Binding to Any ESCRT Can Mediate Ubiquitin-Independent Cargo Sorting

Abstract

We constructed fusion proteins that bind to endosomes and interact with ESCRTs. The trafficking analysis of these proteins showed that binding to ESCRTs caused either recycling (mimicking ESCRT machinery) or mediated ubiquitin-independent sorting into the MVB pathway (mimicking cargo). Furthermore, efficient MVB sorting of soluble proteins was observed. The ESCRT (endosomal sorting complex required for transport) machinery is known to sort ubiquitinated transmembrane proteins into vesicles that bud into the lumen of multivesicular bodies (MVBs). Although the ESCRTs themselves are ubiquitinated they are excluded from the intraluminal vesicles and recycle back to the cytoplasm for further rounds of sorting. To obtain insights into the rules that distinguish ESCRT machinery from cargo we analyzed the trafficking of artificial ESCRT-like protein fusions. These studies showed that lowering ESCRT-binding affinity converts a protein from behaving like ESCRT machinery into cargo of the MVB pathway, highlighting the close relationship between machinery and the cargoes they sort. Furthermore, our findings give insights into the targeting of soluble proteins into the MVB pathway and show that binding to any of the ESCRTs can mediate ubiquitin-independent MVB sorting. © 2013 John Wiley & Sons A/S.