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Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers
Published on: December 6, 2019
Endosomal sorting complex required for transport (ESCRT) complexes induce phase-separated microdomains in supported
Evzen Boura1, Vassili Ivanov, Lars-Anders Carlson
1Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
The Journal of Biological Chemistry
|June 22, 2012
Summary
Human ESCRT-II protein self-assembles into clusters on cell membranes, requiring cholesterol. These functional clusters bind ubiquitin and ESCRT-III, potentially aiding membrane budding in cellular transport.
Area of Science:
- Cell Biology
- Biochemistry
- Membrane Trafficking
Background:
- The endosomal sorting complex required for transport (ESCRT) machinery mediates ubiquitinated cargo trafficking to lysosomes.
- ESCRT function involves membrane budding reactions directed away from the cytosol.
Purpose of the Study:
- To investigate the self-assembly and membrane interaction properties of human ESCRT-II.
- To determine the functional significance of ESCRT-II clustering in membrane trafficking.
Main Methods:
- Utilized supported lipid bilayers to study ESCRT-II self-assembly.
- Investigated ESCRT-II binding to ubiquitin and VPS20 using nanomolar concentration assays.
- Analyzed the role of cholesterol in ESCRT-II cluster formation and membrane domain organization.
Main Results:
- Human ESCRT-II self-assembles into molecular clusters (10-100 molecules) on lipid bilayers.
- These clusters functionally bind ubiquitin and ESCRT-III (VPS20) with observed solution/crystal stoichiometries.
- Cluster formation is dependent on cholesterol (>10 mol %) and induces ordered membrane domains, excluding specific dyes.
Conclusions:
- ESCRT-II clustering on membranes is cholesterol-dependent and functionally relevant.
- ESCRT complexes can induce lateral lipid phase separation, a property that may facilitate ESCRT-mediated membrane budding.
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