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.
Highlights
IntroductionThe endosomal sorting complexes required for transport (ESCRTs) are membrane remodeling machinery that mediate diverse fundamental cellular processes, including the biogenesis of multivesicular body (MVB) during receptor down-regulation (Katzmann et al, 2001), enveloped virus budding (Garrus et al, 2001), cytokinesis (Carlton and Martin-Serrano, 2007), plasma membrane repair (Jimenez et al, 2014), nuclear pore complex assembly (Webster et al, 2014), and nuclear envelope reformation (Olmos et al, 2015; Vietri et al, 2015)
The endosomal sorting complexes required for transport (ESCRTs)-III machinery plays a critical role in numerous fundamental cellular processes, including multivesicular body (MVB) biogenesis, viral budding and cytokinesis, indicating an ancient and conserved membrane remodeling mechanism
The importance of understanding this mechanism is bolstered by the fact that this is so distinct from all other well characterized membrane budding processes, which invariantly bud into the cytoplasm
Summary
The endosomal sorting complexes required for transport (ESCRTs) are membrane remodeling machinery that mediate diverse fundamental cellular processes, including the biogenesis of multivesicular body (MVB) during receptor down-regulation (Katzmann et al, 2001), enveloped virus budding (Garrus et al, 2001), cytokinesis (Carlton and Martin-Serrano, 2007), plasma membrane repair (Jimenez et al, 2014), nuclear pore complex assembly (Webster et al, 2014), and nuclear envelope reformation (Olmos et al, 2015; Vietri et al, 2015). ESCRT-II sets the architecture and initiates the assembly of the ESCRT-III complex, which together with Vps is responsible for remodeling endosomal membranes (Henne et al, 2011; Hurley and Hanson, 2010). ESCRT-III is a unique protein complex in that it is metastable and conformationally dynamic, forming hetero-oligomeric filaments of multiple subunits on membranes (Saksena et al, 2009; Teis et al, 2008). Its subunits are inactive monomers in the cytoplasm, which activate and assemble
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