Arf GTPase-activating protein ASAP1 interacts with Rab11 effector FIP3 and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome.
ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment.
Inoue
Hiroki
H
Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Ha
Vi Luan
VL
Prekeris
Rytis
R
Randazzo
Paul A
PA
D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D018385:Centrosome / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D020727:ADP-Ribosylation Factors; D048868:Adaptor Proteins, Signal Transducing; D020690:GTPase-Activating Proteins; D051550:I-kappa B Kinase; D034741:RNA, Small Interfering; D011990:Receptors, Transferrin; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C117891:ASAP1 protein, human; C491731:IKBKG protein, human