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Vol. 273, Issue 4, 2445-2451, January 23, 1998

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

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.

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