Structural characterization of the MIT domain from human Vps4b

The microtubule interacting and trafficking (MIT) domain is a small protein module of unknown function that is conserved in proteins of diverse function, such as Vps4, sorting nexin 15 (SNX15), and spastin. One non-synonymous single nucleotide polymorphism was reported, which results in a Ile58-to-Met (I58M) substitution in hVps4b. Here, we have determined the solution structure of the MIT domain isolated from the NH(2)-terminus of human Vps4b, an AAA-ATPase involved in multivesicular body formation. The MIT domain adopts an 'up-and-down' three-helix bundle. Comparison with the sequences of other MIT domains clearly shows that the residues involved in inter-helical contacts are well conserved. The Ile58-to-Met substitution resulted a substantial thermal instability. In addition, we found a shallow crevice between helices A and C that may serve as a protein-binding site. We propose that the MIT domain serves as a putative adaptor domain for the ESCRT-III complex involved in endosomal trafficking.     

The Linker Region Plays a Regulatory Role in Assembly and Activity of the Vps4 AAA ATPase

The AAA-type ATPase Vps4 functions with components of the ESCRT (endosomal sorting complex required for transport) machinery in membrane fission events that are essential for endosomal maturation, cytokinesis, and the formation of retroviruses. A key step in these events is the assembly of monomeric Vps4 into the active ATPase complex, which is aided in part by binding of Vps4 via its N-terminal MIT (microtubule interacting and trafficking) domain to its substrate ESCRT-III. We found that the 40-amino acid linker region between the MIT and the ATPase domain of Vps4 is not required for proper function but plays a role in regulating Vps4 assembly and ATPase activity. Deletion of the linker is expected to bring the MIT domains into close proximity to the central pore of the Vps4 complex. We propose that this localization of the MIT domain in linker-deleted Vps4 mimics a repositioning of the MIT domain normally caused by binding of Vps4 to ESCRT-III. This structure would allow the Vps4 complex to engage ESCRT-III subunits with both the pore and the MIT domain simultaneously, which might be essential for the ATP-driven disassembly of ESCRT-III.     

Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity

The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function.     

Structural and functional organization of the ESCRT-I trafficking complex

The endosomal sorting complex required for transport (ESCRT) complexes are central to receptor downregulation, lysosome biogenesis, and budding of HIV. The yeast ESCRT-I complex contains the Vps23, Vps28, and Vps37 proteins, and its assembly is directed by the C-terminal steadiness box of Vps23, the N-terminal half of Vps28, and the C-terminal half of Vps37. The crystal structures of a Vps23:Vps28 core subcomplex and the Vps23:Vps28:Vps37 core were solved at 2.1 and 2.8 A resolution. Each subunit contains a structurally similar pair of helices that form the core. The N-terminal domain of Vps28 has a hydrophobic binding site on its surface that is conformationally dynamic. The C-terminal domain of Vps28 binds the ESCRT-II complex. The structure shows how ESCRT-I is assembled by a compact core from which the Vps23 UEV domain, the Vps28 C domain, and other domains project to bind their partners.     

Structural Basis of Molecular Recognition between ESCRT-III-like Protein Vps60 and AAA-ATPase Regulator Vta1 in the Multivesicular Body Pathway

The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128–186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128–186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128–186) interacts with Vta1NTD through helices α4′ and α5′, extending over Vta1NTD MIT2 domain helices 1–3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.     

Relief of Autoinhibition Enhances Vta1 Activation of Vps4 via the Vps4 Stimulatory Element

The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.     

Cryo-EM structure of dodecameric Vps4p and its 2:1 complex with Vta1p

The type I AAA (ATPase associated with a variety of cellular activities) ATPase Vps4 and its co-factor Vta1p/LIP5 function in membrane remodeling events that accompany cytokinesis, multivesicular body biogenesis, and retrovirus budding, apparently by driving disassembly and recycling of membrane-associated ESCRT (endosomal sorting complex required for transport)-III complexes. Here, we present electron cryomicroscopy reconstructions of dodecameric yeast Vps4p complexes with and without their microtubule interacting and transport (MIT) N-terminal domains and Vta1p co-factors. The ATPase domains of Vps4p form a bowl-like structure composed of stacked hexameric rings. The two rings adopt dramatically different conformations, with the “upper” ring forming an open assembly that defines the sides of the bowl and the lower ring forming a closed assembly that forms the bottom of the bowl. The N-terminal MIT domains of the upper ring localize on the symmetry axis above the cavity of the bowl, and the binding of six extended Vta1p monomers causes additional density to appear both above and below the bowl. The structures suggest models in which Vps4p MIT and Vta1p domains engage ESCRT-III substrates above the bowl and help transfer them into the bowl to be pumped through the center of the dodecameric assembly.     

The beta domain is required for Vps4p oligomerization into a functionally active ATPase

Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a beta domain, which protrudes from the AAA domain, and a final C-terminal alpha-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the beta domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the beta domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.     

Vps4 regulates a subset of protein interactions at the multivesicular endosome

During endocytic transport, specific integral membrane proteins are sorted into intraluminal vesicles that bud from the limiting membrane of the endosome. This process, known as multivesicular body (MVB) sorting, is important for several important biological processes. Moreover, components of the MVB sorting machinery are implicated in virus budding. During MVB sorting, a cargo protein recruits components of the MVB sorting machinery from cytoplasmic pools and these sequentially assemble on the endosome. Disassembly of these proteins and recycling into the cytoplasm is critical for MVB sorting. Vacuolar protein sorting 4 (Vps4) is an AAA (ATPase associated with a variety of cellular activities) ATPase which has been proposed to play a critical role in disassembly of the MVB sorting machinery. However, the mechanism by which it disassembles the complex is not clear. Vps4 contains an N-terminal microtubule interacting and trafficking (MIT) domain, which has previously been shown to be required for recruitment to endosomes, and a single AAA ATPase domain, the activity of which is required for Vps4 function. In this study we have systematically characterized the interaction of Vps4 with other components of the MVB sorting machinery. We demonstrate that Vps4 interacts directly with Vps2 and Bro1. We also show that a subset of Vps4 interactions is regulated by ATP hydrolysis, and one interaction is regulated by ATP binding. Finally, we show that most proteins interact with the Vps4 MIT domain. Our studies indicate that the MIT domain has a dual role in substrate binding and recruitment to endosomes and indicate that Vps4 disassembles the MVB sorting machinery by direct effects on multiple proteins.     

