Zn7metallothionein-3 and the synaptic vesicle cycle:: Interaction of metallothionein-3 with the small GTPase Rab3A

In the central nervous system, a large amount of chelatable Zn2+ is sequestered in presynaptic vesicles of certain glutamatergic nerve terminals. The exo-endocytotic cycle of synaptic vesicles is strictly linked to the small GTPase Rab3A. Metallothionein-3 (Zn7MT-3) has been proposed to be involved in the intracellular trafficking of Zn2+ in zinc-containing neurons, but its role in this process is not understood. By using affinity precipitation and surface plasmon resonance analysis, we show that Zn7MT-3 binds reversibly to Rab3A(.)GDP (K-D = 2.6 muM), but not to Rab3A(.)GTP. The binding of Zn7MT-3 to Rab3A(.)GDP is specific as no binding was observed with the metal-free form of MT-3. Mutational studies of Rab3A mapped the interaction site to the effector binding site of the protein. This location is further supported by the kinetics of GDP exchange, which was found to be unaffected by binding of Zn7MT-3 to Rab3A(.)GDP. The interaction of Zn7MT-3 with Rab3A indicates that Zn7MT-3 is not merely a cellular Zn2+ buffer, but actively participates in synaptic vesicle trafficking upstream of vesicle fusion.