The AAA (ATPase associated with various cellular activities) proteins participate in membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA Vps (vacuolar protein sorting) 4 is central to traffic to lysosomes, retroviral budding and mammalian cell division. It dissociates ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes, enabling their recycling to the cytosol, and plays a role in fission of intraluminal vesicles within MVBs (multivesicular bodies). The mechanism of Vps4-catalysed disassembly of ESCRT networks is unknown; however, it requires interaction between Vps4 and ESCRT-III subunits. The 30 C-terminal residues of Vps2 and Vps46 (Did2) subunits are both necessary and sufficient for interaction with the Vps4 N-terminal MIT (microtubule-interacting and transport) domain, and the crystal structure of the Vps2 C-terminus in a complex with the Vps4 MIT domain shows that MIT helices α2 and α3 recognize a (D/E)XXLXXRLXXL(K/R) MIM (MIT-interacting motif). These Vps2–MIT interactions are essential for vacuolar sorting and for Vps4-catalysed disassembly of ESCRT-III networks in vitro. Electron microscopy of ESCRT-III filaments assembled in vitro has enabled us to identify surfaces of the Vps24 subunit that are critical for protein sorting in vivo. The ESCRT-III–Vps4 interaction predates the divergence of Archaea and Eukarya. The Crenarchaea have three classes of ESCRT-III-like subunits, and one of these subunits interacts with an archaeal Vps4-like protein in a manner closely related to the human Vps4–human ESCRT-III subunit Vps20 interaction. This archaeal Vps4–ESCRT-III interaction appears to have a fundamental role in cell division in the Crenarchaea.
Endosomes facilitate transport of membrane proteins between the plasma membrane, the trans-Golgi network and the lysosome/vacuole . Sorting to the lysosome/vacuole compartment involves late endosomal structures known as MVBs (multivesicular bodies), containing ILVs (intraluminal vesicles) formed by invagination of the endosomal limiting membrane into the endosome lumen . Ubiquitination serves as the primary signal for sorting membrane proteins into MVBs [3,4]. Four ESCRTs (endosomal sorting complexes required for transport), ESCRT-0, -I, -II and -III, regulate protein sorting into ILVs, and the ESCRT subunits are among a group of proteins that, when deleted, cause enlarged class E compartments and sorting defects . The AAA (ATPase associated with various cellular activities), Vps (vacuolar protein sorting) 4, catalyses dissociation of the ESCRT machinery from endosomal membranes and is essential for MVB biogenesis and function [6–8]. ESCRTs were first characterized by their role in transport of ubiquitinated transmembrane proteins to lysosomes via MVBs. Subsequently, it was shown that many ESCRT components are also important for viral budding and more recently for cytokinesis [9,10].
The structures of most of the components of the ESCRT machinery have been determined. The most challenging current goal is to understand how these complexes assemble on endosomal membranes and catalyse internal budding and abscission.
ESCRT-III assemblies in vitro
The ESCRT-III subunits are thought to have a critical role in membrane budding and abscission. All of the ESCRT-III-like subunits are likely to have a similar fold, consisting of a long helical α1/α2 hairpin packed against a shorter α3/α4 helical hairpin . These subunits are metastable monomers capable of forming extensive assemblies on membranes. The stoichiometry and structures of these assemblies are still largely unknown. Nevertheless, recent studies of polymeric complexes of ESCRT-III subunits in vitro suggest interactions that are likely to be important for these assemblies. Among the core yeast ESCRT-III subunits, Vps32 (Snf7) and Vps24 spontaneously form ordered homopolymeric filaments in vitro , and this has led to the 25 Å (1 Å=0.1 nm) resolution structure of helical filaments of yeast Vps24 . Weissenhorn and colleagues have shown that human Vps2 [CHMP (charged multivesicular body protein) 2A] and Vps24 (CHMP3) can co-assemble into heteropolymeric tubules in vitro . A human Vps32 (CHMP4B) overexpressed in mammalian cells gives rise to helical filaments that have been visualized by deep-etch electron microscopy to form circular assemblies in cells .
Structure of the yeast Vps24 homopolymeric filaments
Although recombinant full-length yeast Vps24 is eluted on gel filtration as monomers or dimers, when concentrated, it forms filaments with a predominant diameter of approx. ∼15 nm (Figure 1A). The most abundant filaments are made of two strands wrapped around each other. Owing to the structural heterogeneity of the filaments, the IHRSR (iterative helical real-space reconstruction) approach was essential .
Yeast Vps24 homopolymers
Figure 1(B) illustrates the helical reconstruction of yeast Vps24 filaments . The high-resolution crystal structure of the core of human Vps24 (CHMP3)  was fitted manually into the density. The longest axis of the subunit makes an angle of ∼60° with respect to the z-axis. The loop of the α1/α2 hairpin points towards the centre of the filament, with the C-termini pointing toward the surface and contributing to the density of the ‘knuckles’ (Figure 1C). A yeast Vps24-deletion variant lacking the C-terminus is also able to form filaments, although they are more irregular and have a reduced diameter (∼11 nm).
Helix α4 (residues 125–138) and the α3/α4 loop (residues 118–124) are central to packing between molecules within a strand. Site-specific mutagenesis of residues involved in intermolecular contacts using these elements are essential for filament formation, although CD observations suggest that they are not essential for the protein folding. Mutations of these elements also produce CPS (carboxypeptidase S) sorting defects and result in class E compartments .
