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

Elife. 2017 Oct 11;6:e31652. doi: 10.7554/eLife.31652.

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.

Keywords: 3D tracking; ESCRT; S. cerevisiae; cell biology; electron tomography; endosomes; high pressure freeze substitution; lattice light sheet microscopy; membrane budding.

MeSH terms

  • Adenosine Triphosphatases / metabolism*
  • Electron Microscope Tomography
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Endosomes / metabolism*
  • Intracellular Membranes / metabolism*
  • Microscopy, Fluorescence
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Saccharomyces cerevisiae Proteins
  • VPS4 protein, S cerevisiae
  • Adenosine Triphosphatases