Purification and properties of rabconnectin-3

Rab3A, a member of the Rab3 small GTP-binding protein (G protein) family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). We) have found a novel protein, named rabconnectin-3, that is coimmunoprecipitated with Rab3 GEP or GAP from the crude synaptic vesicle fraction of rat brain. Rabconnectin-3 constitutes a subunit structure consisting of alpha and beta subunits and localizes at synaptic vesicles. Overexpression of the C-terminal fragment of rabconnectin-3alpha inhibits Ca(2+)-dependent exocytosis from PC12 cells. We describe the purification method for native rabconnectin-3alpha and -3beta from rat brain and the functional properties of rabconnectin-3alpha in Ca(2+)-dependent exocytosis by use of human growth hormone coexpression assay system of PC12 cells.