Structure and functional interactions of the Tsg101 UEV domain

Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended beta-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the beta-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein-protein interactions required for HIV budding and VPS.     

The trihelical bundle subdomain of the GGA proteins interacts with multiple partners through overlapping but distinct sites

The Golgi-localized, gamma-adaptin ear-containing, ARF-binding (GGA) proteins are monomeric clathrin adaptors that mediate the sorting of cargo at the trans-Golgi network and endosomes. The GGAs contain four different domains named Vps27, Hrs, Stam (VHS); GGAs and TOM1 (GAT); hinge; and gamma-adaptin ear (GAE). The VHS domain recognizes transmembrane cargo, whereas the hinge and GAE regions bind clathrin and accessory proteins, respectively. The GAT domain is a polyfunctional module that interacts with various partners including the small GTPase ARF, the endosomal fusion regulator Rabaptin-5, ubiquitin, and the product of the tumor susceptibility gene 101 (TSG101). Previous x-ray crystallographic analyses showed that the GAT region is composed of two subdomains, an N-terminal helix-loop-helix containing the ARF binding site, and a C-terminal triple alpha-helical (trihelical) bundle. In this study, we define the Rabaptin-5 binding site on the GGA1-GAT domain and its relationship to the binding sites for ubiquitin and TSG101. Our observations show that Rabaptin-5, ubiquitin, and TSG101 bind to overlapping but distinct binding sites on the trihelical bundle. The different GAT binding partners engage in both competitive and cooperative interactions that may be important for the function of the GGAs in protein sorting.     

Spongiform neurodegeneration-associated E3 ligase Mahogunin ubiquitylates TSG101 and regulates endosomal trafficking

A null mutation in the gene encoding the putative E3 ubiquitin-protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.     

An HRD/DER-independent ER quality control mechanism involves Rsp5p-dependent ubiquitination and ER-Golgi transport

There is increasing evidence that ubiquitination of receptors provides an important endosomal sorting signal. Here we report that mammalian class E vacuolar protein-sorting (vps) proteins recognize ubiquitin. Both tumor susceptibility gene 101 (TSG101)/human VPS (hVPS)28 and hepatocyte growth factor receptor substrate (Hrs) cytosolic complexes bind ubiquitin-agarose. TSG101 and hVPS28 are localized to endosomes that contain internalized EGF receptor and label strongly for ubiquitinated proteins. Microinjection of anti-hVPS28 specifically retards EGF degradation and leads to endosomal accumulation of ubiquitin-protein conjugates. Likewise, depletion of TSG101 impairs EGF trafficking and causes dramatic relocalization of ubiquitin to endocytic compartments. Similar defects are found in cells overexpressing Hrs, further emphasizing the links between class E protein function, receptor trafficking, and endosomal ubiquitination.     

Structural insights into endosomal sorting complex required for transport (ESCRT-I) recognition of ubiquitinated proteins

The endosomal sorting complex required for transport (ESCRT-I) is a 350-kDa complex of three proteins, Vps23, Vps28, and Vps37. The N-terminal ubiquitin-conjugating enzyme E2 variant (UEV) domain of Vps23 is required for sorting ubiquitinated proteins into the internal vesicles of multivesicular bodies. UEVs are homologous to E2 ubiquitin ligases but lack the conserved cysteine residue required for catalytic activity. The crystal structure of the yeast Vps23 UEV in a complex with ubiquitin (Ub) shows the detailed interactions made with the bound Ub. Compared with the solution structure of the Tsg101 UEV (the human homologue of Vps23) in the absence of Ub, two loops that are conserved among the ESCRT-I UEVs move toward each other to grip the Ub in a pincer-like grasp. The contacts with the UEV encompass two adjacent patches on the surface of the Ub, one containing several hydrophobic residues, including Ile-8(Ub), Ile-44(Ub), and Val-70(Ub), and the second containing a hydrophilic patch including residues Asn-60(Ub), Gln-62(Ub), Glu-64(Ub). The hydrophobic Ub patch interacting with the Vps23 UEV overlaps the surface of Ub interacting with the Vps27 ubiquitin-interacting motif, suggesting a sequential model for ubiquitinated cargo binding by these proteins. In contrast, the hydrophilic patch encompasses residues uniquely interacting with the ESCRT-I UEV. The structure provides a detailed framework for design of mutants that can specifically affect ESCRT-I-dependent sorting of ubiquitinated cargo without affecting Vps27-mediated delivery of cargo to endosomes.     

Abnormal regulation of TSG101 in mice with spongiform neurodegeneration

Spongiform neurodegeneration is characterized by the appearance of vacuoles throughout the central nervous system. It has many potential causes, but the underlying cellular mechanisms are not well understood. Mice lacking the E3 ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) develop age-dependent spongiform encephalopathy. We identified an interaction between a “PSAP” motif in MGRN1 and the ubiquitin E2 variant (UEV) domain of TSG101, a component of the endosomal sorting complex required for transport I (ESCRT-I), and demonstrate that MGRN1 multimonoubiquitinates TSG101. We examined the in vivo consequences of loss of MGRN1 on TSG101 expression and function in the mouse brain. The pattern of TSG101 ubiquitination differed in the brains of wild-type mice and Mgrn1 null mutant mice: at 1 month of age, null mutant mice had less ubiquitinated TSG101, while in adults, mutant mice had more ubiquitinated, insoluble TSG101 than wild-type mice. There was an associated increase in epidermal growth factor receptor (EGFR) levels in mutant brains. These results suggest that loss of MGRN1 promotes ubiquitination of TSG101 by other E3s and may prevent its disassociation from endosomal membranes or cause it to form insoluble aggregates. Our data implicate loss of normal TSG101 function in endo-lysosomal trafficking in the pathogenesis of spongiform neurodegeneration in Mgrn1 null mutant mice.     

