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

TLDR

The ESCRT machinery mediates reverse membrane scission. The authors propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT‑III filaments. The model predicts that Vps4‑driven assembly of ESCRT‑III filaments causes cargo crowding, inward membrane buckling, bud neck constriction, and ILV formation by vesicle fission. Live‑cell imaging and EM reveal that ESCRT‑III subunits polymerize rapidly on yeast endosomes with at least two Vps4 hexamers, productive budding requires additional Vps4 hexamers, the process involves continuous stochastic exchange, ATPase‑dependent membrane budding, an all‑or‑none release step, and is halted by acute Vps4 recruitment disruption.

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.