Abstract
The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.
Highlights
The endosomal sorting complex required for transport (ESCRT)-0 complex proteins, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and signaltransducing adaptor molecule (STAM), cooperatively recognize ubiquitylated endosomal cargo
Hrs is targeted to endosomes independently of STAM binding and dissociates from endosomes in a STAM bindingdependent manner
We have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes
Summary
The ESCRT-0 complex proteins, Hrs and STAM, cooperatively recognize ubiquitylated endosomal cargo. The ESCRT-0 complex, which consists of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) 1/2 proteins in metazoa, recognizes ubiquitylated cargo and concentrates it into endosomal microdomains via its interaction with clathrin [7]. A reconstitution study revealed that ESCRT-0 forms a cluster with ubiquitylated cargo in endosomal microdomains [6] The recruitment of this cluster to the bud necks via its interaction with ESCRT-I causes the confinement of cargo proteins within the buds [6]. Endosomal accumulation of overexpressed Hrs causes a severe impairment in cargo degradation [13, 14] These experimental data suggest the functionally significant recruitment and dissociation cycle of ESCRT-0 on endosomes during cargo sorting. The behavior of ESCRT-0 described in this study is required for the maintenance of normal endosomal morphology and the effective degradation of membrane proteins
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
