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

5 years ago

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

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

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 second 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.

Publisher URL: https://elifesciences.org/articles/31652

DOI: 10.7554/eLife.31652