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J. Cell Biol.,
Volume 143, Number 7, December 28, 1998 1845-1857
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892
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.
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