The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release

Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical "boomerang" folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.     

Recruitment of UBPY and ESCRT Exchange Drive HD-PTP-Dependent Sorting of EGFR to the MVB

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Highlights? HD-PTP is essential for sorting EGFR to the MVB lumen ? HD-PTP binds the ESCRT-0 subunit STAM2 at two sites ? CHMP4B and UBPY displace EGFR from ESCRT-0 and drive ESCRT-III association ? UBPY is important for sorting EGFR to the MVB lumen     

HD-PTP and Alix share some membrane-traffic related proteins that interact with their Bro1 domains or proline-rich regions

Mammalian Alix is a multifunctional adaptor protein involved in cell death, receptor endocytosis, endosomal protein sorting and cell adhesion by associating with various proteins such as ALG-2, CIN85/Rukl/SETA, endophilins, CHMP4s and TSG101. HD-PTP is a paralog of Alix and a putative protein tyrosine phosphatase (PTP) that contains a Bro1 domain, coiled-coils, a proline-rich region (PRR) in addition to a PTP domain. We investigated interactions between HD-PTP and Alix-binding proteins. In the yeast two-hybrid assay, HD-PTP showed positive interactions with CHMP4b/Shax1, TSG101, endophilin A1 and ALG-2 but not with either RabGAPLP or CIN85. We confirmed the interactions in a mammalian system by Strep-pulldown assays in which pulldown products from the lysates of HEK293T cells expressing either Strep-tagged HD-PTP alone or co-expressing with epitope-tagged proteins were analyzed by Western blotting using specific antibodies. While Alix associated with both ALG-2 and TSG101 in a Ca2+-dependent manner, HD-PTP interacted with ALG-2 Ca2+-dependently but with TSG101 Ca2+-independently.     

Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central “V” domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.     

Brox, a novel farnesylated Bro1 domain-containing protein that associates with charged multivesicular body protein 4 (CHMP4)

Human Brox is a newly identified 46 kDa protein that has a Bro1 domain-like sequence and a C-terminal thioester-linkage site of isoprenoid lipid (CAAX motif) (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid). Mammalian Alix and its yeast ortholog, Bro1, are known to associate with charged multivesicular body protein 4 (CHMP4), a component of endosomal sorting complex required for transport III, via their Bro1 domains and to play roles in sorting of ubiquitinated cargoes. We investigated whether Brox has an authentic Bro1 domain on the basis of its capacity for interacting with CHMP4s. Both Strep Tactin binding sequence (Strep)-tagged wild-type Brox (Strep-Brox(WT)) and Strep-tagged farnesylation-defective mutant (Cys-->Ser mutation; Strep-Brox(C408S)) pulled down FLAG-tagged CHMP4b that was coexpressed in HEK293 cells. Treatment of cells with a farnesyltransferase inhibitor, FTI-277, caused an electrophoretic mobility shift of Strep-Brox(WT), and the mobility coincided with that of Strep-Brox(C408S). The inhibitor also caused a mobility shift of endogenous Brox detected by western blotting using polyclonal antibodies to Brox, suggesting farnesylation of Brox in vivo. Fluorescence microscopic analyses revealed that Strep-Brox(WT) exhibited accumulation in the perinuclear area and caused a punctate pattern of FLAG-CHMP4b that was constitutively expressed in HEK293 cells. On the other hand, Strep-Brox(C408S) showed a diffuse pattern throughout the cell, including the nucleus, and did not cause accumulation of FLAG-CHMP4b. Fluorescent signals of monomeric green fluorescent protein (mGFP)-fused Brox(WT) merged partly with those of Golgi markers and with those of abnormal endosomes induced by overexpression of a dominant negative mutant of AAA type ATPase SKD1/Vps4B in HeLa cells, but such colocalization was less efficient for mGFP-Brox(C408S). These results suggest a physiological significance of farnesylation of Brox in its subcellular distribution and efficient interaction with CHMP4s in vivo.     

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.     

Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X)nL Motif-Containing Target Proteins

Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.     

Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner

Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release-mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT.     

Structural basis for endosomal targeting by the Bro1 domain

Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.     

Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding

ALIX/AIP1 functions in enveloped virus budding, endosomal protein sorting, and many other cellular processes. Retroviruses, including HIV-1, SIV, and EIAV, bind and recruit ALIX through YPX(n)L late-domain motifs (X = any residue; n = 1-3). Crystal structures reveal that human ALIX is composed of an N-terminal Bro1 domain and a central domain that is composed of two extended three-helix bundles that form elongated arms that fold back into a "V." The structures also reveal conformational flexibility in the arms that suggests that the V domain may act as a flexible hinge in response to ligand binding. YPX(n)L late domains bind in a conserved hydrophobic pocket on the second arm near the apex of the V, whereas CHMP4/ESCRT-III proteins bind a conserved hydrophobic patch on the Bro1 domain, and both interactions are required for virus budding. ALIX therefore serves as a flexible, extended scaffold that connects retroviral Gag proteins to ESCRT-III and other cellular-budding machinery.     

Two distinct binding modes define the interaction of Brox with the C-terminal tails of CHMP5 and CHMP4B

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Highlights? CHMP5 binds and recruits Brox to membrane fractions through its C-terminal tail ? The CHMP5 C-terminal tail adopts a tandem β-hairpin structure ? The CHMP4B C-terminal tail α helix binds at a concave surface of Brox ? The CHMP5 β-hairpins and CHMP4B α helix bind the same surface at Brox     

Structure of the Bro1 domain protein BROX and functional analyses of the ALIX Bro1 domain in HIV-1 budding

Background

Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC^Gag protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements.

Methodology/Principal Findings

We report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding.

Conclusions/Significance

Our studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.     

Trafficking and developmental signaling: Alix at the crossroads

Alix is a phylogenetically conserved protein that participates in mammals in programmed cell death in association with ALG-2, a penta-EF-hand calciprotein. It contains an N-terminal Bro1 domain, a coiled-coil region and a C-terminal proline-rich domain containing several SH3- and WW-binding sites that contribute to its scaffolding properties. Recent data showed that by virtue of its Bro1 domain, Alix is functionally associated to the ESCRT complexes involved in the biogenesis of the multivesicular body and sorting of transmembrane proteins within this specific endosomal compartment. In Dictyostelium, an alx null strain shows a markedly perturbed starvation-induced morphogenetic program while ALG-2 disruptants remain unaffected. This review summarizes Dictyostelium data on Alix and ALG-2 homologues and evaluates whether known functions of Alix in other organisms can account for the developmental arrest of the alx null mutant and how Dictyostelium studies can substantiate the current understanding of the function(s) of this versatile and conserved signaling molecule.     

Divergent Bro1 Domains Share the Capacity To Bind Human Immunodeficiency Virus Type 1 Nucleocapsid and To Enhance Virus-Like Particle Production

