Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for 
reverse membrane budding

Alonso Y Adell, Manuel; Migliano, Simona M.; Upadhyayula, Srigokul; Bykov, Yury S.; Sprenger, Simon; Pakdel, Mehrshad; Vogel, Georg F.; Jih, Gloria; Skillern, Wesley; Behrouzi, Reza; Babst, Markus; Schmidt, Oliver; Hess, Michael W.; Briggs, John A. G.; Kirchhausen, Tomas; Teis, David

doi:10.7554/eLife.31652

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Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

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Pakdel, Mehrshad
von Blume, Julia / Molecular Basis of Protein Trafficking, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Alonso Y Adell, M., Migliano, S. M., Upadhyayula, S., Bykov, Y. S., Sprenger, S., Pakdel, M., et al. (2017). Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding. eLife, 6: e31652. doi:10.7554/eLife.31652.

Cite as: https://hdl.handle.net/11858/00-001M-0000-002E-968A-A

Abstract

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.