Conner S, Wessel G M
Department of Molecular and Cellular Biology & Biochemistry, Brown University, Providence, Rhode Island, 02912, USA.
Dev Biol. 1998 Nov 15;203(2):334-44. doi: 10.1006/dbio.1998.9057.
Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane 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 associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are 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 leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking.
海胆受精时的卵子激活会导致约15,000个停靠在质膜上的皮质颗粒发生胞吐作用。此前，我们报道过，除了ras超家族的一个小GTP酶rab3外，在SNARE假说中构建模型的几种整合膜蛋白，即突触结合蛋白、VAMP和 syntaxin，也存在于皮质颗粒上（康纳，S.，利夫，D.，和韦塞尔，G.，《分子生殖与发育》48卷，1 - 13页，1997年）。在此我们报道，rab3在整个卵子发生过程中、皮质颗粒转运期间以及停靠在卵质膜上时都与皮质颗粒相关联。然而，在皮质颗粒胞吐作用之后，rab3会与另一群不同的囊泡重新结合，其中至少一些囊泡是内吞起源的。由于其在卵子和卵母细胞中与皮质颗粒的选择性关联，我们推测rab3在皮质颗粒胞吐作用中发挥作用。为了验证这一假说，我们采用了一种策略，即通过与其效应结构域（特定rab类型中的一个保守区域）进行肽竞争来干扰rab3的功能。我们首先在多棘海胆卵中鉴定出效应结构域序列，发现其与紫球海胆中rab3的效应结构域序列有94%的同源性。然后，利用针对rab3蛋白不同区域的合成肽，我们发现注射了效应结构域肽的卵子中皮质颗粒胞吐作用受到抑制，但注射高变区肽或打乱的效应肽的卵子则不受影响。此外，注射了效应肽的卵子再注射IP3后，其皮质颗粒胞吐能力受到阻碍，这表明抑制作用直接作用于膜融合事件，而非干扰导致钙释放的信号转导机制的结果。我们将这些结果解释为rab3在囊泡停靠后对皮质颗粒胞吐作用的调节中发挥作用。
