Rabphilin-3A is associated with synaptic vesicles through a vesicle protein in a manner independent of Rab3A

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein, which is implicated in regulated secretion, particularly in neurotransmitter release. Rabphilin-3A is associated with synaptic vesicles, although it has no transmembrane segment. Here we have studied how rabphilin-3A is associated with synaptic vesicles. Treatment of the synaptic vesicles isolated from rat brain with 1 M NaCl completely solubilized rabphilin-3A from the vesicles. These vesicles deprived of rabphilin-3A still contained Rab3A and synaptophysin. Exogenous rabphilin-3A bound to the vesicles deprived of endogenous rabphilin-3A in dose-dependent and saturable manners. The concentration of exogenous rabphilin-3A giving a half-maximal binding was about 50 nM and maximally 5 +/- 1 molecules of exogenous rabphilin-3A bound to one vesicle. Addition of exogenous Rab3A bound to one vesicle. Addition of exogenous Rab3A or removal of endogenous Rab3A by the action of Rab GDI did not affect the binding of exogenous rabphilin-3A to the vesicles. However, treatment of the vesicles with trypsin completely abolished the binding of exogenous rabphilin-3A. These results suggest that rabphilin-3A is associated with synaptic vesicles at least through a vesicle protein in a manner independent of Rab3A.