Stahl B, von Mollard G F, Walch-Solimena C, Jahn R
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1994 Oct 7;269(40):24770-6.
Neurotransmitter release from presynaptic nerve terminals is a highly regulated form of exocytosis. Small GTP-binding proteins of the Rab family have been proposed to act as central regulators in this process that cycle between a GTP- and GDP-bound form. Previous work has shown that the synaptic vesicle protein Rab3A undergoes a membrane association-dissociation cycle that is associated with neurotransmitter release. Using isolated nerve terminals as our model system, we have now analyzed the GDP/GTP status of Rab3A. Synaptic vesicle-bound Rab3A was almost exclusively in the GTP form whereas cytosolic Rab3A contained only GDP. Approximately equal amounts of GTP and GDP were found in the pool of Rab3A localized to a membrane fraction containing plasma membrane-synaptic vesicle complexes. In contrast to Rab3A, Rab5 (an endosomal G-protein) was predominantly GDP-bound in all analyzed compartments. To analyze whether Rab3A-bound GTP is cleaved during exocytosis, synaptosomes were stimulated with alpha-latrotoxin, the active component of black widow spider venom. This resulted in massive exocytosis. A significant increase of the GDP/GTP ratio of Rab3A was observed under these conditions that was not due to a nonspecific loss of high energy nucleotides. Our findings suggest that cleavage of Rab3A-bound GTP is a crucial step in regulated exocytosis of synaptic vesicles.
神经递质从突触前神经末梢释放是一种受到高度调控的胞吐形式。Rab家族的小GTP结合蛋白被认为在此过程中作为核心调节因子，在结合GTP和GDP的形式之间循环。先前的研究表明，突触囊泡蛋白Rab3A经历与神经递质释放相关的膜结合-解离循环。以分离的神经末梢作为我们的模型系统，我们现在分析了Rab3A的GDP/GTP状态。与突触囊泡结合的Rab3A几乎完全处于GTP形式，而胞质中的Rab3A仅含有GDP。在定位于包含质膜-突触囊泡复合物的膜部分的Rab3A池中，发现GTP和GDP的量大致相等。与Rab3A相反，Rab5（一种内体G蛋白）在所有分析的区室中主要结合GDP。为了分析Rab3A结合的GTP在胞吐过程中是否被裂解，用黑寡妇蜘蛛毒液的活性成分α-拉毒素刺激突触体。这导致大量胞吐。在这些条件下观察到Rab3A的GDP/GTP比率显著增加，这并非由于高能核苷酸的非特异性损失。我们的研究结果表明，Rab3A结合的GTP的裂解是突触囊泡受调控胞吐的关键步骤。
