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Published on: March 14, 2019
ESCRT-0 assembles as a heterotetrameric complex on membranes and binds multiple ubiquitinylated cargoes
Jonathan R Mayers1, Ian Fyfe, Amber L Schuh
1Department of Biomolecular Chemistry, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706, USA.
The Journal of Biological Chemistry
|January 4, 2011
Summary
The ESCRT-0 complex, comprising Hrs and STAM, binds ubiquitin non-cooperatively. Hrs exhibits higher ubiquitin affinity, suggesting its primary role in cargo recruitment for multivesicular endosome biogenesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Biochemistry
Background:
- The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is crucial for multivesicular endosome (MVE) biogenesis.
- ESCRT-0, composed of Hrs and STAM, interacts with ubiquitylated substrates targeted for lysosomal degradation.
Purpose of the Study:
- To comprehensively analyze ESCRT-0:ubiquitin interactions and quantify binding affinities.
- To elucidate the assembly and structural organization of ESCRT-0 on endosomal membranes.
Main Methods:
- Isothermal titration calorimetry (ITC) to measure ubiquitin-binding domain (UBD) affinities.
- Atomic force microscopy (AFM) to visualize ESCRT-0 assembly on lipid bilayers.
- Hydrodynamic analysis to determine the quaternary structure of endogenous ESCRT-0.
Main Results:
- Ubiquitin binding to ESCRT-0 UBDs is non-cooperative.
- The Hrs double ubiquitin-interacting motif (DUIM) shows over 2-fold higher affinity for ubiquitin than STAM UBDs.
- ESCRT-0 forms heterodimers and heterotetramers on membranes, existing primarily as heterotetramers in vivo.
Conclusions:
- Hrs is the major ubiquitin-binding protein within the ESCRT-0 complex.
- A revised model for ESCRT-0 function in endosomal cargo recruitment and concentration is proposed.
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