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

Abstract

The endosomal sorting complexes required for transport (ESCRT) pathway mediates cellular membrane remodeling and fission reactions. The pathway comprises five core complexes: ALIX, ESCRT-I, ESCRT-II, ESCRT-III, and Vps4. These soluble complexes are typically recruited to target membranes by site-specific adaptors that bind one or both of the early-acting ESCRT factors: ALIX and ESCRT-I/ESCRT-II. These factors, in turn, nucleate assembly of ESCRT-III subunits into membrane-bound filaments that recruit the AAA ATPase Vps4. Together, ESCRT-III filaments and Vps4 remodel and sever membranes. Here, we review recent advances in our understanding of the structures, activities, and mechanisms of the ESCRT-III and Vps4 machinery, including the first high-resolution structures of ESCRT-III filaments, the assembled Vps4 enzyme in complex with an ESCRT-III substrate, the discovery that ESCRT-III/Vps4 complexes can promote both inside-out and outside-in membrane fission reactions, and emerging mechanistic models for ESCRT-mediated membrane fission.

Keywords: AAA ATPase; ESCRT pathway; ESCRT-III; Vps4; membrane fission; membrane remodeling.

Figure 1. ESCRT-dependent membrane fission reactions.

(a) Cellular processes proposed to be ESCRT dependent are labeled in bold, cellular structures are labeled in normal font, membrane-specific adaptor proteins that recruit early-acting ESCRT factors are labeled in red (with human protein names in all caps and yeast protein names, where provided, in parentheses), and a stylized membrane receptor and its ligand are shown in blue/turquoise. Sites of ESCRT membrane remodeling are denoted by stylized double-stranded ESCRT-III filaments (green, with the membrane-associated strand in dark green) and rings of Vps4 (violet) or the related meiotic clade AAA ATPase, SPASTIN (dark purple). Box 1 shows the site of endocytic vesicle formation, with stylized BAR domain/dynamin assemblies (orange) formed about the neck of an endocytic vesicle. Box 2 shows the site of enveloped virus budding, with double-stranded ESCRT-III filaments stabilizing a negatively curved membrane tubule (see panel b). Box 3 shows a site of endosomal vesicle formation, with double-stranded ESCRT-III filaments stabilizing a positively curved membrane tubule (see panel c). Abbreviations: ILV, intralumenal vesicle; MVB, multivesicular body; NPC, nuclear pore complex. (b) Filaments containing the ESCRT-III protein CHMP4A can stabilize flat membranes and negatively curved plasma membrane tubules and thereby promote inside-out membrane fission reactions. The left and middle panels show electron micrographs of human cells overexpressing CHMP4A under conditions of reduced VPS4 ATPase activity (Hanson et al. 2008, McCullough et al. 2015). These electron micrographs show that cytoplasmic filaments containing CHMP4A can assemble on flat membranes (left panel) and can promote membrane tubule extrusion and coat the interior of the negatively curved tubules, consistent with a role in inside-out fission reactions (middle panel). The right panel shows a schematic depiction of the middle panel (side view), (c) Filaments containing the ESCRT-III protein CHMP1B can stabilize flat membranes and positively curved plasma membrane tubules and thereby promote outside-in membrane fission reactions (Cashikar et al. 2014, Hanson et al. 2008, McCullough et al. 2015). The three subpanels in panel c are analogous to those in panel b, except that CHMP1B promotes membrane tubule invagination and coats the exterior of the positively curved tubules. Panels b and c are adapted from McCullough et al. (2015) and reprinted with permission from AAAS.

Figure 2. Structures and binding partners of the ESCRT-III proteins.

(a) Secondary structure showing the five conserved helices that organize ESCRT-III proteins in the closed conformation. The terminal ligand-binding tail (red) is helical in most ESCRT-III complexes but can alternatively adopt a β-strand secondary structure in some cases (see panel c). ESCRT-III ligands and their approximate binding sites are shown above the secondary structure, (b) Structures of ESCRT-III subunits in their open and closed configurations. (Left to right) Crystal structure of the ESCRT-III subunit IST1_NTD in the closed conformation (from Bajorek et al. 2009), cryo-EM structure of CEIMP1B in the open conformation (from McCullough et al. 2015), and superposition of the closed (lighter shades, modeled) and open (darker shades) conformations of CHMP1B (from McCullough et al. 2015, Talledge et al. 2018). The N-terminal helical hairpin remains intact (and is extended upon opening), while the remaining helices either pack against the hairpin (closed conformation) or open to pack against other subunits in the CHMP1B filament (open conformation; see Figure 3b). (c) Structures of the C-terminal tails of ESCRT-III proteins (red) in complex with their two major classes of binding partners (blue-gray): BRO domain proteins such as ALIX and BROX (above) and MIT domain proteins such as VPS4, SPASTIN, AMSH, and LIP5 (Vta1) (below). Note the variety of distinct ways in which different ESCRT-III tails can bind BRO and MIT domains [denoted MIT-interacting motifs (MIMs) 1–5 in the MIT case]. Structures above are from McCullough et al. (2008) (left) and Mu et al. (2012) (right). Structures below (from left to right) are from Stuehell-Brereton et al. (2007), Kieffer et al. (2008), Yang et al. (2008), Solomons et al. (2011), and Skalieky et al. (2012).

Figure 3. ESCRT-III filament structures.

