ESCRT-0 marks an APPL1-independent transit route for EGFR between the cell surface and the EEA1-positive early endosome.

ESCRT-0 sorts ubiquitinated EGFR within the early endosome, so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here we employ a suite of software to localise ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whilst ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.