EVIDENCE FOR A FUNCTIONAL-LINK BETWEEN RAB3 AND THE SNARE COMPLEX

Rab3 is a monomeric GTP-binding protein associated with secretory vesi cles which has been implicated in the control of regulated exocytosis. We have exploited Rab3 mutant proteins to investigate the function of Rab3 in the process of neurotransmitter release from Aplysia neurons, A GTPase-deficient Rab3 mutant protein was found to inhibit acetylcho line release suggesting that GTP hydrolysis by Rab3 is rate-limiting i n the exocytosis process, This effect was abolished by a mutation in t he effector domain, and required the association of Rab3 with membrane s, Ln order to determine the step at which Rab3 interferes with the se cretory process, tetanus and botulinum type A neurotoxins were applied to Aplysia neurons pre-injected with the GTPase-deficient Rab3 mutant protein, These neurotoxins are Zn2+-dependent proteases that cleave V AMP/synaptobrevin and SNAP-25, two proteins which can form a ternary c omplex (termed the SNARE complex) with syntaxin and have been implicat ed in the docking of synaptic vesicles at the plasma membrane. The ons et of toxin-induced inhibition of neurotransmitter release was strongl y delayed in these cells, indicating that the mutant Rab3 protein led to the accumulation of a toxin-insensitive component of release. Since tetanus and botulinum type A neurotoxins cannot attach their targets, VAMP/synaptobrevin and SNAP-25, when the latter are engaged in the SN ARE complex, we propose that Rab3 modulates the activity of the fusion machinery by controlling the formation or the stability of the SNARE complex.