NEDD4L overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking PTAP and YPXL late domains

The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6(Gag) bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101- and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.     

Structure of the Tsg101 UEV domain in complex with the PTAP motif of the HIV-1 p6 protein

The structural proteins of HIV and Ebola display PTAP peptide motifs (termed 'late domains') that recruit the human protein Tsg101 to facilitate virus budding. Here we present the solution structure of the UEV (ubiquitin E2 variant) binding domain of Tsg101 in complex with a PTAP peptide that spans the late domain of HIV-1 p6(Gag). The UEV domain of Tsg101 resembles E2 ubiquitin-conjugating enzymes, and the PTAP peptide binds in a bifurcated groove above the vestigial enzyme active site. Each PTAP residue makes important contacts, and the Ala 9-Pro 10 dipeptide binds in a deep pocket of the UEV domain that resembles the X-Pro binding pockets of SH3 and WW domains. The structure reveals the molecular basis of HIV PTAP late domain function and represents an attractive starting point for the design of novel inhibitors of virus budding.     

HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.     

Interactions of TOM1L1 with the multivesicular body sorting machinery

Tom1L1 (Tom1-like1) and related proteins Tom1 (Target of Myb1) and Tom1L2 (Tom1-like2) constitute a new protein family characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion followed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 binds ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). Here we report a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), whereas a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for binding to TSG101 (tumor susceptibility gene 101). Myc epitope-tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In addition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after epidermal growth factor internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together, we propose that Tom1L1 could act as an intermediary between signaling and degradative pathways.     

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.     

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.     

The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. 1) TSG101 bound to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. 2) TSG101 and VPS28 co-immunoprecipitated with VPS37B-FLAG, and the three proteins comigrated together in soluble complexes of the correct size for human ESCRT-I ( approximately 350 kDa). 3) Like TGS101, VPS37B became trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. 4) Finally, VPS37B could recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibited PTAP or VPS28 binding blocked HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity.     

TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes

Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.     

GGA proteins bind ubiquitin to facilitate sorting at the trans-Golgi network

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.     

Structural basis for ubiquitin recognition by the human ESCRT-II EAP45 GLUE domain

The ESCRT-I and ESCRT-II complexes help sort ubiquitinated proteins into vesicles that accumulate within multivesicular bodies (MVBs). Crystallographic and biochemical analyses reveal that the GLUE domain of the human ESCRT-II EAP45 (also called VPS36) subunit is a split pleckstrin-homology domain that binds ubiquitin along one edge of the beta-sandwich. The structure suggests how human ESCRT-II can couple recognition of ubiquitinated cargoes and endosomal phospholipids during MVB protein sorting.     

PPPYVEPTAP motif is the late domain of human T-cell leukemia virus type 1 Gag and mediates its functional interaction with cellular proteins Nedd4 and Tsg101 [corrected

The human T-cell leukemia virus type 1 (HTLV-1) Gag polyprotein contains two adjacent proline-rich motifs (sequence PPPYVEPTAP) in the C terminus of the matrix domain [corrected]. Proline-to-alanine mutations were introduced into either or both motifs of HTLV-1 to determine the effect on the release of HTLV-1 virus-like particles from 293T cells. The release of both single mutants was significantly reduced, whereas a double mutation in both motifs abolished the release of the HTLV-1 particles. Two-hybrid and in vitro binding assays showed that the HTLV-1 Gag polyprotein binds both Tsg101 and Nedd4 proteins. The interaction with HTLV-1 Gag required the central WW domain of Nedd4 and the ubiquitin enzyme variant (UEV) domain of Tsg101. We expressed various fragments of Nedd4 and Tsg101 proteins in 293T cells and tested for their ability to interfere with virion release mediated by the HTLV-1 Gag-Pro polyprotein. Fragments consisting of the N-terminal UEV domain of Tsg101 and the central WW and C-terminal Hect domains of Nedd4 protein all caused transdominant inhibition of HTLV-1 particle release. Similarly, inhibition of the proteasome significantly decreased HTLV-1 particle release. Furthermore, the WW domain overexpression caused an early arrest of HTLV-1 particle morphogenesis before the membrane is deformed into the typical half-shell structure. This result suggests that Nedd4 is involved early in budding of HTLV-1.     

Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation

Monoubiquitylation is a well-characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin-binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded alpha-helices that bind ubiquitin independently, non-cooperatively and with modest affinity. The Vps27 N-terminal UIM engages the Leu8-Ile44-Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8-Ile44-Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin-binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.