Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.     

Ubiquitin interactions of NZF zinc fingers

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.     

Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.     

Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex

ESCRT-II plays a pivotal role in receptor downregulation and multivesicular body biogenesis and is conserved from yeast to humans. The crystal structures of two human ESCRT-II complex structures have been determined at 2.6 and 2.9 A resolution, respectively. The complex has three lobes and contains one copy each of VPS22 and VPS36 and two copies of VPS25. The structure reveals a dynamic helical domain to which both the VPS22 and VPS36 subunits contribute that connects the GLUE domain to the rest of the ESCRT-II core. Hydrodynamic analysis shows that intact ESCRT-II has a compact, closed conformation. ESCRT-II binds to the ESCRT-I VPS28 C-terminal domain subunit through a helix immediately C-terminal to the VPS36-GLUE domain. ESCRT-II is targeted to endosomal membranes by the lipid-binding activities of both the Vps36 GLUE domain and the first helix of Vps22. These data provide a unifying structural and functional framework for the ESCRT-II complex.     

Ubiquitin signals protein trafficking via interaction with a novel ubiquitin binding domain in the membrane fusion regulator,Vps9p

The conserved vacuolar protein-sorting (Vps) pathway controls the trafficking of proteins to the vacuole/lysosome. Both the internalization of ubiquitylated cargo from the plasma membrane and its sorting at the late endosome via the Vps pathway depend on ubiquitin (Ub) binding motifs present in trafficking regulators. Here we report that Ub controls yet a third step in the Vps pathway. Vps9p, which promotes endosomal and Golgi-derived vesicle fusion, binds directly to Ub via a Cue1p-homologous (CUE) domain. The CUE domain is structurally related to the Ub-associated (UBA) domain. In an assay for vacuolar delivery of a transmembrane receptor fused to Ub, a Ub mutation impairing interaction with Vps9p led to a cytoplasmic block in receptor trafficking. This block resembled that of a receptor fused to wild-type Ub but expressed in a vps9-null background. Strikingly, this trafficking defect caused by a mutant Ub was rescued by deletion of the Vps9p CUE domain, indicating that lack of the CUE domain renders Vps9p independent of Ub for activation in vivo. We thus provide evidence for biochemical and genetic interactions between Ub and a novel Ub binding domain in Vps9p. Ub plays a positive role, whereas the CUE domain plays both positive and negative roles in Vps9p function in trafficking.     

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.     

ESCRT-I core and ESCRT-II GLUE domain structures reveal role for GLUE in linking to ESCRT-I and membranes

ESCRT complexes form the main machinery driving protein sorting from endosomes to lysosomes. Currently, the picture regarding assembly of ESCRTs on endosomes is incomplete. The structure of the conserved heterotrimeric ESCRT-I core presented here shows a fan-like arrangement of three helical hairpins, each corresponding to a different subunit. Vps23/Tsg101 is the central hairpin sandwiched between the other subunits, explaining the critical role of its "steadiness box" in the stability of ESCRT-I. We show that yeast ESCRT-I links directly to ESCRT-II, through a tight interaction of Vps28 (ESCRT-I) with the yeast-specific zinc-finger insertion within the GLUE domain of Vps36 (ESCRT-II). The crystal structure of the GLUE domain missing this insertion reveals it is a split PH domain, with a noncanonical lipid binding pocket that binds PtdIns3P. The simultaneous and reinforcing interactions of ESCRT-II GLUE domain with membranes, ESCRT-I, and ubiquitin are critical for ubiquitinated cargo progression from early to late endosomes.     

Structural insight into the ESCRT-I/-II link and its role in MVB trafficking

ESCRT (endosomal sorting complex required for transport) complexes orchestrate efficient sorting of ubiquitinated transmembrane receptors to lysosomes via multivesicular bodies (MVBs). Yeast ESCRT-I and ESCRT-II interact directly in vitro; however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, we have characterised the ESCRT-I/-II supercomplex and determined the crystal structure of its interface. The link is formed by the vacuolar protein sorting (Vps)28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiquitinated cargo. We also show that a new component of ESCRT-I, Mvb12 (YGR206W), engages ESCRT-I directly with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. Our data suggest a complex regulatory mechanism for the ESCRT-I/-II link in yeast.     

Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting "receptors," the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.     

DOA1/UFD3 plays a role in sorting ubiquitinated membrane proteins into multivesicular bodies

Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs.     

Avian sarcoma virus and human immunodeficiency virus, type 1 use different subsets of ESCRT proteins to facilitate the budding process

Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in avian sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (Deltap2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/Deltap2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/Deltap2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin-tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements.     

The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.     

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co‐expression of affinity‐tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in‐vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non‐ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9‐GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.     

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.     

Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.     

Structural basis for specific recognition of Lys 63‐linked polyubiquitin chains by NZF domains of TAB2 and TAB3

TAB2 and TAB3 activate the Jun N‐terminal kinase and nuclear factor‐κB pathways through the specific recognition of Lys 63‐linked polyubiquitin chains by its Npl4 zinc‐finger (NZF) domain. Here we report crystal structures of the TAB2 and TAB3 NZF domains in complex with Lys 63‐linked diubiquitin at 1.18 and 1.40 Å resolutions, respectively. Both NZF domains bind to the distal ubiquitin through a conserved Thr‐Phe dipeptide that has been shown to be important for the interaction of the NZF domain of Npl4 with monoubiquitin. In contrast, a surface specific to TAB2 and TAB3 binds the proximal ubiquitin. Both the distal and proximal binding sites of the TAB2 and TAB3 NZF domains recognize the Ile 44‐centred hydrophobic patch on ubiquitin but do not interact with the Lys 63‐linked isopeptide bond. Mutagenesis experiments show that both binding sites are required to enable binding of Lys 63‐linked diubiquitin. We therefore propose a mechanism for the recognition of Lys 63‐linked polyubiquitin chains by TAB2 and TAB3 NZF domains in which diubiquitin units are specifically recognized by a single NZF domain.     

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.     

ESCRT-II, an endosome-associated complex required for protein sorting: crystal structure and interactions with ESCRT-III and membranes

ESCRT-I, -II, and -III protein complexes are sequentially recruited to endosomal membranes, where they orchestrate protein sorting and MVB biogenesis. In addition, they play a critical role in retrovirus budding. Structural understanding of ESCRT interaction networks is largely lacking. The 3.6 A structure of the yeast ESCRT-II core presented here reveals a trilobal complex containing two copies of Vps25, one copy of Vps22, and the C-terminal region of Vps36. Unexpectedly, the entire ESCRT-II core consists of eight repeats of a common building block, a "winged helix" domain. Two PPXY-motifs from Vps25 are involved in contacts with Vps22 and Vps36, and their mutation leads to ESCRT-II disruption. We show that purified ESCRT-II binds directly to the Vps20 component of ESCRT-III. Surprisingly, this binding does not require the protruding N-terminal coiled-coil of Vps22. Vps25 is the chief subunit responsible for Vps20 recruitment. This interaction dramatically increases binding of both components to lipid vesicles in vitro.     

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.     

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.