To promote the release of infectious virions, human immunodeficiency virus type 1 (HIV-1) exploits the endosomal sorting complex required for transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in p6 Gag. An LYPx_nL motif in p6 serves as docking site for the central V domain of ALIX and is required for its ability to stimulate HIV-1 budding. Additionally, the nucleocapsid (NC) domain of Gag binds to the N-terminal Bro1 domain of ALIX, which connects ALIX to the membrane-deforming ESCRT-III complex via its CHMP4 subunits. Since the isolated Bro1 domain of ALIX is sufficient to markedly stimulate virus-like particle (VLP) production in a minimal Gag rescue assay, we examined whether the Bro1 domains of other human proteins possess a similar activity. We now show that the Bro1 domain-only protein Brox and the isolated Bro1 domains of HD-PTP and rhophilin all bind to HIV-1 NC. Furthermore, all shared the capacity to stimulate VLP production by a minimal HIV-1 Gag molecule, and Brox in particular was as potent as the Bro1 domain of ALIX in this assay. Unexpectedly, Brox retained significant activity even if its CHMP4 binding site was disrupted. Thus, the ability to assist in VLP production may be an intrinsic property of the boomerang-shaped Bro1 domain.Retroviruses engage an endosomal budding machinery via so-called late assembly (L) domains in Gag to promote virus budding at the plasma membrane (4, 17, 33). In the case of human immunodeficiency virus type 1 (HIV-1), the C-terminal p6 domain of Gag harbors a conserved P(T/S)AP motif, which binds to the host protein Tsg101 and functions as the primary L domain (18, 29, 44). Additionally, HIV-1 p6 contains an auxiliary L domain of the LYPx_nL type, which serves as a docking site for ALIX (28, 41, 45). Tsg101 and ALIX are both components of a protein network that is required for the biogenesis of multivesicular bodies (MVB) (22, 38). These compartments are formed through the budding of vesicles from the limiting membrane of endosomes into their lumen, a process that is topologically equivalent to virus budding at the plasma membrane. Recently, it emerged that the protein network essential for MVB formation also functions in cytokinesis, which requires a membrane fission event of similar topology (7, 32).Most of the components of the protein network that mediates these events are subunits of heteromeric endosomal sorting complexes required for transport (ESCRT) (3, 22, 38). For instance, Tsg101 is a subunit of the heterotetrameric ESCRT-I complex (22, 38). ESCRT-I and the downstream ESCRT-II are stable complexes, whereas ESCRT-III assembles only upon membrane binding (38). ESCRT-III is formed by the structurally related human CHMP proteins, which exist in an autoinhibited monomeric conformation in the cytosol (40, 46). A conformational change from a closed to an open conformation is thus likely required for the activation of CHMP proteins and the assembly of ESCRT-III. Interestingly, the uncontrolled activation of CHMP proteins through the removal of autoinhibitory C-terminal sequences results in the potent inhibition of HIV-1 budding, indicating a central role for ESCRT-III in retroviral release (46).ALIX consists of a boomerang-shaped N-terminal Bro1 domain, a central ligand binding domain that is shaped like a V, and a C-terminal proline-rich region (16). While ALIX is essential for equine anemia virus budding, its role in HIV-1 budding is less critical than that of Tsg101 (8, 16, 28, 41). However, ALIX can clearly support efficient HIV-1 budding, because its overexpression potently rescues the release defect of Tsg101 binding site mutants (16, 43). This effect of ALIX depends on the interaction between its central V domain and the LYPx_nL motif in HIV-1 p6 (16, 43), confirming that this motif constitutes a functional L domain.The Bro1 domain of ALIX interacts tightly with ESCRT-III subunit CHMP4B and less avidly with CHMP4A and CHMP4C (25, 28, 41, 45). The ability of ALIX to rescue HIV-1 L domain mutants depends on the interaction between its Bro1 domain and CHMP4, indicating that CHMP4 is of particular importance in viral budding (16, 43). Interestingly, human CHMP4A assembles into membrane-attached filaments if overexpressed in mammalian cells, and these filaments can be induced to form circular arrays that drive the formation of buds and tubules with the same topology as that of a retroviral bud (21). Also, the single yeast ortholog of the mammalian CHMP4 proteins forms homo-oligomeric filaments on endosomes that appear to drive MVB sorting and biogenesis (42).By binding to membranes with its convex surface, the Bro1 domain of ALIX could also contribute directly to the generation of negative curvature required for budding away from the cytosol. In support of this notion, we recently observed that the isolated Bro1 domain of ALIX can potently enhance the formation of virus-like particles (VLP) by a minimal HIV-1 Gag construct that retains the primary L domain but lacks certain assembly domains and thus is presumably defective in its ability to deform membranes (37). We also observed that the Bro1 domain of ALIX physically interacts with the nucleocapsid (NC) region of HIV-1 Gag and that mutations in NC that interfere with the interaction induce a phenotype that resembles that of L domain mutants (37).Despite limited sequence homology between human ALIX and a yeast counterpart, the structures of their Bro1 domains are largely superimposable (16, 26), suggesting that all Bro1 domains have a shape that would be compatible with a membrane-deforming function. We therefore asked whether the ability to stimulate VLP production is unique to the Bro1 domain of ALIX or a property of Bro1 domains in general. We now show that widely divergent Bro1 domains share the ability to associate with HIV-1 Gag in an NC-dependent manner and to enhance VLP production by a minimal Gag molecule. In particular, a human Bro1 domain-only protein termed Brox (23) was as potent as the ALIX Bro1 domain in stimulating VLP production, and even forms of Brox that did not bind to CHMP4 retained significant activity. We thus propose that Bro1 domains are inherently capable of promoting budding events away from the cytosol.     

