Rabphilin dissociated from Rab3 promotes endocytosis through interaction with Rabaptin-5

Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3, We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpressio n of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis, By contrast, Rabphilin V 61A, a mutant that is unable to interact with Rab3, enhanced the rate of tr ansferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding, Careful analysi s of the properties of the two mutants revealed that Rabphilin V61A and Rab philin L83A are both targeted to secretory vesicles, have stimulatory activ ity on exocytosis, and bind equally well to alpha -actinin, However, Rabphi lin L83A fails to interact with Rabaptin-5, an important component of the e ndocytotic machinery, These results indicate that Rabphilin promotes recept or-mediated endocytosis and that its action is negatively modulated by Rab3 , We propose that the hydrolysis of GTP that is coupled to the exocytotic e vent disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rab aptin-5 at the fusion site. Our data show that immediately after internaliz ation the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the com ponents of the secretory vesicle.