GTP CLEAVAGE BY THE SMALL GTP-BINDING PROTEIN RAB3A IS ASSOCIATED WITH EXOCYTOSIS OF SYNAPTIC VESICLES INDUCED BY ALPHA-LATROTOXIN

Neurotransmitter release from presynaptic nerve terminals is a highly regulated form of exocytosis. Small GTP-binding proteins of the Rab fa mily have been proposed to act as central regulators in this process t hat cycle between a GTP- and GDP-bound form. Previous work has shown t hat the synaptic vesicle protein Rab3A undergoes a membrane associatio n-dissociation cycle that is associated with neurotransmitter release. Using isolated nerve terminals as our model system, we have now analy zed the GDP/GTP status of Rab3A. Synaptic vesicle-bound Rab3A was almo st 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, synapto somes were stimulated with alpha-latrotoxin, the active component of b lack widow spider venom. This resulted in massive exocytosis. A signif icant increase of the GDP/GTP ratio of Rab3A was observed under these conditions that was not due to a nonspecific loss of high energy nucle otides. Our findings suggest that cleavage of Rab3A-bound GTP is a cru cial step in regulated exocytosis of synaptic vesicles.