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.     

A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5

Disassembly of the endosomal sorting complex required for transport (ESCRT) machinery from biological membranes is a critical final step in cellular processes that require the ESCRT function. This reaction is catalyzed by VPS4, an AAA-ATPase whose activity is tightly regulated by a host of proteins, including LIP5 and the ESCRT-III proteins. Here, we present structural and functional analyses of molecular interactions between human VPS4, LIP5, and the ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly inhibits the stimulation. Both of these observations are distinct from what was previously described for homologous yeast proteins. The crystal structure of LIP5NTD in complex with the MIT (microtubule-interacting and transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, was determined at 1 Å resolution. It reveals an ESCRT-III binding induced moderate conformational change in LIP5NTD, which results from insertion of a conserved CHMP5 tyrosine residue (Tyr¹⁸²) at the core of LIP5NTD structure. Mutation of Tyr¹⁸² partially relieves the inhibition displayed by CHMP5. Together, these results suggest a novel mechanism of VPS4 regulation in metazoans, where CHMP5 functions as a negative allosteric switch to control LIP5-mediated stimulation of VPS4.     

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.     

Trapping of a transcription complex using a new nucleotide analogue: AMP aluminium fluoride

Mechanochemical proteins rely on ATP hydrolysis to establish the different functional states required for their biological output. Studying the transient functional intermediate states these proteins adopt as they progress through the ATP hydrolysis cycle is key to understanding the molecular basis of their mechanism. Many of these intermediates have been successfully ‘trapped’ and functionally characterised using ATP analogues. Here, we present a new nucleotide analogue, AMP-AlF_x, which traps PspF, a bacterial enhancer binding protein, in a stable complex with the σ⁵⁴-RNA polymerase holoenzyme. The crystal structure of AMP-AlF_x•PspF_1-275 provides new information on protein-nucleotide interactions and suggests that the β and γ phosphates are more important than the α phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AlF_x establish distinct roles for the conserved catalytic AAA⁺ (ATPases associated with various cellular activities) residues, suggesting that AMP-AlF_x is a powerful new tool to study AAA⁺ protein family members and, more generally, Walker motif ATPases.     

Two distinct modes of ESCRT-III recognition are required for VPS4 functions in lysosomal protein targeting and HIV-1 budding

The ESCRT pathway mediates membrane remodeling during enveloped virus budding, cytokinesis, and intralumenal endosomal vesicle formation. Late in the pathway, a subset of membrane-associated ESCRT-III proteins display terminal amphipathic "MIM1" helices that bind and recruit VPS4 ATPases via their MIT domains. We now report that VPS4 MIT domains also bind a second, "MIM2" motif found in a different subset of ESCRT-III subunits. The solution structure of the VPS4 MIT-CHMP6 MIM2 complex revealed that MIM2 elements bind in extended conformations along the groove between the first and third helices of the MIT domain. Mutations that block VPS4 MIT-MIM2 interactions inhibit VPS4 recruitment, lysosomal protein targeting, and HIV-1 budding. MIT-MIM2 interactions appear to be common throughout the ESCRT pathway and possibly elsewhere, and we suggest how these interactions could contribute to a mechanism in which VPS4 and ESCRT-III proteins function together to constrict the necks of budding vesicles.     

Structural basis for autoinhibition of ESCRT-III CHMP3

Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family—cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.     

The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.     

Structural and mechanistic studies of VPS4 proteins

VPS4 ATPases function in multivesicular body formation and in HIV-1 budding. Here, we report the crystal structure of monomeric apo human VPS4B/SKD1 (hVPS4B), which is composed of five distinct elements: a poorly ordered N-terminal MIT domain that binds ESCRT-III substrates, large (mixed alpha/beta) and small (alpha) AAA ATPase domains that closely resemble analogous domains in the p97 D1 ATPase cassette, a three-stranded antiparallel beta domain inserted within the small ATPase domain, and a novel C-terminal helix. Apo hVPS4B and yeast Vps4p (yVps4p) proteins dimerized in solution, and assembled into larger complexes (10-12 subunits) upon ATP binding. Human and yeast adaptor proteins (LIP5 and yVta1p, respectively) bound the beta domains of the fully assembled hVPS4B and yVps4p proteins. We therefore propose that Vps4 proteins cycle between soluble, inactive low molecular weight complexes and active, membrane-associated double-ring structures that bind ATP and coassemble with LIP5/Vta1. Finally, HIV-1 budding was inhibited by mutations in a loop that projects into the center of the modeled hVPS4B rings, suggesting that hVPS4B may release the assembled ESCRT machinery by pulling ESCRT-III substrates up into the central pore.     

