Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments

Elife. 2015 Dec 15;4:e12548. doi: 10.7554/eLife.12548.

Abstract

The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30 Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling.

Keywords: ESCRT-III; PDS spectroscopy; S. cerevisiae; Snf7; X-ray crystal structure; biophysics; cell biology; protofilament; structural biology; structural rearrangement.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Endosomal Sorting Complexes Required for Transport / chemistry*
  • Endosomal Sorting Complexes Required for Transport / genetics
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Endosomes / metabolism*
  • Membranes / metabolism*
  • Models, Molecular
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Binding
  • Protein Conformation
  • Protein Multimerization*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spectrum Analysis

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Mutant Proteins
  • SNF7 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins