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 n eurotransmitter release. Rabphilin-3A is associated with synaptic vesi cles, although it has no transmembrane segment. Here we have studied h ow rabphilin-3A is associated with synaptic vesicles. Treatment of the synaptic vesicles isolated from rat brain with 1 M NaCl completely so lubilized rabphilin-3A from the vesicles. These vesicles deprived of r abphilin-3A still contained Rab3A and synaptophysin. Exogenous rabphil in-3A bound to the vesicles deprived of endogenous rabphilin-3A in dos e-dependent and saturable manners. The concentration of exogenous rabp hilin-3A giving a half maximal binding was about 50 nM and maximally 5 +/- 1 molecules of exogenous rabphilin-3A bound to one vesicle. Addit ion of exogenous Rab3A or removal of endogenous Rab3A by the action of Rab GDI did not affect the binding of exogenous rabphilin-3A BA to th e vesicles. However, treatment of the vesicles with trypsin completely abolished the binding of exogenous rabphilin-3A. These results sugges t that rabphilin-3A is associated with synaptic vesicles at least thro ugh a vesicle protein in a manner independent of Rab3A.