SNX15 links clathrin endocytosis to the PtdIns(3)P early endosome independent of the APPL1 endosome

Sorting nexins (SNXs) are key regulators of the endosomal network. In designing an RNAi-mediated loss-of-function screen, we establish that of thirty human SNXs only SNX3, SNX5, SNX9, SNX15 and SNX21 appear to regulate EGF receptor degradative sorting. Suppression of SNX15 results in a delay in receptor degradation arising from a defect in movement of newly internalised EGF receptor-labelled vesicles into early endosomes. Besides a PtdIns(3)P- and PX domain-dependent association to early endosomes, SNX15 also associates with clathrin-coated pits and clathrin-coated vesicles via direct binding to clathrin through a non-canonical clathrin-binding box. From live cell imaging, the activated EGF receptor enters distinct sub-populations of SNX15- and APPL1-labelled peripheral endocytic vesicles, which do not undergo heterotypic fusion. The SNX15-decorated receptor-containing sub-population does however undergo direct fusion with the Rab5-labelled early endosome. Our data is consistent with a model in which the EGF receptor enters the early endosome following clathrin-mediated endocytosis through at least two parallel pathways: maturation via an APPL1-intermediate compartment and an alternative more direct fusion between SNX15 decorated endocytic vesicles and the Rab5-positive early endosome.