How the ESCRTs package ubiquitinated membrane proteins for destruction

Professor James H. Hurley
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, US Department of Health & Human Services
27 September 2012 - 12:00pm
Rountree Room 356, Level 3, Biological Sciences Building

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The ESCRT complexes catalyze one of the most unusual membrane remodeling events in cell biology. ESCRT-mediated membrane neck cleavage is critical for the biogenesis of multivesicular bodies (MVBs), key intermediates in lysosomal sorting; cytokinesis; and the detachment of HIV-1 and other enveloped viruses from the plasma membrane of infected cells. Moreover, ESCRTs are responsible for ubiquitinated cargo sorting and membrane budding into multivesicular bodies. The biogenesis of MVBs was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, I, II, and III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. ESCRT-0 forms domains of clustered cargo but does not deform membranes.

ESCRT-I and II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction.

Structural analysis of the ESCRT-I and –II supercomplex in solution offers hints as how this complex-of-complexes might stablize bud necks and integrate the process from cargo sorting through membrane scission. A computational analysis will be presented that describes the role of lipid domain separation, together with experimental evidence that ESCRTs can promote lateral segregation of membrane lipids. Finally, the similarities and differences between the functions of ESCRTs in endosomal budding and the release of HIV-1 particles will be considered.