We have continued our combined biophysical and biochemical research on the molecular mechanisms of membrane fusion during exocytosis, the critical step in insulin, neurotransmitter, and immunoglobulin secretion, and in fertilization. First,expression library screening with a monoclonal antibody to mammalian SNAP-25 was used to identify a S.purpuratus SNAP-25 homologue. A single isoform of sea urchin ovary SNAP-25 was cloned. The clone encodes a 212 amino acid protein (Gen Bank accession no. AF061750). This SNAP-25 is highly conserved, throughout the whole amino acid sequence when aligned to SNAP-25 from several species. The recombinant protein has been expressed in E.coli as a fusion construct, purified, and used as a standard in quantitative evaluation of the amount of SNAP-25 in cortical vesicles. The preliminary data show that ~1000 copies of SNAP-25 are present per vesicle. Second, syntaxin and VAMP have been immunoprecipated from cortical vesicles and the peptide maps analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The syntaxin immunoprecipated and purified from CV had peptide compositions after digestion that matched those predicted from the cDNA for urchin ovary syntaxin. VAMP from CV was isolated and the tryptic peptides were examined. Although the peptide map of VAMP contained many of the peptides predicted from the cDNA sequence, a major mass peak was present that did not correspond to the peptides predicted for urchin ovary VAMP. A stage-specific urchin ovary cDNA library was rescreened for a VAMP clone which had an amino terminal peptide consistent with the masses seen with post source decay (PSD)fragmentation. Several positive clones were isolated which contained an amino terminal sequence consistent with that obtained from the isolated CV VAMP. The sequence of the sea urchin ovary VAMP was determined (Gen Bank accession no. AF151536). In addition, the cDNA library was screened for sequences of the amino terminal of VAMP corresponding to that predicted for the cDNA sequence reported in the database. No positive clones were obtained using that sequence, indicating that there is no urchin VAMP that matches the published amino terminal sequence. Further analysis of this tryptic peptide by PSDfragmentation was used to determine the amino acid sequence and composition. PSD analysis indicated that the peptide could be the amino terminal peptide, since the peptide contained an amino terminal N-acetyl alanine. Although the amino terminal sequence of the peptide was distinct from the cDNA clone, the C-terminal sequence corresponded to that predicated from the cDNA clone. This discrepancy between the published VAMP cDNA sequence and the amino acid sequence of the isolated VAMP points out the importance of confirming the structure of the expressed protein, and not relying only on cloning data for sequence determination. Third, the fusion of CV to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescence microscopy in combination with electrical recordings of membrane conductance. A strong binding of CV to protein-free planar membrane was observed in the absence of calcium. Calcium-induced fusion of CV was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy; and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a content-specific dye. The results indicate that CV have sufficient calcium-sensitive proteins and docking machinery for fusion to lipid membranes, and in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites access to new membranes.
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