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

1998, Journal of Biological Chemistry

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 (Ϸ70 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 [Ca 2؉ ] free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca 2؉-triggering step, blocks complex disruption by Ca 2؉. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca 2؉-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca 2؉ 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 Ca 2؉ binding removes a "fusion-clamp." Membrane fusion is the fundamental cellular process by which exocytotic secretion, enveloped virus entry, intracellular trafficking, and fertilization occur. Recently, conceptual advances have been made in how we think of the proteins involved in intracellular membrane trafficking and exocytosis (1-3), due in part to the discovery of a series of interactions between homologous proteins known to be required for membrane trafficking in vitro, yeast secretion in vivo, and synaptic transmission at the neuromuscular junction. These interactions are thought to contribute to the formation of a protein complex that is postulated to mediate the targeting, docking, and subsequent fusion of membranes (2).