Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly.
ABSTRACT: ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.
Project description:The endosomal sorting complexes required for transport (ESCRTs) function in a variety of membrane remodeling processes including multivesicular body sorting, abscission during cytokinesis, budding of enveloped viruses, and repair of the plasma membrane. Vps4 ATPase activity modulates ESCRT function and is itself modulated by its cofactor Vta1 and its substrate ESCRT-III. The carboxyl-terminal Vta1/SBP-1/Lip5 (VSL) domain of Vta1 binds to the Vps4 ?-domain to promote Vps4 oligomerization-dependent ATP hydrolysis. Additionally, the Vps4 stimulatory element (VSE) of Vta1 contributes to enhancing Vps4 oligomer ATP hydrolysis. The VSE is also required for Vta1-dependent stimulation of Vps4 by ESCRT-III subunits. However, the manner by which the Vta1 VSE contributes to Vps4 activation is unknown. Existing structural data were used to generate a model of the Vta1 VSE in complex with Vps4. This model implicated residues within the small ATPase associated with various activities (AAA) domain, specifically ?-helices 7 and 9, as relevant contact sites. Rational generation of Vps4 mutants defective for VSE-mediated stimulation, as well as intergenic compensatory mutations, support the validity of this model. These findings have uncovered the Vps4 surface responsible for coordinating ESCRT-III-stimulated Vta1 input during ESCRT function and identified a novel mechanism of Vps4 stimulation.
Project description:Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 'walks' along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.
Project description:The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.
Project description:Vps4 is a member of AAA+ ATPase (adenosine triphosphatase associated with diverse cellular activities) that operates as an oligomer to disassemble ESCRT-III (endosomal sorting complex required for transport III) filaments, thereby catalyzing the final step in multiple ESCRT-dependent membrane remodeling events. We used electron cryo-microscopy to visualize oligomers of a hydrolysis-deficient Vps4 (vacuolar protein sorting-associated protein 4) mutant in the presence of adenosine 5'-triphosphate (ATP). We show that Vps4 subunits assemble into an asymmetric hexameric ring following an approximate helical path that sequentially stacks substrate-binding loops along the central pore. The hexamer is observed to adopt an open or closed ring configuration facilitated by major conformational changes in a single subunit. The structural transition of the mobile Vps4 subunit results in the repositioning of its substrate-binding loop from the top to the bottom of the central pore, with an associated translation of 33 Å. These structures, along with mutant-doping experiments and functional assays, provide evidence for a sequential and processive ATP hydrolysis mechanism by which Vps4 hexamers disassemble ESCRT-III filaments.
Project description:The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced ?-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.
Project description:The hexameric AAA ATPase Vps4 drives membrane fission by remodeling and disassembling ESCRT-III filaments. Building upon our earlier 4.3 Å resolution cryo-EM structure (Monroe et al., 2017), we now report a 3.2 Å structure of Vps4 bound to an ESCRT-III peptide substrate. The new structure reveals that the peptide approximates a ?-strand conformation whose helical symmetry matches that of the five Vps4 subunits it contacts directly. Adjacent Vps4 subunits make equivalent interactions with successive substrate dipeptides through two distinct classes of side chain binding pockets formed primarily by Vps4 pore loop 1. These pockets accommodate a wide range of residues, while main chain hydrogen bonds may help dictate substrate-binding orientation. The structure supports a 'conveyor belt' model of translocation in which ATP binding allows a Vps4 subunit to join the growing end of the helix and engage the substrate, while hydrolysis and release promotes helix disassembly and substrate release at the lagging end.
Project description:The AAA+ ATPase Vps4 disassembles ESCRT-III and is essential for HIV-1 budding and other pathways. Vps4 is a paradigmatic member of a class of hexameric AAA+ ATPases that disassemble protein complexes without degradation. To distinguish between local displacement versus global unfolding mechanisms for complex disassembly, we carried out hydrogen/deuterium exchange during Saccharomyces cerevisiae Vps4 disassembly of a chimeric Vps24-2 ESCRT-III filament. EX1 exchange behavior shows that Vps4 completely unfolds ESCRT-III substrates on a time scale consistent with the disassembly reaction. The established unfoldase ClpX showed the same pattern, thus demonstrating a common unfolding mechanism. Vps4 hexamers containing a single cysteine residue in the pore loops were cross-linked to ESCRT-III subunits containing unique cysteines within the folded core domain. These data support a mechanism in which Vps4 disassembles its substrates by completely unfolding them and threading them through the central pore.
Project description:The ESCRT complexes are required for multivesicular body biogenesis, macroautophagy, cytokinesis, and the budding of HIV-1. The final step in the ESCRT cycle is the disassembly of the ESCRT-III lattice by the AAA+ ATPase Vps4. Vps4 assembles on its membrane-bound ESCRT-III substrate with its cofactor, Vta1. The crystal structure of the dimeric VSL domain of yeast Vta1 with the small ATPase and the betadomains of Vps4 was determined. Residues involved in structural interactions are conserved and are required for binding in vitro and for Cps1 sorting in vivo. Modeling of the Vta1 complex in complex with the lower hexameric ring of Vps4 indicates that the two-fold axis of the Vta1 VSL domain is parallel to within approximately 20 degrees of the six-fold axis of the hexamer. This suggests that Vta1 might not crosslink the two hexameric rings of Vps4, but rather stabilizes an array of Vps4-Vta1 complexes for ESCRT-III disassembly.
Project description:The cellular ESCRT-III (endosomal sorting complex required for transport-III) and Vps4 (vacuolar protein sorting 4) comprise a common machinery that mediates a variety of membrane remodelling events. Vps4 is essential for the machinery function by using the energy from ATP hydrolysis to disassemble the ESCRT-III polymer into individual proteins. Here, we report the structures of the ATP-bound Vps4E233Q hexamer and its complex with the cofactor Vta1 (vps twenty associated 1) at resolutions of 3.9 and 4.2 Å, respectively, determined by electron cryo-microscopy. Six Vps4E233Q subunits in both assemblies exhibit a spiral-shaped ring-like arrangement. Locating at the periphery of the hexameric ring, Vta1 dimer bridges two adjacent Vps4 subunits by two different interaction modes to promote the formation of the active Vps4 hexamer during ESCRT-III filament disassembly. The structural findings, together with the structure-guided biochemical and single-molecule analyses, provide important insights into the process of the ESCRT-III polymer disassembly by Vps4.
Project description:Vps4 is a key enzyme that functions in endosomal protein trafficking, cytokinesis, and retroviral budding. Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III, where the protein oligomerizes into an active ATPase. The recruitment and oligomerization steps are mediated by a complex network of at least 12 distinct interactions between Vps4, ESCRT-III, Ist1, Vta1, and Did2. The order of events leading to active, ESCRT-III-associated Vps4 is poorly understood. In this study we present a systematic in vivo analysis of the Vps4 interaction network. The data demonstrated a high degree of redundancy in the network. Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others. The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7. In our model we propose the formation of a recruitment complex in the cytoplasm that is composed of Did2-Ist1-Vps4, which upon binding to ESCRT-III recruits Vta1. Vta1 in turn is predicted to cause a rearrangement of the Vps4 interactions that initiates the assembly of the active Vps4 oligomer.