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.

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Adell MAY, Migliano SM, Upadhyayula S, Bykov YS, Sprenger S, Pakdel M, Vogel GF, Jih G, Skillern W, Behrouzi R, ... Show all

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