Sprouty 2 binds ESCRT-II factor Eap20 and facilitates HIV-1 gag release

The four ESCRT (endocytic sorting complexes required for transport) complexes (ESCRT-0, -I, -II, and -III) normally operate sequentially in the trafficking of cellular cargo. HIV-1 Gag trafficking and release as virus-like particles (VLPs) require the participation of ESCRTs; however, its use of ESCRTs is selective and nonsequential. Specifically, Gag trafficking to release sites on the plasma membrane does not require ESCRT-0 or -II. It is known that a bypass of ESCRT-0 is achieved by the direct linkage of the ESCRT-I component, Tsg101, to the primary L domain motif (PTAP) in Gag and that bypass of ESCRT-II is achieved by the linkage of Gag to ESCRT-III through the adaptor protein Alix. However, the mechanism by which Gag suppresses the interaction of bound ESCRT-I with ESCRT-II is unknown. Here we show (i) that VLP release requires the steady-state level of Sprouty 2 (Spry2) in COS-1 cells, (ii) that Spry2 binds the ESCRT-II component Eap20, (iii) that binding Eap20 permits Spry2 to disrupt ESCRT-I interaction with ESCRT-II, and (iv) that coexpression of Gag with a Spry2 fragment that binds Eap20 increases VLP release. Spry2 also facilitated release of P7L-Gag (i.e., release in the absence of Tsg101 binding). In this case, rescue required the secondary L domain (YPX(n)L) in HIV-1 Gag that binds Alix and the region in Spry2 that binds Eap20. The results identify Spry2 as a novel cellular factor that facilitates release driven by the primary and secondary HIV-1 Gag L domains.     

Backbone 1H, 13C, and 15N assignments for the tandem ubiquitin binding domains of signal transducing adapter molecule 1

Signal transducing adapter molecule (STAM) forms the endosomal sorting complex required for transport-0 (ESCRT-0) complex with hepatocyte growth factor-regulated substrate (Hrs) to sort the ubiquitinated cargo proteins from the early endosomes to the ESCRT-1 complex. ESCRT-0 complex, STAM and Hrs, contains multiple ubiquitin binding domains, in which STAM has two ubiquitin binding domains, Vps27/Hrs/Stam (VHS) and ubiquitin interacting motif (UIM) at its N-terminus. By the cooperation of the multiple ubiquitin binding domains, the ESCRT-0 complex recognizes poly-ubiquitin, especially Lys63-linked ubiquitin. Here, we report the backbone resonance assignments and the secondary structure of the N-terminal 191 amino acids of the human STAM1 which includes the VHS domain and UIM. The {¹H}-¹⁵N heteronuclear NOE experiments revealed that an unstructured and flexible loop region connects the VHS domain and UIM. Our work provides the basic information for the further NMR investigation of the interaction between STAM1 and poly-ubiquitin.     

UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting

Endosomal sorting complexes required for transport (ESCRTs) regulate several events involving membrane invagination, including multivesicular body (MVB) biogenesis, viral budding, and cytokinesis. In each case, upstream ESCRTs combine with additional factors, such as Bro1 proteins, to recruit ESCRT-III and the ATPase VPS4 in order to drive membrane scission. A clue to understanding how such diverse cellular processes might be controlled independently of each other has been the identification of ESCRT isoforms. Mammalian ESCRT-I comprises TSG101, VPS28, VPS37A-D, and MVB12A/B. These could generate several ESCRT-I complexes, each targeted to a different compartment and able to recruit distinct ESCRT-III proteins. Here we identify a novel ESCRT-I component, ubiquitin-associated protein 1 (UBAP1), which contains a region conserved in MVB12. UBAP1 binds the endosomal Bro1 protein His domain protein tyrosine phosphatase (HDPTP), but not Alix, a Bro1 protein involved in cytokinesis. UBAP1 is required for sorting EGFR to the MVB and for endosomal ubiquitin homeostasis, but not for cytokinesis. UBAP1 is part of a complex that contains a fraction of total cellular TSG101 and that also contains VPS37A but not VPS37C. Hence, the presence of UBAP1, in combination with VPS37A, defines an endosome-specific ESCRT-I complex.     

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.