RAB3 MEDIATES CORTICAL GRANULE EXOCYTOSIS IN THE SEA-URCHIN EGG

Egg activation at fertilization in the sea urchin results in the exocy tosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membra ne proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel,; G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is ass ociated with cortical granules throughout oogenesis, during cortical g ranule translocation, and while docked at the egg plasma membrane, fol lowing cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocy tic origin. Because of its selective association with cortical granule s in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of int erfering with rab3 function by peptide competition with its effector d omain, a conserved region within specific rab types. We first identifi ed the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglo centrotus purpuratus. Then, with synthetic peptides to different regio ns of the rab3 protein, we find that cortical granule exocytosis is in hibited in eggs injected with effector domain peptides, but not with p eptides from the hypervariable region or with a scrambled effector pep tide. Additionally, effector-peptide-injected eggs injected with IP3 a re blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism lead ing to calcium release. We interpret these results to mean that rab3 f unctions in the regulation of cortical granule exocytosis following ve sicle docking, (C) 1998 Academic Press.