title: Arf6 exchange factor EFA6 and endophilin directly interact at the plasma membrane to control clathrin-mediated endocytosis.
abstract: Members of the Arf family of small G proteins are involved in membrane traffic and organelle structure. They control the recruitment of coat proteins, and modulate the structure of actin filaments and the lipid composition of membranes. The ADP-ribosylation factor 6 (Arf6) isoform and the exchange factor for Arf6 (EFA6) are known to regulate the endocytic pathway of many different receptors. To determine the molecular mechanism of the EFA6/Arf6 function in vesicular transport, we searched for new EFA6 partners. In a two-hybrid screening using the catalytic Sec7 domain as a bait, we identified endophilin as a new partner of EFA6. Endophilin contains a Bin/Amphiphysin/Rvs (BAR) domain responsible for membrane bending, and an SH3 domain responsible for the recruitment of dynamin and synaptojanin, two proteins involved, respectively, in the fission and uncoating of clathrin-coated vesicles. By using purified proteins, we confirmed the direct interaction, and identified the N-BAR domain as the binding motif to EFA6A. We showed that endophilin stimulates the catalytic activity of EFA6A on Arf6. In addition, we observed that the Sec7 domain competes with flat but not with highly curved lipid membranes to bind the N-BAR. In cells, expression of EFA6A recruits endophilin to EFA6A-positive plasma membrane ruffles, whereas expression of endophilin rescues the EFA6A-mediated inhibition of transferrin internalization. Overall, our results support a model whereby EFA6 recruits endophilin on flat areas of the plasma membrane to control Arf6 activation and clathrin-mediated endocytosis.
authors: Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 7275, Centre National de la Recherche Scientifique, Université de Nice Sophia Antipolis, 06560 Valbonne, France; and.
authors: Sonia
authors: S
authors: Boulakirba
authors: Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 7275, Centre National de la Recherche Scientifique, Université de Nice Sophia Antipolis, 06560 Valbonne, France; and.
authors: Eric
authors: E
authors: Macia
authors: Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 7275, Centre National de la Recherche Scientifique, Université de Nice Sophia Antipolis, 06560 Valbonne, France; and.
authors: Mariagrazia
authors: M
authors: Partisani
authors: Centre Commun de Microscopie Appliquée, Université de Nice Sophia Antipolis, 06103 Nice, France.
authors: Sandra
authors: S
authors: Lacas-Gervais
authors: Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 7275, Centre National de la Recherche Scientifique, Université de Nice Sophia Antipolis, 06560 Valbonne, France; and.
authors: Frédéric
authors: F
authors: Brau
authors: Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 7275, Centre National de la Recherche Scientifique, Université de Nice Sophia Antipolis, 06560 Valbonne, France; and.
authors: Frédéric
authors: F
authors: Luton
authors: Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 7275, Centre National de la Recherche Scientifique, Université de Nice Sophia Antipolis, 06560 Valbonne, France; and franco@ipmc.cnrs.fr.
authors: Michel
authors: M
authors: Franco
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D003001:Cloning, Molecular; D004705:Endocytosis / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014966:Xanthenes
chemical_list: D000089982:ADP-Ribosylation Factor 6; D048868:Adaptor Proteins, Signal Transducing; D002966:Clathrin; D020662:Guanine Nucleotide Exchange Factors; C504474:Psd protein, mouse; D011994:Recombinant Proteins; C498434:Sh3gl2 protein, mouse; D014966:Xanthenes; C034657:Texas red; C000715727:Arf6 protein, mouse
keywords: membrane curvature; small GTP-binding proteins; vesicular trafficking