Calcium Can Disrupt the SNARE Protein Complex on Sea Urchin Egg Secretory Vesicles without Irreversibly Blocking Fusion

doi:10.1074/jbc.273.50.33667

Calcium Can Disrupt the SNARE Protein Complex on Sea Urchin Egg Secretory Vesicles without Irreversibly Blocking Fusion*

From the ^‡Laboratory of Cellular and Molecular Biophysics, NICHD, National Institutes of Health, Bethesda, Maryland 20892, the ^¶Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, United Kingdom, and the ^‖Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University Medical Center, Stanford, California 94305

Abstract

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (≈70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca²⁺]_free that trigger maximal fusion.N-Ethylmaleimide, which blocks fusion at or before the Ca²⁺-triggering step, blocks complex disruption by Ca²⁺. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca²⁺-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca²⁺ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion (“zippers-up”) after Ca²⁺binding removes a “fusion-clamp.”

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Present address: Osaka University Medical School, Osaka, Japan.
↵** To whom correspondence should be addressed. Tel.: 301-496-6571; Fax: 301-594-0813; E-mail: joshz{at}helix.nih.gov.
↵2 M. Tahara, J. R. Coorssen, and J. Zimmerberg, unpublished observations.
↵3 K. Timmers, I. Kolosova, D. Kingsley, and J. Zimmerberg, GenBank accession no. AF061750.
↵4 J. D. Sasaki, J. R. Schulz, and V. D. Vacquier, accession no. AF036902.
Abbreviations:

SNAP

soluble NSF attachment protein

SNARE

SNAP receptor

NSF

N-ethylmaleimide-sensitive factor

VAMP

vesicle-associated membrane protein

NEM

N-ethylmaleimide

PAGE

polyacrylamide gel electrophoresis

DTT

dithiothreitol

CV

cortical vesicle(s)

LPC

lysophosphatidylcholine

PIPES

1,4-piperazinediethanesulfonic acid

BAPTA

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

AEBSF

4-(2-aminoethyl)benzensulfonyl fluoride.
- Received July 8, 1998.
- Revision received September 3, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.