ESCRT-III family members stimulate Vps4 ATPase activity directly or via Vta1

The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.     

Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode.     

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.     

ESCRT complexes and the biogenesis of multivesicular bodies

Multivesicular bodies (MVBs) are crucial intermediates in the trafficking of ubiquitinated receptors and other cargo destined for lysosomes. The formation of MVBs by invagination of the endosomal limiting membrane is catalyzed by the endosomal sorting complex required for transport (ESCRT) complexes, a process that has recently been visualized in three-dimensional detail by electron tomography. Structural and biochemical analysis of the upstream components, Vps27-Hse1, ESCRT-I, and ESCRT-II, shows how these complexes assemble and cluster cargo. Rapid progress has been made in understanding the assembly and disassembly of the ESCRT-III complex and the interactions of its subunits with MIT domain and other proteins. A key role for deubiquitination in the regulation of the system has been demonstrated. One central question remains largely unanswered, which is how the ESCRTs actually promote the invagination of the endosomal membrane.     

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate.     

Global analysis of yeast endosomal transport identifies the vps55/68 sorting complex

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process.     

Identical domains of Yarrowia lipolytica Vps23 are required for both ESCRT and Rim pathways, but the latter needs an interaction between the Vps23 UEV domain and Rim8/PalF

A conserved pathway, called Rim or Pal, transduces the ambient pH signal in ascomycetous yeasts and fungi, respectively. This pathway requires most of the components of the endosomal sorting complex required for transport (ESCRT) pathway. In the yeast Yarrowia lipolytica, a functional analysis of the ESCRT-I subunit Vps23 was carried out by in-frame deletions of each of the conserved domains to test whether Vps23 functions in the Rim and ESCRT pathways could be separated. These two pathways were shown to necessitate both the coiled-coil domain and the C-terminal steadiness box. However, the central proline-rich region seems to be required for neither of them. Both pathways involve the N-terminal ubiquitin E2 variant (UEV) domain. Thus, identical domains of YlVps23 are required for both Rim and ESCRT pathways, but the UEV domain was shown to bind the arrestin-like protein Rim8/PalF in the Rim pathway, whereas it binds Vps27 in the ESCRT pathway. Vps23 is therefore required to link pH signalling and endocytosis.     

Dictyostelium Tom1 Participates to an Ancestral ESCRT-0 Complex

Sorting of ubiquitinated proteins to multivesicular bodies (MVBs) in mammalian cells relies on proteins with a Vps27/Hrs/STAM (VHS) domain. Here, we show that the amoeba Dictyostelium presents only one protein with a VHS domain: DdTom1. We demonstrate that the VHS domain of DdTom1 is followed by a Golgi-localized, γ-ear-containing, ADP-ribosylation-factor-binding and Tom1 (GAT) domain that binds ubiquitin, and by a non-conserved C-terminal domain that can recruit clathrin, EGFr pathway substrate 15 and tumor susceptibility gene 101, a component of the MVB biogenesis machinery [endosomal complexes required for transport (ESCRT) complexes]. Both VHS and GAT domains interact with phospholipids and therefore could ensure the recruitment of DdTom1 to endosomal membranes. We propose that DdTom1 participates in an ancestral ESCRT-0 complex implicated in the sorting of ubiquitinated proteins into MVBs.     

Structural basis for endosomal recruitment of ESCRT-I by ESCRT-0 in yeast

The ESCRT‐0 and ESCRT‐I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85‐Å crystal structure of interacting regions of the yeast ESCRT‐0 and ESCRT‐I complexes reveals that PSDP motifs of the Vps27 ESCRT‐0 subunit bind to a novel electropositive N‐terminal site on the UEV domain of the ESCRT‐I subunit Vps23 centred on Trp16. This novel site is completely different from the C‐terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT‐0 and HIV‐1 Gag. Disruption of the novel PSDP‐binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome‐related class E compartments in yeast cells. However, this site is non‐essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms.     

Structure of Vps26B and mapping of its interaction with the retromer protein complex

Retromer is a heteromeric protein complex with important roles in endosomal membrane trafficking, most notably in the retrograde transport of lysosomal hydrolase receptors from endosomes to the Golgi. The core of retromer is composed of three subunits vacuolar protein sorting (Vps)35, Vps26 and Vps29, and in mammals, there are two paralogues of the medium subunit Vps26A and Vps26B. We find that both Vps26A and Vps26B bind to Vps35/Vps29 with nanomolar affinity and compete for a single-binding site to define distinct retromer complexes in vitro and in vivo. We have determined the crystal structure of mouse Vps26B and compare this structure with that of Vps26A. Vps26 proteins have a striking similarity to the arrestin family of proteins that regulate the signalling and endocytosis of G-protein-coupled receptors, although we observe that surface residues involved in arrestin function are not conserved in Vps26. Using structure-based mutagenesis, we show that both Vps26A and Vps26B are incorporated into retromer complexes through binding of Vps35 to a highly conserved surface patch within the C-terminal subdomain and that this interaction is required for endosomal recruitment of the proteins.