UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes

Eps15 is an endocytic adaptor protein that stimulates clathrin-mediated endocytosis. Among other interactions, Eps15 binds ubiquitin via UIM domains, recruiting ubiquitinated cargo into clathrin-coated vesicles. In EGF-treated cells, Eps15 also localizes to endosomes. The basis of this localization is not known.

We show for the first time that ubiquitin is sufficient for Eps15 recruitment to endosomes. We speculate that Eps15 recruitment to ubiquitin-rich endosomes may reduce the level of Eps15 at the plasma membrane, slowing endocytosis to allow time for processing of ubiquitinated cargo in endosomes.

We show that accumulation of ubiquitinated cargo can recruit Eps15 to endosomes via UIM domain interactions. First, treatment of SK-Br-3 breast cancer cells, which overexpress the EGFR family member ErbB2, with geldanamycin to promote receptor ubiquitination and endosomal transport, recruited FLAG-Eps15 to endosomes. Two in-frame ubiquitin constructs, PM-GFP-Ub (retained in endosomes after endocytosis), and GFP-FYVE-UbΔGG (targeted directly to endosomes) also recruited Eps15 to endosomes, as did slowing endosome maturation with constitutively-active Rab5-Q79L. Endosomal recruitment required the UIM domains, but not the N-terminal EH domains or central coiled-coil domains, of Eps15. Silencing of the endosomal Eps15 binding partner Hrs did not affect recruitment of Eps15 to ubiquitin-enriched endosomes. In fact, Hrs silencing itself modestly recruited Eps15 to endosomes, probably by accumulating endogenous ubiquitinated cargo. Eps15 silencing did not affect lysosomal degradation of ubiquitinated ErbB2; however, GFP-FYVE-UbΔGG overexpression inhibited internalization of EGFR and transferrin receptor. Conclusions: We show for the first time that ubiquitin is sufficient for Eps15 recruitment to endosomes. We speculate that Eps15 recruitment to ubiquitin-rich endosomes may reduce the level of Eps15 at the plasma membrane, slowing endocytosis to allow time for processing of ubiquitinated cargo in endosomes.