Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system i n which to correlate the biochemistry and physiology of membrane fusion, Ho mologues 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 (approximate to 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, an d syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolys is by tetanus toxin, All criteria identify the SNARE complex as that descri bed 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-E thylmaleimide, which blocks fusion at or before the Ca2+-triggering step, b locks complex disruption by Ca2+. However, disruption is not blocked by lys ophosphatidylcholine, which transiently arrests a late stage of fusion, Sin ce removal of lysophosphatidylcholine from Ca2+-treated CV is known to allo w fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably c oiled coil SNARE interactions are not needed at the time of fusion, These f indings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion clamp."