The adaptor complex AP-2 regulates post-endocytic trafficking through the non-clathrin Arf6-dependent endocytic pathway.
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 beta1 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 beta1 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.
Lau
Alan W
AW
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Chou
Margaret M
MM
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D045744:Cell Line, Tumor; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D060149:Tetraspanin 30; D014162:Transfection; D052118:Lysosomal-Associated Membrane Protein 1
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D015703:Antigens, CD; D002966:Clathrin; D015395:Histocompatibility Antigens Class I; D019012:Integrin beta1; D051907:Lysosomal Membrane Proteins; D018942:Macrolides; D010980:Platelet Membrane Glycoproteins; D034741:RNA, Small Interfering; D060149:Tetraspanin 30; C554586:CD63 protein, human; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C057620:bafilomycin A; C000715348:ARF6 protein, human