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Arf6 exchange factor EFA6 and endophilin directly interact at the plasma membrane to control clathrin-mediated endocytosis

  1. Michel Francoa,1
  1. aInstitut 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
  2. bCentre Commun de Microscopie Appliquée, Université de Nice Sophia Antipolis, 06103 Nice, France

  1. Edited by Martha Vaughan, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, and approved May 27, 2014 (received for review January 22, 2014)

Significance

The small G protein ADP-ribosylation factor 6 (Arf6) and the exchange factor for Arf6 (EFA6) are involved in endocytic vesicular transport, but their precise functions remain unclear. The Bin/Amphiphysin/Rvs (BAR) domain containing endophilin is known to couple fission to uncoating of the clathrin-coated vesicles. Here, we identified endophilin as a direct interactor of EFA6. We analyzed in vitro the effect of this interaction on EFA6 guanine nucleotide exchange factor activity, and on endophilin lipid binding and remodeling activities. We then studied in vivo the role of the two proteins in transferrin receptor endocytosis. Our results suggest a model in which EFA6 recruits endophilin on flat areas of endocytic zones of the plasma membrane, where endophilin cooperates with EFA6 to activate Arf6 and regulate 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.

Footnotes

  • ↵1To whom correspondence should be addressed. E-mail: franco{at}ipmc.cnrs.fr.

  • Author contributions: F.L. and M.F. designed research; S.B., E.M., M.P., and S.L.-G. performed research; S.B., E.M., F.B., F.L., and M.F. analyzed data; M.F. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1401186111/-/DCSupplemental.

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