| Endocytosis is the internalization of material from the extracellular environment and cell periphery. There are multiple types of endocytotic pathways, however, they can be simplified based on their requirement for the coat protein, clathrin. Clathrin-dependent pathways are well characterized and a great deal is known about the molecular machinery involved in its functioning. On the other hand, little is known about proteins that are important in cargo sorting, cargo sequestering, and vesicle formation in clathrin-independent trafficking.;We show here that the adaptor protein, AP-2, functions in Arf6-mediated clathrin-independent trafficking by promoting the recycling of cargo. While depletion of AP-2 or clathrin has no effect on internalization of Arf6 cargo, it alters the subcellular localization of both MHCI and beta1 Integrin to a perinuclear locale. However, only knockdown of AP-2 enhances targeting of Arf6 cargo to the lysosome for degradation. In alpha-adaptin knockdown cells, total levels of MHCI are lower and can be rescued with the lysosomal inhibitor Bafilomycin A1. We also show that the half-life of MHCI is reduced in AP-2-knockdown cells. Furthermore, co-localization of beta1 Integrin with the lysosomal marker CD63 is enhanced in cells depleted of AP-2.;Activation of the Arf6 GTPase is important in regulating many cellular processes. Recent work by our laboratory identified TRE17 as a novel regulator of Arf6. TRE17's TBC domain binds Arf6-GDP and induces activation of Arf6 by an unknown mechanism. We report here that TRE17/Arf6 binding is required for TRE17-mediated activation of Arf6. A triple point mutant of TRE17 deficient in binding Arf6 (TRE17/Arf6-) is significantly impaired in its ability to activate Arf6. This perturbation in interaction is not caused by a gross mis-folding of the protein as TRE17/Arf6- is still capable of binding Calmodulin. Furthermore, we show that ubiquitination is not required for binding or activation of Arf6 by TRE17.;In summary, this work gives further insight into non-clathrin endocytosis, in particular the endocytic pathway mediated by Arf6. Furthermore, it begins to shed some light on mechanisms of regulating this GTPase. |
