Hrs and STAM function synergistically to bind ubiquitin-modified cargoes in vitro.

The turnover of integral membrane proteins requires a specialized transport pathway mediated by components of the endosomal sorting complex required for transport (ESCRT) machinery. In most cases, entry into this pathway requires that cargoes undergo ubiquitin-modification, thereby facilitating their sequestration on endosomal membranes by specific, ubiquitin-binding ESCRT subunits. However, requirements underlying initial cargo recognition of mono-ubiquitinated cargos remain poorly defined. In this study, we determine the capability of each ESCRT complex that harbors a ubiquitin-binding domain to bind a reconstituted integral membrane cargo (VAMP2), which has been covalently linked to mono-ubiquitin. We demonstrate that ESCRT-0, but not ESCRT-I or ESCRT-II, is able to associate stably with the mono-ubiquitinated cargo within a lipid bilayer. Moreover, we show that the ubiquitin-binding domains in both Hrs and STAM must be intact to enable cargo binding. These results indicate that the two subunits of ESCRT-0 function together to bind and sequester cargoes for downstream sorting into intralumenal vesicles.