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.

ESCRT-0 Protein Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate (Hrs) Is Targeted to Endosomes Independently of Signal-transducing Adaptor Molecule (STAM) and the Complex Formation with STAM Promotes Its Endosomal Dissociation

“…Previous studies have reported that cooperative ubiquitin binding by the Hrs⅐STAM complex is required for endosomal sorting of the ubiquitylated cargo (8,14). Moreover, there is a report that in Caenorhabditis elegans Hrs and STAM assemble into a heterotetrameric complex on the membrane both in vitro and in vivo (22). To investigate the effect of STAM co-expression on Hrs accumulation and endosome morphology, FLAGtagged STAM1 was overexpressed in 2HP67 cells.…”

Reciprocal cross-regulation between RNF41 and USP8 controls cytokine receptor sorting and processing

“…ESCRT-0 assembles as a heterotetrameric complex on the endosomal membrane, providing ten ubiquitin-binding domains (UBD) (Lange et al, 2012;Mayers et al, 2011), which recognize and sort ubiquitylated cargo. However, the ESCRT-0 complex can also mediate ubiquitin-independent trafficking.…”

Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

“…C. elegans FCHO-1, EHS-1, and ITSN-1 antibodies were raised in rabbits, which were immunized with either a GST fusion to a fragment of FCHO-1 (amino acids 1-274), a GST fusion to a fragment of ITSN-1 (amino acids 1-283), or a polyhistidine-tagged form of full length EHS-1 produced in Escherichia coli and affinity purified. Antibodies directed against HGRS-1, STAM-1, EEA-1, APA-2, and RAB-5 have been described (40,41). Confocal images were acquired on a sweptfield confocal microscope (Nikon Ti-E), and TIRF imaging was conducted on a Nikon TE2000E equipped with a TIRF illuminator.…”

“…These findings also raise the possibility that ubiquitin-mediated signaling during pit maturation is coupled to ESCRT-0 recruitment. Because membrane-associated ESCRT-0 is capable of binding at least eight ubiquitin molecules simultaneously (40), the localized enrichment of ubiquitin-modified cargoes at a subset of coated pits may help to specify the timing and/or site of its recruitment. Furthermore, the additional presence of endocytic adaptors, acidic phospholipids, and clathrin, all of which can associate with ESCRT-0, may further promote a level of coincidence detection that is necessary to enhance targeting to sites of clathrin-mediated endocytosis.…”