Reconstitution of Calcium-triggered Membrane Fusion Using “Reserve” Granules*
- Valery V. Chestkov‡§,
- Sergey P. Radko§,
- Myoung-Soon Cho§,
- Andreas Chrambach§ and
- Steven S. Vogel§¶‖**
- From the ‡Medical Genetics Center, Russian Academy of Medical Sciences, Moscow, Russia, §Laboratory of Cellular and Molecular Biophysics, NICHD and ¶Synaptic Mechanisms Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892, and ‖Medical College of Georgia, Institute for Molecular Medicine and Genetics, Augusta, Georgia 30912-2630
Abstract
Calcium-gated secretion of proteins involves the transfer of “reserve” granules, exocytotic vesicles that are cytoplasmic and, hence, plasma membrane-naive, from the cell interior to the surface membrane where they dock prior to fusion. Docking and subsequent priming steps are thought to require cytoplasmic factors. These steps are believed to induce fusion competence. We have tested this hypothesis by isolating reserve granules from sea urchin eggs and determining under which conditions these granules will fuse. We find that isolated reserve granules, lacking soluble cofactors, support calcium-dependent membrane fusion in vitro. Preincubation with adenosine 5′-3-O-(thio)triphosphate and guanosine 5′-3-O-(thio)triphosphate did not prevent fusion. Thus, isolated reserve granules have all the necessary components required for calcium-gated fusion prior to docking.
Footnotes
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↵* 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.
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↵** To whom all correspondence should be addressed: Medical College of Georgia, Institute for Molecular Medicine and Genetics, 1120 15th St. CB 2803, Augusta, GA 30912-2630. Tel.: 706-721-0713; Fax: 706-721-7915; E-mail: stevev{at}immag.mcg.edu.
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↵1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ATPγS, adenosine 5′-3-O-(thio)triphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; NSF,N-ethylmaleimide-sensitive factor.
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- Received August 7, 1997.
- Revision received October 9, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