(a) End-on view of a turn of the N-terminal ESCRT-III domain of an IST1_NTD/CHMP1B filament surrounding a stylized lipid bilayer. IST1_NTD subunits are shown in red, CHMP1B subunits are shown in rainbow colors, and the lipid bilayer is shown in gray. The structure is from McCullough et al. (2015). (b) Side view of a segment of the IST1_NTD/CHMP1B filament (viewed from the membrane and corresponding to the wedge highlighted in panel a). Seven interacting CHMP1B subunits are shown, with just a single associated IST_NTD subunit shown for clarity. (c) Equivalent view showing a linear strand of Snf7_12–150 and emphasizing the equivalent packing of N-terminal helical hairpins in the two ESCRT-III strands. The structure is from Tang et al. (2015).

Figure 4. Models for stabilization of curved and flat membranes by BAR domain and ESCRT-III proteins.

(a) Illustrations showing how changing the angle between the end-associated BAR domain dimers (blue and orange subunits) can stabilize a continuum of differentially curved membranes (gray). Pairs of dimers from continuous BAR domain assemblies (end-on views) are shown in each case. Structural models are based on Mim & Unger (2012) and Mim et al. (2012) for the N-BAR case, Shimada et al. (2007) and Frost et al. (2008) for the F-BAR case, Guerrier et al. (2009) and Sporny et al. (2017) for the IF-BAR case, and Pykalainen et al. (2011) for the PINK-BAR case, (b) Illustrations showing how changes in intrinsic filament curvature could similarly allow ESCRT-III filaments (green) to stabilize a continuum of differentially curved membranes. (Top to bottom) A CHMP1B strand from the IST1_NTD/CHMP1B filament bound to a stylized membrane (from McCullough et al. 2015), structure of the linear strand of Snf7_12–150 (CHMP4) from a crystal lattice (from Tang et al. 2015) bound to a stylized membrane, and a hypothetical strand of Snf7_12–150 (CHMP4) subunits bound to a negatively curved membrane. The Snf7_12—150 (CHMP4) strand in the bottom panel was modeled by altering the angle between each successive subunit in the strand shown in the middle panel.

Figure 5. Structure, assembly, and mechanism of the Vps4 ATPase.

(a) Domain structure of a Vps4 monomer in complex with ATP. Structures are reproduced from Han et al. (2017) (ATPase cassette) and Obita et al. (2007) (MIT domain), (b) Top view of the asymmetric yeast Vps4 ring hexamer (subunits A–F) with associated dimeric VSL domains from Vta1 (beige), ESCRT-III peptide (green), and nucleotides (ATP, pink; ADP, dark red), (c) Same as panel b, viewed from the lower side and with subunit F and the VSL domains removed for clarity. (d) Type 1 binding pockets for alternating odd-numbered substrate amino acids, formed along the central channel of the Vps4 hexamer. Bound substrate amino acid side chains are shown explicitly, as are the Vps4 residues that compose the pocket (fully labeled in the second pocket). Note that the four intact amino acid binding sites form a helix around the bound ESCRT-III substrate in the central channel. Vps4 structure and color coding are the same as in panels b and c. (e) Type 2 binding pockets for alternating even-numbered substrate amino acids, formed along the central channel of the Vps4 hexamer. Bound substrate amino acid side chains are shown explicitly, as are the Vps4 residues that compose these pockets. Note that these binding sites form a second helix around the bound ESCRT-III substrate in the central channel. Vps4 structure and color coding are the same as in panels b and c. (f) Proposed mechanism of ESCRT-III translocation by Vps4. This panel shows the central translocation pore with representative Vps4 residues from the type 1 pocket (W206) and the type 2 pocket (M207) and with the ESCRT-III substrate (green) passing through the pore. Substrate translocation is proposed to occur as Vps4 subunits progress through states A to E while maintaining contacts with their respective substrate dipeptides. ATP hydrolysis at subunit D destabilizes the D/E interface and promotes displacement of subunit E toward the transitioning subunit F configuration, which allows for displacement of ADP and full release of the F subunit from the substrate (lower red arrow). Subsequent ATP binding allows subunit F to rejoin to the top of the helix (upper red arrow), where it packs against subunit A, binds the next substrate dipeptide, and assumes the subunit A configuration. Gray arrows show the relative direction of substrate peptide translocation. Panels b–f adapted from Han et al. (2017).

Figure 6. ESCRT-III filament topologies and membrane remodeling.

(a) Cone formation by double-stranded filaments formed by the N-terminal ESCRT-III domain of IST1 (IST1_NTD) and CHMP1B. (Left to right) Double-stranded IST1_NTD/CHMP1B filaments wrapping about a conical membrane; reconstructed cone comprising double-stranded IST1_NTD/CHMP1B filaments; schematic depiction of the structures shown in the left panel, with the IST1_NTD strand shown in light green, the CELMP1B strand shown in dark green, and the internal lipid bilayer shown in gray, (b–d) Illustrations of how membrane deformation, constriction, and fission could be driven by heteromeric and dynamic ESCRT-III filaments that adopt different topologies in response to a variety of conditions, including (b) sequential copolymerization of different ESCRT-III subunits with distinct intrinsic curvatures, (c) dynamic exchange of subunits with high intrinsic curvature into tubes of less curved filaments, and (d) out-of-plane buckling induced by the accumulation of elastic stress arising from growth of a spiraling filament beyond its preferred radius of curvature. For simplicity, panels b–d show single long filaments, but analogous principles could also apply to arrays of shorter, close-packed filaments. Illustrations in panel a were adapted from McCullough et al. (2015) and reproduced with permission from AAAS. Illustrations in panels b–d were adapted from Chiaruttini & Roux (2017).