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. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to alpha-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.
Coppola
T
T
Institut de Biologie Cellulaire et de Morphologie, University of Lausanne, Switzerland.
Hirling
H
H
Perret-Menoud
V
V
Gattesco
S
S
Catsicas
S
S
Joberty
G
G
Macara
I G
IG
Regazzi
R
R
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D033921:Vesicular Transport Proteins; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism*
D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins