The adaptor complex AP-2 regulates post-endocytic trafficking through the non-clathrin Arf6-dependent endocytic pathway

Abstract

The ADP-ribosylation factor 6 (Arf6) GTPase functions as a key regulator of endocytic trafficking, participating in clathrin-independent endocytosis in most cell types. Unexpectedly, we found that siRNA-mediated depletion of clathrin or of adaptor protein 2 (AP-2)-complex subunits alters trafficking of Arf6 pathway cargo proteins, such as major histocompatibility complex class I (MHCI) and β1 integrin. Internalization of these cargoes from the plasma membrane was not affected in cells depleted of clathrin, but was modestly delayed in cells lacking AP-2. Furthermore, depletion of clathrin or AP-2 altered the intracellular distribution of MHCI and β1 integrin, inducing clustering in a perinuclear region. Despite this altered localization in both depleted populations, enhanced lysosomal targeting of MHCI was observed uniquely in cells that lack AP-2. Total levels of MHCI were modestly but consistently reduced in AP-2-depleted cells, and restored by the lysosomal inhibitor bafilomycin A. Furthermore, the half-life of surface-derived MHCI was reduced in AP-2-depleted cells. Consistent with enhanced degradative sorting, colocalization of Arf6 cargo with the late endosome and lysosome markers CD63 and Lamp1 was increased in cells depleted of AP-2 but not clathrin. These studies indicate a role for AP-2 in maintaining normal post-endocytic trafficking through the Arf6-regulated, non-clathrin pathway, and reveal pervasive effects of clathrin and AP-2 depletion on the endosomal and lysosomal system.

Numb Regulates Post-endocytic Trafficking and Degradation of Notch1

“…This suggests that the Numb interaction with AP-2 is important for the post-endocytic trafficking of Notch1. In keeping with this notion, a role for AP-2 in the post-endocytic trafficking of cargo through a clathrin-independent pathway has recently been described (61).…”

Tissue‐ and development‐dependent expression of the small GTPase Arf6 in mice

“…In contrast, Arf6 mainly localizes at the plasma membrane, where it is involved in a wide variety of cellular events. These cellular events include actin cytoskeleton reorganization (Song et al, 1998;Honda et al, 1999), regulation of adherens junctions (Palacios et al, 2001), clathrin-mediated endocytosis (Radhakrishna and Donaldson, 1997;Altschuler et al, 1999;Radhakrishna et al, 1999), phagocytosis (Zhang et al, 1998;Uchida et al, 2001), exocytosis (Galas et al, 1997;Vitale et al, 2002), and post-endocytic vesicle trafficking (Lau and Chou, 2008). Thus, cellular events, in which Arf6 is involved, differ from those regulated by other members of the Arf family.…”

Internalization of Coxsackievirus A9 Is Mediated by β2-Microglobulin, Dynamin, and Arf6 but Not by Caveolin-1 or Clathrin

“…Recent studies have shown that some of these pathways differ only slightly from each other, and certain endocytic components can participate in more than just one pathway (35,41,55). Most of the research carried out on enterovirus endocytosis has been done with echovirus 1 (EV1), coxsackievirus B3 (CBV3), and poliovirus (PV).…”