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Updated: Jun 3, 2026

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells
Published on: June 30, 2021
Decoupling of activation and effector binding underlies ARF6 priming of fast endocytic recycling
Guillaume Montagnac1, Hélène de Forges, Elizabeth Smythe
1Centre de Recherche, Institut Curie, CNRS, UMR 144, 26 rue d'Ulm, 75248 Paris Cedex 05, France. guillaume.montagnac@curie.fr
Abstract:
The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.
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