title: ESCRT-0 assembles as a heterotetrameric complex on membranes and binds multiple ubiquitinylated cargoes simultaneously.
abstract: The ESCRT machinery consists of multiple protein complexes that collectively participate in the biogenesis of multivesicular endosomes (MVEs). The ESCRT-0 complex is composed of two subunits, Hrs and STAM, both of which can engage ubiquitinylated substrates destined for lysosomal degradation. Here, we conduct a comprehensive analysis of ESCRT-0:ubiquitin interactions using isothermal titration calorimetry and define the affinity of each ubiquitin-binding domain (UBD) within the intact ESCRT-0 complex. Our data demonstrate that ubiquitin binding is non-cooperative between the ESCRT-0 UBDs. Additionally, our findings show that the affinity of the Hrs double ubiquitin interacting motif (DUIM) for ubiquitin is more than 2-fold greater than that of UBDs found in STAM, suggesting that Hrs functions as the major ubiquitin-binding protein in ESCRT-0. In vivo, Hrs and STAM localize to endosomal membranes. To study recombinant ESCRT-0 assembly on lipid bilayers, we used atomic force microscopy. Our data show that ESCRT-0 forms mostly heterodimers and heterotetramers of Hrs and STAM when analyzed in the presence of membranes. Consistent with these findings, hydrodynamic analysis of endogenous ESCRT-0 indicates that it exists largely as a heterotetrameric complex of its two subunits. Based on these data, we present a revised model for ESCRT-0 function in cargo recruitment and concentration at the endosome.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706, USA.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Mayers
authors.forename: Ian
authors.initials: I
authors.lastname: Fyfe
authors.forename: Amber L
authors.initials: AL
authors.lastname: Schuh
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
authors.forename: Anjon
authors.initials: A
authors.lastname: Audhya
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008051:Lipid Bilayers; D025801:Ubiquitin