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

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.DOI: http://dx.doi.org/10.7554/eLife.12548.001

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes

“…In the ALIX case, the N-terminal BRO domain binds directly to a C-terminal CHMP4 helix located immediately downstream of the ordered ESCRT-III core domain (Figure 2). ALIX (Bro1) binding may thereby weaken autoinhibitory interactions, consistent with the observation that CHMP4 autoinhibition can be relieved by removing the terminal helix (Lata et al 2008b, 2009; Tang et al 2015, 2016). ALIX can dimerize (Pires et al 2009), and both ESCRT-II and ALIX may, therefore, help activate ESCRT-III subunits to form two filaments or even double-stranded filaments (Henne et al 2012, McCullough et al 2015, Mierzwa et al 2017, Teis et al 2010).…”

“…Structures are available for model ESCRT-III filaments visualized in two different contexts: ( a ) a high-resolution cryo-EM reconstruction of the double-stranded heteropolymeric filaments formed by CHMP1B and the ordered N-terminal ESCRT-III domain of IST1 (IST1 NTD ) (both free and in complex with nucleic acids) (McCullough et al 2015, Talledge et al 2018), and ( b ) linear filaments of truncated homologs of CHMP4 from S. cerevisiae (Snf7) and Drosophila (Shrub) that crystallized with similar lattice packing interactions, both of which recapitulate interactions seen in the CHMP1B strand of the IST1 NTD /CHMP1B filament (McMillan et al 2016, Tang et al 2015). Both sets of structures are informative but also suffer from some limitations.…”

“…Many ESCRT-III subunits can interconvert between two conformations: a closed, monomeric soluble state and an open, polymeric, membrane-bound state (Lata et al 2008a, Lin et al 2005, McCullough et al 2015, McMillan et al 2016, Tang et al 2015) (Figure 2 a , b ). Crystal structures of CHMP3 and IST1 demonstrate that the closed state is organized by a long N-terminal helical hairpin that spans the entire domain (Bajorek et al 2009, Muziol et al 2006, Xiao et al 2009).…”

“…Open conformations have been visualized in structures of human CHMP1B (McCullough et al 2015, Talledge et al 2018) and truncated CHMP4 homologs from S. cerevisiae (Snf7) and Drosophila (Shrub) (McMillan et al 2016, Tang et al 2015). The open conformations resemble an arm, with closed helices 1–3 rearranging to form an extended helical hairpin that defines the upper arm; helix 4 and helix A combining to form the helical forearm; and helix 5 (missing in the two CHMP4 structures) forming the hand.…”

Exosomes in developmental signalling

“…ESCRT-I and ESCRT-II, which also contain ubiquitin-interaction domains, cooperate with ESCRT-0 to generate a sorting domain with high avidity for ubiquitylated cargo at the site of ILV formation (Hurley, 2010;Raiborg and Stenmark, 2009). At the same time, they recruit ESCRT-III (Babst et al, 2002), which drives membrane deformation and constricts the neck of the resultant invagination (Babst et al, 2002;Chiaruttini et al, 2015;Henne et al, 2013;Tang et al, 2015;Wollert et al, 2009). De-ubiquitylating enzymes (DUBs) then remove ubiquitin from cargo (Agromayor and Martin-Serrano, 2006) and the ATPase VPS4, along with its co-factor VTA1, disassemble the ESCRT complex (Hurley, 2010), allowing its subunits to be recycled.…”