The ability of the AAA Vps4 to interact with ESCRT-III subunits is critically dependent on the N-terminal MIT (microtubule-interacting and transport) domain of Vps4. MIT domains are three-helix bundles with a TPR (tetratricopeptide-like repeat)-like architecture (reviewed by ). The C-termini of ESCRT-III subunits Vps2 and Vps46 (Did2) contain a consensus sequence (D/E)XXLXXRLXXL(K/R) that is recognized by the N-terminal MIT domain of Vps4 [16–20]. Vps4 forms a dodecamer or tetradecamer, consisting of two stacked rings [21,22–24]. This double-ring structure is stabilized by interactions with the Vta1 (Vps20-associated 1 homologue)/LIP5 (LYST-interacting protein 5) protein [22,25–28]. Vta1 also has a MIT domain that can recruit some ESCRT-III subunits [29,30]. Vps4 rings enclose a conserved central pore, and it has been suggested that ESCRTs may be drawn through this central pore during Vps4-mediated disassembly of ESCRT networks . Structures of complexes of the Vps4 MIT domains with the Vps2 subunits [17,18] show that the Vps2 MIM (MIT-interacting motif) 1 forms a helix that interacts with the MIT three-helix TPR-like bundle by slotting between helices α2 and α3, although with a direction that does not continue the solenoid architecture of TPR-like domains (Figure 2).
A chimaeric ESCRT-III subunit consisting of the helices α1–α5 of yeast Vps24 and the C-terminal, Vps4-binding region of yeast Vps2 forms filaments that are indistinguishable from the wild-type yeast Vps24 filaments. In the presence of ATP, Vps4 can disassemble these chimaeric filaments, as indicated by the absence of filaments in EM (electron microscopy) and sedimentation assays. Vps4 mutated in a residue of MIT helix α3 (L64D), which makes essential contacts with ESCRT-III, is unable to disassemble the chimaeric filaments.
More recently, it has become apparent that the Vps4 MIT domain is capable of making diverse interactions. Whereas Vps2 and Vps46A (CHMP1A) have MIM1s that form helices that slot between MIT helices α2 and α3, the human Vps20 (CHMP6) subunit has a C-terminal motif that interacts between MIT helices α1 and α3  (Figure 2B). This alternative MIM has been referred to as a MIM2. In contrast with the MIM1 sequences of Vps2 and Vps46, the Vps20 MIM2 has an extended conformation in its complex with the MIT domain rather than the helical conformation that is characteristic of Vps2 and Vps46.
Evolution of the ESCRT-III–Vps4 interaction
The ESCRTs have an ancient origin and appear to have been present in the common ancestor of all eukaryotes . Although there are individual members of eukaryotic clades that lack some types of ESCRT subunit, Vps4 and ESCRT-III subunits are present in all members. The structure of the MIT domain of an archaeal ATPase from Sulfolobus solfataricus (KEGG accession number SSO0909; Swiss-Prot accession number Q97ZJ7) is closely related to the Vps4 MIT domain . However, the archaeal MIT domain lacks the specificity determinants important for MIM1 binding in the α2/α3 slot. Interestingly, all archaea that have a Vps4-like ATPase also have homologues of ESCRT-III-like subunits [17,33], although apparently not ESCRT-0, -I or -II subunits. The crenarchaeal Vps4 gene is located within an operon with an ESCRT-III homologue  and a third protein predicted to contain a coiled-coil structure . Analogous to the multiple ESCRT-III subunits present in eukaryotic species, the crenarchaea appear to have three ESCRT-III-like classes, primarily on the basis of their distinct C-terminal sequences. The ESCRT-III-like subunits have a core that is probably similar to the core of the eukaryotic ESCRT-III subunits; however, sequence analysis does not suggest a one-to-one correspondence between members of the archaeal subunits and members of the eukaryotic subunits. The minimal region that interacts with the archaeal Vps4 corresponds to residues 183–193 that precedes a C-terminal extension characteristic of this class of archaeal ESCRT-III . The structure of a complex of the Vps4 (KEGG accession number Saci_1372) MIT domain with this region of the Saci_1373 ESCRT-III-like subunit (Figure 2A) shows that it has an extended conformation that slots between MIT helices α1 and α3 . This interaction is analogous to the way human Vps20 (CHMP6) MIM2 interacts with the human Vps4 MIT domain .
Eukaryotic ESCRT-III and Vps4 subunits are associated with vesicle budding, with maturation and release of some enveloped viruses such as HIV and with cell division. Surprisingly, archaeal ESCRT-III-like subunits have been shown to be associated with released Sulfolobus vesicles , incorporated into a virus, STIV (Sulfolobus turreted icosahedral virus), that infects Sulfolobus , and associated with cell division . The role of ESCRT-III-like subunits in archaeal cell division is reinforced by the observation that the Saci_1373 ESCRT-III subunit localizes to the site of membrane pinching during archaeal cell division . The role of the ESCRT proteins in cell division may pre-date the divergence of the crenarchaeal and eukaryotic lineages and thus be reflective of the ancestral role of this complex.
This work was supported by a studentship from Trinity College Cambridge (to S.G.T.), by a European Molecular Biology Organization Fellowship (to A.V.P.) [grant number ALTF 165-2007], by the Wellcome Trust [grant number 083639/Z/07/Z] (to R.L.W.), by EPA Trust (to S.D.B.) and the Medical Research Council (to R.L.W. and S.D.B.).
ESCRTs: from Cell Biology to Pathogenesis: Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 26–28 August 2008. Organized and Edited by Katherine Bowers (University College London, U.K.), Juan Martin-Serrano (King's College London, U.K.) and Paul Whitley (Bath, U.K.).
ATPase associated with various cellular activities
charged multivesicular body protein
endosomal sorting complex required for transport
microtubule-interacting and transport
vacuolar protein sorting
Vps20-associated 1 homologue