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Membrane scission by the ESCRT-III complex

Abstract

The endosomal sorting complex required for transport (ESCRT) system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals.

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Figure 1: ESCRT-III and Vps4 bind to GUV membranes.
Figure 2: Uptake of the bulk phase and vesicle scission by ESCRT-III.
Figure 3: Three-dimensional reconstruction of an ESCRT-III-treated GUV.
Figure 4: Contributions of individual ESCRT-III subunits to ILV formation.
Figure 5: Vps4 and ATP induce a second cycle of ILV formation.
Figure 6: A simple hypothesis for the mechanism of the ESCRT-III-Vps4 membrane scission cycle.

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Acknowledgements

We thank V. Schram and the NICHD imaging core facility for use of the LSM5 LiveDuo microscope, D. Yang and G. Patterson for providing purified Vps4 and GFP, respectively, B. Beach for assistance with subcloning, E. Tyler for generating Fig. 6 and Supplementary Movie 3, Y. Im for assistance with Supplementary Fig. 1, C. Biertümpfel and W. Yang for use of their light-scattering instrument, and members of the Hurley and Lippincott-Schwartz laboratories for discussions. This research was supported by the Intramural Program of the National Institutes of Health, NICHD (J.L.-S.), NIDDK (J.H.H.) and IATAP (J.H.H. and J.L.-S.). T.W. is an EMBO long-term fellow.

Author Contributions T.W. and C.W. prepared GUVs and carried out confocal microscopy; T.W. purified and labelled proteins; C.W. carried out the three-dimensional reconstruction; T.W., C.W., J.L.-S. and J.H.H. analysed data; J.H.H. designed the study and wrote the manuscript.

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Correspondence to James H. Hurley.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S3 with Legends and Supplementary Movies Legends S1-S3 (PDF 1670 kb)

Supplementary Movie S1

This file shows 3D reconstruction of a GUV (see file s1 for full legend). (MOV 14472 kb)

Supplementary Movie S2

The file shows ILV dynamics (see file s1 for full legend). (MOV 1444 kb)

Supplementary Movie S3

The file shows animation of model for ILV scission (see file s1 for full legend). (MOV 8429 kb)

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Wollert, T., Wunder, C., Lippincott-Schwartz, J. et al. Membrane scission by the ESCRT-III complex. Nature 458, 172–177 (2009). https://doi.org/10.1038/nature07836

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