SNX15 links clathrin endocytosis to the PtdIns3P early endosome independently 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 30 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 phosphatidylinositol 3-phosphate- and PX-domain-dependent association to early endosomes, SNX15 also associates with clathrin-coated pits and clathrin-coated vesicles by direct binding to clathrin through a non-canonical clathrin-binding box. From live-cell imaging, it was identified that 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 are consistent with a model in which the EGF receptor enters the early endosome following clathrin-mediated endocytosis through at least two parallel pathways: maturation through an APPL1-intermediate compartment and an alternative more direct fusion between SNX15-decorated endocytic vesicles and the Rab5-positive early endosome.