Calcium-triggered membrane fusion proceeds independently of specific presynaptic proteins.
Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca2+-triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca2+ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca2+ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.
Szule
Joseph A
JA
Department of Physiology and Biophysics, Cellular and Molecular Neurobiology Research Group, University of Calgary, Health Sciences Centre, Faculty of Medicine, Alberta T2N 4N1, Canada.
Jarvis
Scott E
SE
Hibbert
Julie E
JE
Spafford
J David
JD
Braun
Janice E A
JE
Zamponi
Gerald W
GW
Wessel
Gary M
GM
Coorssen
Jens R
JR
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins; D005089:Exocytosis / Q000502:physiology; D005260:Female; D008561:Membrane Fusion / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011485:Protein Binding; D050600:SNARE Proteins; D012617:Sea Urchins; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium