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.
Highlights
The ESCRT machinery mediates ‘reverse’ membrane budding in distinct but topologically related processes, including formation of intraluminal vesicles (ILVs) in multivesicular bodies (MVBs), plasma-membrane repair and microvesicle formation, abscission in cytokinesis, budding of certain viruses, and nuclear membrane resealing (Christ et al, 2017) as well as membrane deformation towards the cytosol (McCullough et al, 2015)
Direct fusion of eGFP to the C-terminus of ESCRT-III subunits interferes with function (Teis et al, 2008), probably because of incompatibility with the periodicity of ESCRT-III polymers (Tang et al, 2015) (Shen et al, 2014)
The models invoke the properties of ESCRT-III polymerization linked to a membrane surface as the driving force for membrane deformation; free energy input from ATP hydrolysis by Vps4 re-sets the system by recycling the ESCRT-III components
Summary
The ESCRT (endosomal sorting complexes required for transport) machinery mediates ‘reverse’ membrane budding in distinct but topologically related processes, including formation of intraluminal vesicles (ILVs) in multivesicular bodies (MVBs), plasma-membrane repair and microvesicle formation, abscission in cytokinesis, budding of certain viruses, and nuclear membrane resealing (Christ et al, 2017) as well as membrane deformation towards the cytosol (McCullough et al, 2015). Budding requires recruitment of ESCRT-III heteropolymers and the type I AAA+ (ATPase associated with a variety of cellular activities) ATPase, Vps (Babst et al, 2002). ESCRT-III includes a major structural component, Snf, core subunits Vps, Vps, and Vps, and accessory proteins Did, Vps and Ist (McCullough et al, 2013). Interaction of Vps with ESCRT-II triggers membrane recruitment of Snf, which in turn recruits Vps and Vps. Vps recruits Vps to these filaments and together with Vps may restrict Snf polymerization (Babst et al, 2002; Teis et al, 2008; Saksena et al, 2009; Teis et al, 2010). Snf alone or together with Vps and Vps, forms curved filaments (Henne et al, 2012; Mierzwa et al, 2017; Schoneberg et al, 2017)
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