MITD1 is recruited to midbodies by ESCRT-III and participates in cytokinesis

Diverse cellular processes, including multivesicular body formation, cytokinesis, and viral budding, require the sequential functions of endosomal sorting complexes required for transport (ESCRTs) 0 to III. Of these multiprotein complexes, ESCRT-III in particular plays a key role in mediating membrane fission events by forming large, ring-like helical arrays. A number of proteins playing key effector roles, most notably the ATPase associated with diverse cellular activities protein VPS4, harbor present in microtubule-interacting and trafficking molecules (MIT) domains comprising asymmetric three-helical bundles, which interact with helical MIT-interacting motifs in ESCRT-III subunits. Here we assess comprehensively the ESCRT-III interactions of the MIT-domain family member MITD1 and identify strong interactions with charged multivesicular body protein 1B (CHMP1B), CHMP2A, and increased sodium tolerance-1 (IST1). We show that these ESCRT-III subunits are important for the recruitment of MITD1 to the midbody and that MITD1 participates in the abscission phase of cytokinesis. MITD1 also dimerizes through its C-terminal domain. Both types of interactions appear important for the role of MITD1 in negatively regulating the interaction of IST1 with VPS4. Because IST1 binding in turn regulates VPS4, MITD1 may function through downstream effects on the activity of VPS4, which plays a critical role in the processing and remodeling of ESCRT filaments in abscission.     

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.     

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.     

Mechanism of inhibition of retrovirus release from cells by interferon-induced gene ISG15

Budding of retroviruses from cell membranes requires ubiquitination of Gag and recruitment of cellular proteins involved in endosome sorting, including endosome sorting complex required for transport III (ESCRT-III) protein complex and vacuolar protein sorting 4 (VPS4) and its ATPase. In response to infection, a cellular mechanism has evolved that blocks virus replication early and late in the budding process through expression of interferon-stimulated gene 15 (ISG15), a dimer homologue of ubiquitin. Interferon treatment of DF-1 cells blocks avian sarcoma/leukosis virus release, demonstrating that this mechanism is functional under physiological conditions. The late block to release is caused in part by a loss in interaction between VPS4 and its coactivator protein LIP5, which is required to promote the formation of the ESCRT III-VPS4 double-hexamer complex to activate its ATPase. ISG15 is conjugated to two different LIP5-ESCRT-III-binding charged multivesicular body proteins, CHMP2A and CHMP5. Upon ISGylation of each, interaction with LIP5 is no longer detected. Two other ESCRT-III proteins, CHMP4B and CHMP6, are also conjugated to ISG15. ISGylation of CHMP2A, CHMP4B, and CHMP6 weakens their binding directly to VPS4, thereby facilitating the release of this protein from the membrane into the cytosol. The remaining budding complex fails to release particles from the cell membrane. Introducing a mutant of ISG15 into cells that cannot be conjugated to proteins prevents the ISG15-dependent mechanism from blocking virus release. CHMP5 is the primary switch to initiate the antiviral mechanism, because removal of CHMP5 from cells prevents ISGylation of CHMP2A and CHMP6.     

Biochemical Analyses of Human IST1 and Its Function in Cytokinesis

The newly described yeast endosomal sorting complexes required for transport (ESCRT) protein increased sodium tolerance-1 (Ist1p) binds the late-acting ESCRT proteins Did2p/charged MVB protein (CHMP) 1 and Vps4p and exhibits synthetic vacuolar protein sorting defects when combined with mutations in the Vta1p/LIP5–Vps60p/CHMP5 complex. Here, we report that human IST1 also functions in the ESCRT pathway and is required for efficient abscission during HeLa cell cytokinesis. IST1 binding interactions with VPS4, CHMP1, LIP5, and ESCRT-I were characterized, and the IST1–VPS4 interaction was investigated in detail. Mutational and NMR spectroscopic studies revealed that the IST1 terminus contains two distinct MIT interacting motifs (MIM1 and MIM2) that wrap around and bind in different groves of the MIT helical bundle. IST1, CHMP1, and VPS4 were recruited to the midbodies of dividing cells, and depleting either IST1 or CHMP1 proteins blocked VPS4 recruitment and abscission. In contrast, IST1 depletion did not inhibit human immunodeficiency virus-1 budding. Thus, IST1 and CHMP1 act together to recruit and modulate specific VPS4 activities required during the final stages of cell division.     

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.     

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.     

Charged multivesicular body protein 2B (CHMP2B) of the endosomal sorting complex required for transport-III (ESCRT-III) polymerizes into helical structures deforming the plasma membrane

The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation.     

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.     

Calpain-7 binds to CHMP1B at its second α-helical region and forms a ternary complex with IST1

Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.