over synaptobrevin. Their formation increased upon Ca(2+)-stimulated exocytosis; notably, the 2D protocol proved much superior to 1D SDS-PAGE for the detection of large complexes and revealed that for forms with relative molecular mass greater than 100,000 stimulated induction was more significant than for smaller species. MgATP enhanced Ca(2+)-triggered catecholamine release but reduced the content of complexes. By contrast, botulinum neurotoxin type A inhibited exocytosis and altered the stoichiometry of the SNAP-25:syntaxin binary association, without lowering its abundance. The individual SNAREs were protected against trypsin proteolysis to varying extents in binary and ternary complexes of different sizes, suggestive of distinct folding intermediates. Our data suggest that Ca(2+) triggers an early stage of SNARE complex formation causing an accumulation of partially folded intermediates, especially of binary forms, as well as their maturation into smaller, more protease resistant states. In addition, botulinum neurotoxin A inhibits exocytosis by perturbing the syntaxin:SNAP-25 ratio in binary intermediates.
Lawrence
Gary W
GW
Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK.
Dolly
J Oliver
JO
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009465:Neuromuscular Agents / Q000378:metabolism; D020033:Protein Isoforms; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins
D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; D020033:Protein Isoforms; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D019274:Botulinum Toxins, Type A; D002118:Calcium