SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis.
The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.
Banerjee
A
A
Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Kowalchyk
J A
JA
DasGupta
B R
BR
Martin
T F
TF
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D009498:Neurotoxins / Q000494:pharmacology*; D016716:PC12 Cells; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D013329:Structure-Activity Relationship; D013572:Synaptic Vesicles / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D000255:Adenosine Triphosphate; D001905:Botulinum Toxins; D002118:Calcium