title: Hrs and STAM function synergistically to bind ubiquitin-modified cargoes in vitro.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom.
authors.forename: Hirohide
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Mayers
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom.
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin-Madison School of Medicine and Public Health, Madison, Wisconsin. Electronic address: audhya@wisc.edu.
authors.forename: Anjon
authors.initials: A
authors.lastname: Audhya
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D051379:Mice; D018625:Microscopy, Atomic Force; D025801:Ubiquitin / Q000737:chemistry*; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry*
chemical_list: D029742:Caenorhabditis elegans Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008051:Lipid Bilayers; D008081:Liposomes; D025801:Ubiquitin; D050988:Vesicle-Associated Membrane Protein 2; C494808:vesicle-associated membrane protein 2, mouse