Current Biology
Volume 21, Issue 7, 12 April 2011, Pages 574-579
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Decoupling of Activation and Effector Binding Underlies ARF6 Priming of Fast Endocytic Recycling

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Summary

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.

Highlights

► ARF6 accumulates in clathrin-coated pits (CCPs) in a GTP-dependent manner ► CCP-localized ARF6 is accessible to downstream effectors after membrane uncoating ► ARF6 does not control clathrin-dependent endocytosis but regulates fast recycling ► An ARF6/JIP4 interaction is required for fast recycling

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