Association of the endosomal sorting complex ESCRT-II with the Vps20 subunit of ESCRT-III generates a curvature-sensitive complex capable of nucleating ESCRT-III filaments

The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.     

Plasma membrane deformation by circular arrays of ESCRT-III protein filaments

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used "deep-etch" electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis-deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.     

Molecular Mechanisms of the Membrane Sculpting ESCRT Pathway

The endosomal sorting complexes required for transport (ESCRT) drive multivesicular body (MVB) biogenesis and cytokinetic abscission. Originally identified through genetics and cell biology, more recent work has begun to elucidate the molecular mechanisms of ESCRT-mediated membrane remodeling, with special focus on the ESCRT-III complex. In particular, several light and electron microscopic studies provide high-resolution imaging of ESCRT-III rings and spirals that purportedly drive MVB morphogenesis and abscission. These studies highlight unifying principles to ESCRT-III function, in particular: (1) the ordered assembly of the ESCRT-III monomers into a heteropolymer, (2) ESCRT-III as a dynamic complex, and (3) the role of the AAA ATPase Vps4 as a contributing factor in membrane scission. Mechanistic comparisons of ESCRT-III function in MVB morphogenesis and cytokinesis suggest common mechanisms in membrane remodeling.     

Structure and disassembly of filaments formed by the ESCRT-III subunit Vps24

The ESCRT machinery mediates sorting of ubiquitinated transmembrane proteins to lysosomes via multivesicular bodies (MVBs) and also has roles in cytokinesis and viral budding. The ESCRT-III subunits are metastable monomers that transiently assemble on membranes. However, the nature of these assemblies is unknown. Among the core yeast ESCRT-III subunits, Snf7 and Vps24 spontaneously form ordered polymers in vitro. Single-particle EM reconstruction of helical Vps24 filaments shows both parallel and head-to-head subunit arrangements. Mutations of regions involved in intermolecular assembly in vitro result in cargo-sorting defects in vivo, suggesting that these homopolymers mimic interactions formed by ESCRT-III heteropolymers during MVB biogenesis. The C terminus of Vps24 is at the surface of the filaments and is not required for filament assembly. When this region is replaced by the MIT-interacting motif from the Vps2 subunit of ESCRT-III, the AAA-ATPase Vps4 can both bundle and disassemble the chimeric filaments in a nucleotide-dependent fashion.     

Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central “V” domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.     

Regulators of Vps4 ATPase Activity at Endosomes Differentially Influence the Size and Rate of Formation of Intralumenal Vesicles

Recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomes regulates selective inclusion of transmembrane proteins into the lumenal vesicles of multivesicular bodies (MVBs). ESCRT-0, -I, and -II bind directly to ubiquitinated transmembrane cargoes of the MVB pathway, whereas polymerization of ESCRT-III at endosomes is thought to bend the membrane and/or provide the energetic force that drives membrane scission and detachment of vesicles into the endosome lumen. Disassembly of the ESCRT-III polymer and dissociation of its subunits from endosomes requires the Vps4 ATPase, the activity of which is controlled in vivo by regulatory proteins. We identify distinct spatiotemporal roles for Vps4-regulating proteins through examinations of subcellular localization and endosome morphology. Did2 plays a unique role in the regulation of MVB lumenal vesicle size, whereas Vtal and Vps60 promote efficient membrane scission and delivery of membrane to the endosome lumen. These morphological effects probably result from Vps4-mediated manipulations of ESCRT-III, because we show dissociation of ESCRT-0, -I, and -II from endosomes is not directly dependent on Vps4 activity.     

Activation of Human VPS4A by ESCRT-III Proteins Reveals Ability of Substrates to Relieve Enzyme Autoinhibition

VPS4 proteins are AAA⁺ ATPases required to form multivesicular bodies, release viral particles, and complete cytokinesis. They act by disassembling ESCRT-III heteropolymers during or after their proposed function in membrane scission. Here we show that purified human VPS4A is essentially inactive but can be stimulated to hydrolyze ATP by ESCRT-III proteins in a reaction that requires both their previously defined MIT interacting motifs and ∼50 amino acids of the adjacent sequence. Importantly, C-terminal fragments of all ESCRT-III proteins tested, including CHMP2A, CHMP1B, CHMP3, CHMP4A, CHMP6, and CHMP5, activated VPS4A suggesting that it disassembles ESCRT-III heteropolymers by affecting each component protein. VPS4A is thought to act as a ring-shaped cylindrical oligomer like other AAA⁺ ATPases, but this has been difficult to directly demonstrate. We found that concentrating His₆-VPS4A on liposomes containing Ni²⁺-nitrilotriacetic acid-tagged lipid increased ATP hydrolysis, confirming the importance of inter-subunit interactions for activity. We also found that mutating pore loops expected to line the center of a cylindrical oligomer changed the response of VPS4A to ESCRT-III proteins. Based on these data, we propose that ESCRT-III proteins facilitate assembly of functional but transient VPS4A oligomers and interact with sequences inside the pore of the assembled enzyme. Deleting the N-terminal MIT domain and adjacent linker from VPS4A increased both basal and liposome-enhanced ATPase activity, indicating that these elements play a role in autoinhibiting VPS4A until it encounters ESCRT-III proteins. These findings reveal new ways in which VPS4 activity is regulated and specifically directed to ESCRT-III polymers.     

Coordinated binding of Vps4 to ESCRT-III drives membrane neck constriction during MVB vesicle formation

Five endosomal sorting complexes required for transport (ESCRTs) mediate the degradation of ubiquitinated membrane proteins via multivesicular bodies (MVBs) in lysosomes. ESCRT-0, -I, and –II interact with cargo on endosomes. ESCRT-II also initiates the assembly of a ringlike ESCRT-III filament consisting of Vps20, Snf7, Vps24, and Vps2. The AAA–adenosine triphosphatase Vps4 disassembles and recycles the ESCRT-III complex, thereby terminating the ESCRT pathway. A mechanistic role for Vps4 in intraluminal vesicle (ILV) formation has been unclear. By combining yeast genetics, biochemistry, and electron tomography, we find that ESCRT-III assembly on endosomes is required to induce or stabilize the necks of growing MVB ILVs. Yet, ESCRT-III alone is not sufficient to complete ILV biogenesis. Rather, binding of Vps4 to ESCRT-III, coordinated by interactions with Vps2 and Snf7, is coupled to membrane neck constriction during ILV formation. Thus, Vps4 not only recycles ESCRT-III subunits but also cooperates with ESCRT-III to drive distinct membrane-remodeling steps, which lead to efficient membrane scission at the end of ILV biogenesis in vivo.     

Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and gamma 2-adaptin

Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor gamma 2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role gamma 2-adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess gamma 2-adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed gamma 2-adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of gamma 2-adaptin.     

Molecular Simulation of Mechanical Properties and Membrane Activities of the ESCRT-III Complexes

The endosomal sorting complex required for transport (ESCRT) machinery carries out the membrane scission reactions that are required for many biological processes throughout cells. How ESCRTs bind and deform cellular membranes and ultimately produce vesicles has been a matter of active research in recent years. In this study, we use fully atomistic molecular dynamics simulations to scrutinize the structural details of a filament composed of Vps32 protomers, a major component of ESCRT-III complexes. The simulations show that both hydrophobic and electrostatic interactions between monomers help maintain the structural stability of the filament, which exhibits an intrinsic bend and twist. Our findings suggest that the accumulation of bending and twisting stresses as the filament elongates on the membrane surface likely contributes to the driving force for membrane invagination. The filament exposes a large cationic surface that senses the negatively charged lipids in the membrane, and the N-terminal amphipathic helix of the monomers not only acts as a membrane anchor but also generates significant positive membrane curvature. Taking all results together, we discuss a plausible mechanism for membrane invagination driven by ESCRT-III.     

Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast

Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.     

Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins - hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/CHMP6, and hSnf7-1/CHMP4a - to each other, studying the effects of deleting predicted alpha-helical domains on their behavior in transfected cells. Surprisingly, removing approximately 40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA+ adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default 'closed' state and an activated 'open' state under control of sequences at their C-terminus and associated factors.     

The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis

Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.     

Novel interactions of ESCRT-III with LIP5 and VPS4 and their implications for ESCRT-III disassembly

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2-1, and CHMP3/hVps24 but not CHMP4A/hSnf7-1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with "MIT interacting motifs" (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.     

Computational Model of Membrane Fission Catalyzed by ESCRT-III

ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We suggest and analyze a novel mechanism of membrane fission by the mammalian ESCRT-III subunits CHMP2 and CHMP3. We propose that the CHMP2-CHMP3 complexes self-assemble into hemi-spherical dome-like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein-membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2-CHMP3 assemblies in vitro. We predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission.     

ESCRT-III protein requirements for HIV-1 budding

Two early-acting components of the cellular ESCRT pathway, ESCRT-I and ALIX, participate directly in HIV-1 budding. The membrane fission activities of ESCRT-III subunits are also presumably required, but humans express 11 different CHMP/ESCRT-III proteins whose functional contributions are not yet clear. We therefore depleted cells of each of the different CHMP proteins and protein families and examined the effects on HIV-1 budding. Virus release was profoundly inhibited by codepletion of either CHMP2 or CHMP4 family members, resulting in ≥100-fold titer reductions. CHMP2A and CHMP4B proteins bound one another, and this interaction was required for budding. By contrast, virus release was reduced only modestly by depletion of CHMP3 and CHMP1 proteins (2- to 8-fold titer reductions) and was unaffected by depletion of other human ESCRT-III proteins. HIV-1 budding therefore requires only a subset of the known human ESCRT-III proteins, with the CHMP2 and CHMP4 families playing key functional roles.     

Electron cryotomography of ESCRT assemblies and dividing Sulfolobus cells suggests that spiraling filaments are involved in membrane scission

The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT–CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.     

Structural Basis of Ist1 Function and Ist1–Did2 Interaction in the Multivesicular Body Pathway and Cytokinesis

The ESCRT machinery functions in several important eukaryotic cellular processes. The AAA-ATPase Vps4 catalyzes disassembly of the ESCRT-III complex and may regulate membrane deformation and vesicle scission as well. Ist1 was proposed to be a regulator of Vps4, but its mechanism of action was unclear. The crystal structure of the N-terminal domain of Ist1 (Ist1NTD) reveals an ESCRT-III subunit-like fold, implicating Ist1 as a divergent ESCRT-III family member. Ist1NTD specifically binds to the ESCRT-III subunit Did2, and cocrystallization of Ist1NTD with a Did2 fragment shows that Ist1 interacts with the Did2 C-terminal MIM1 (MIT-interacting motif 1) via a novel MIM-binding structural motif. This arrangement indicates a mechanism for intermolecular ESCRT-III subunit association and may also suggest one form of ESCRT-III subunit autoinhibition via intramolecular interaction.     

Assembly and disassembly of the ESCRT-III membrane scission complex

The ESCRT (endosomal sorting complex required for transport) pathway promotes the final membrane scission step at the end of cytokinesis, assists viral budding and generates multivesicular bodies (MVBs). These seemingly unrelated processes require a topologically similar membrane deformation and scission event that buds membranes/vesicles out of the cytoplasm. The topology of this budding reaction is 'opposite' to reactions that bud endocytic and secretory vesicles into the cytoplasm. Here we summarize recent findings that help to understand how the ESCRT machinery, in particular the ESCRT-III complex, assembles on its target membranes, executes membrane scission and is disassembled by the AAA-ATPase Vps4.