MEMBERS OF THE SNARE HYPOTHESIS ARE ASSOCIATED WITH CORTICAL GRANULE EXOCYTOSIS IN THE SEA-URCHIN EGG

Cortical granule exocytosis is important for the block to polyspermy a t fertilization in the eggs of most vertebrates and many invertebrates . Cortical granules are poised at the cell surface and exocytose in re sponse to sperm stimulation. Following exocytosis, the cortical granul e contents modify the extracellular environment of the egg, the major result of which is to block additional sperm binding. Here we show tha t proteins homologous to members of the SNARE hypothesis-a molecular m odel designed to explain the trafficking, docking, and exocytosis of v esicles in the secretory compartment-are present in eggs at the right time and place to be involved in the regulation of cortical granule ex ocytosis. Using polymerase chain reaction (PCR) screens we have found homologues of synaptobrevin/VAMP, syntaxin, synaptotagmin, and rab3. A ntibodies generated to fusion proteins or to synthetic peptides encode d by the cloned cDNAs were used in an immunofluorescence assay to show that each of the cognate proteins are present in the cortex of the eg g. A synaptobrevin/VAMP homologue appears to be specifically associate d with the membrane of cortical granules before fertilization and, fol lowing cortical granule exocytosis, is incorporated into the plasma me mbrane of the zygote. A rab3 homologue is also associated with cortica l granules specifically but, following fertilization, the protein reas sociates with different, yet undefined, vesicles throughout the cytopl asm of the zygote. Homologues of synaptotagmin and syntaxin are also p resent at the egg cortex but, in contrast to rab3 and VAMP, appear to be associated with the plasma membrane. Following fertilization, synta xin and tagmin remain associated with the plasma membrane and are more readily immunolabeled, presumably due to an increased accessibility o f the antibodies to the target protein domains. We also show by immuno blotting experiments that the cognate proteins are of the sizes predic ted for these homologues. These results suggest that at least some ste ps in the biology of cortical granules may be mediated by SNARE homolo gues, and this finding, along with the unique biology of cortical gran ules, should facilitate examination of specific events of the fertiliz ation reaction and the mechanism of stimulus dependent exocytosis. (C) 1997 Wiley-Liss, Inc.