Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca2+ sensitivity.
Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, <n\>Max, was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both <n\>Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion.
Coorssen
J R
JR
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. jensc@nih.gov
Blank
P S
PS
Tahara
M
M
Zimmerberg
J
J
D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D004527:Egg Proteins / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000187:drug effects*; D008565:Membrane Proteins / Q000502:physiology*; D008954:Models, Biological; D009865:Oocytes / Q000166:cytology; D015388:Organelles / Q000187:drug effects / Q000502:physiology*; D050600:SNARE Proteins; D012617:Sea Urchins; D013324:Strontium / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D004527:Egg Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D001464:Barium; D002118:Calcium; D013324:Strontium