© The Rockefeller University Press,
0021-9525/1998//1845 $5.00
The Journal of Cell Biology, Volume 143, Number 7,
, 1998 1845-1857
Biochemical and Functional Studies of Cortical Vesicle Fusion: The SNARE Complex and Ca2+ Sensitivity
Jens R. Coorssen,
Paul S. Blank,
Masahiro Tahara, and
Joshua Zimmerberg
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.
Key Words: calcium cytoplasmic vesicles exocytosis membrane fusion secretion
Abbreviations used in this paper: [Ca2+]free, free Ca2+ concentration; CSC, cell surface complex; CV, cortical vesicles; LPC, lysophosphatidylcholine; <n>, average number of active fusion complexes; NEM, N-ethylmaleimide; NSF, N-ethylmaleimide-sensitive factor; PM, plasma membrane; pMe2+, –log[divalent cation]free; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; VAMP, vesicle-associated membrane protein.
M. Tahara's present address is Department of Obstetrics and Gynecology, Kaizuka Municipal Hospital, Osaka 597-0015, Japan.

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-
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-
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-
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-
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-
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-
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[Full Text]
-
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[Full Text]
-
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276: 12813-12821
[Abstract]
[Full Text]
-
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