STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif

Conjugation with ubiquitin acts as a sorting signal for proteins in the endocytic and biosynthetic pathways at the endosome. Signal-transducing adaptor molecule (STAM) proteins, STAM1 and STAM2, are associated with hepatocyte growth factor-regulated substrate (Hrs) but their function remains unknown. Herein, we show that STAM proteins bind ubiquitin and ubiquitinated proteins and that the tandemly located VHS (Vps27/Hrs/STAM) domain and ubiquitin-interacting motif serve as the binding site(s). STAM proteins colocalize with Hrs on the early endosome. Overexpression of STAM proteins, but not their mutants lacking the ubiquitin-binding activity, causes the accumulation of ubiquitinated proteins and ligand-activated epidermal growth factor receptor on the early endosome. These results suggest that through interaction with ubiquitinated cargo proteins on the early endosome via the VHS domain and ubiquitin-interacting motif, STAM proteins participate in the sorting of cargo proteins for trafficking to the lysosome.     

ESCRT-0 Protein Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate (Hrs) Is Targeted to Endosomes Independently of Signal-transducing Adaptor Molecule (STAM) and the Complex Formation with STAM Promotes Its Endosomal Dissociation

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.     

STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes

STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes. In this work, we show that STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex. We find that overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors. We propose that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway.     

Association with Hrs is required for the early endosomal localization, stability, and function of STAM

Members of the STAM family of proteins, STAM1 and STAM2, are associated with Hrs through their coiled-coil regions. Both Hrs and STAM bind ubiquitin and are involved in endosomal sorting of ubiquitinated cargo proteins for trafficking to the lysosome. Here we examined the biological significance of STAM binding to Hrs. Endogenous STAM1 and STAM2 were mostly localized on the early endosome, suggesting that they are resident endosomal proteins. A STAM2 mutant that lacks the coiled-coil region and does not bind Hrs, in contrast, mislocalized to the cytoplasm. Deletion of a region located N-terminal to the coiled-coil region and conserved among STAM proteins also severely affected Hrs binding and the endosomal localization of STAM2, suggesting that this region is also involved in these activities. Depletion of endogenous Hrs by RNA interference similarly caused the mislocalization of exogenously expressed STAM2 to the cytoplasm. These results indicate that STAM is localized to the early endosome by binding to Hrs on the target membrane. In addition, the expression level of endogenous STAM proteins was drastically reduced in Hrs-depleted cells, suggesting that STAM is stabilized by binding to Hrs. Finally, STAM2 mutants lacking the Hrs-binding activity were defective in causing the enlargement of early endosomes, accumulating ubiquitinated proteins on this aberrant organelle, and inhibiting the degradation of ligand-activated epidermal growth factor receptors, suggesting that the association with Hrs is a prerequisite for STAM function.     

Cell‐Free Reconstitution of Multivesicular Body Formation and Receptor Sorting

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell‐free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal‐transducing adaptor molecule (STAM), or addition of mutated ATPase‐deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.     

The Hrs/STAM complex in the downregulation of receptor tyrosine kinases

Cell surface receptor proteins that have undergone endocytosis are transported to the endosome. From the endosome, ligand-activated receptor tyrosine kinases are further transported to the lysosome for degradation, a process called "receptor downregulation." By contrast, nutrient receptors, such as those for low-density lipoprotein and transferrin, are recycled back to the plasma membrane. Sorting of these two types of receptors occurs at the endosome, where ubiquitination of receptor proteins serves as the sorting signal. Namely, ubiquitinated receptors are incorporated into the lysosomal degradation pathway, whereas those that are not ubiquitinated are returned to the cell surface. Hrs and STAM are proteins that form a complex on the endosomal membrane. Recent studies have shown that the Hrs/STAM complex binds ubiquitin moieties and acts as sorting machinery that recognizes ubiquitinated receptors and transfers them to further sequential lysosomal sorting/trafficking processes.     

Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p; however, the role of Hrs in lysosomal trafficking is unclear. Here, we report that Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognizes the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor. Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form a approximately 550-kDa complex that excludes EGFR. Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes. By using deletion analysis, we have mapped the binding domains of Hrs and SNX1 that mediate their association. Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis. These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1.     

STAM Adaptor Proteins Interact with COPII Complexes and Function in ER-to-Golgi Trafficking

Signal-transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. In this study, we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells, the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted, the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein–green fluorescent protein trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after Brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with coat protein II (COPII) proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex.     

Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton

In Saccharomyces cerevisiae, the class C vacuole protein sorting (Vps) proteins, together with Vam2p/Vps41p and Vam6p/Vps39p, form a complex that interacts with soluble N-ethylmaleimide-sensitive factor attachment protein receptor and Rab proteins to "tether" vacuolar membranes before fusion. To determine a role for the corresponding mammalian orthologues, we examined the function, localization, and protein interactions of endogenous mVps11, mVps16, mVps18, mVam2p, and mVam6. We found a significant proportion of these proteins localized to early endosome antigen-1 and transferrin receptor-positive early endosomes in Vero, normal rat kidney, and Chinese hamster ovary cells. Immunoprecipitation experiments showed that mVps18 not only interacted with Syntaxin (Syn)7, vesicle-associated membrane protein 8, and Vti1-b but also with Syn13, Syn6, and the Sec1/Munc18 protein mVps45, which catalyze early endosomal fusion events. Moreover, anti-mVps18 antibodies inhibited early endosome fusion in vitro. Mammalian mVps18 also associated with mVam2 and mVam6 as well as with the microtubule-associated Hook1 protein, an orthologue of the Drosophila Hook protein involved in endosome biogenesis. Using in vitro binding and immunofluorescence experiments, we found that mVam2 and mVam6 also associated with microtubules, whereas mVps18, mVps16, and mVps11 associated with actin filaments. These data indicate that the late Vps proteins function during multiple soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated fusion events throughout the endocytic pathway and that their activity may be coordinated with cytoskeletal function.     

The Sec1/Munc18 protein, Vps33p, functions at the endosome and the vacuole of Saccharomyces cerevisiae

The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Delta cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.     

Vps27 recruits ESCRT machinery to endosomes during MVB sorting

Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.     

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.     

Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.     

VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins.

Many of the vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae exhibit severe defects in the sorting of vacuolar proteins but still retain near-normal vacuole morphology. The gene affected in one such mutant, vps21, has been cloned and found to encode a member of the ras-like GTP binding protein family. Sequence comparisons with other known GTP binding proteins indicate that Vps21p is unique but shares striking similarity with mammalian rab5 proteins (> 50% identity and > 70% similarity). Regions with highest similarity are clustered within the putative GTP binding motifs and the proposed effector domains of the Vps21/rab5 proteins. Point mutations constructed within these conserved regions inactivate Vps21p function; the mutant cells missort and secrete the soluble vacuolar hydrolase carboxypeptidase Y (CPY). Cells carrying a complete deletion of the VPS21 coding sequence (i) are viable but exhibit a growth defect at 38 degrees C, (ii) missort multiple vacuolar proteins, (iii) accumulate 40-50 nm vesicles and (iv) contain a large vacuole. VPS21 encodes a 22 kDa protein that binds GTP and fractionates with subcellular membranes. Mutant analysis indicates that the association with a membrane(s) is dependent on geranylgeranylation of the C-terminal cysteine residue(s) of Vps21p. We propose that Vps21p functions in the targeting and/or fusion of transport vesicles that mediate the delivery of proteins to the vacuole.     

Cell-free transport from the trans-golgi network to late endosome requires factors involved in formation and consumption of clathrin-coated vesicles

Transport between the trans-Golgi network (TGN) and late endosome represents a conserved, clathrin-dependent sorting event that separates lysosomal from secretory cargo molecules and is also required for localization of integral membrane proteins to the TGN. Previously, we reported a cell-free reaction that reconstitutes transport from the yeast TGN to the late endosome/prevacuolar compartment (PVC) and requires the PVC t-SNARE Pep12p. Here, we report that factors required both for formation of clathrin-coated vesicles at the TGN (the Chc1p clathrin heavy chain and the Vps1p dynamin homolog) and for vesicle fusion at the PVC (the Vps21p rab protein and Vps45p SM (Sec1/Munc18) protein) are required for cell-free transport. The marker for TGN-PVC transport, Kex2p, is initially present in a clathrin-containing membrane compartment that is competent for delivery of Kex2p to the PVC. A Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC. Antibodies against the Kex2p and Vps10p C-tails selectively block transport of Kex2p and the Kex2-Vps10p chimera. The requirements for factors involved in vesicle formation and fusion, the identification of the donor compartment as a clathrin-containing membrane, and the need for accessibility of C-tail sequences argue that the TGN-PVC transport reaction involves selective incorporation of TGN cargo molecules into clathrin-coated vesicle intermediates. Further biochemical dissection of this reaction should help elucidate the molecular requirements and hierarchy of events in TGN-to-PVC sorting and transport.     

Structure and ubiquitin binding of the ubiquitin-interacting motif

Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules. Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM). We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (Kd = 0.1-1 mm), to free ubiquitin. Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues. NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin. The 1.45 A resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix. Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.     

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 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.     

The Vps27/Hse1 complex is a GAT domain-based scaffold for ubiquitin-dependent sorting

The yeast Vps27/Hse1 complex and the homologous mammalian Hrs/STAM complex deliver ubiquitinated transmembrane proteins to the ESCRT endosomal-sorting pathway. The Vps27/Hse1 complex directly binds to ubiquitinated transmembrane proteins and recruits both ubiquitin ligases and deubiquitinating enzymes. We have solved the crystal structure of the core responsible for the assembly of the Vps27/Hse1 complex at 3.0 A resolution. The structure consists of two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The two GAT domains are connected by an antiparallel coiled coil, forming a 90 A-long barbell-like structure. This structure places the domains of Vps27 and Hse1 that recruit ubiquitinated cargo and deubiquitinating enzymes close to each other. Coarse-grained Monte Carlo simulations of the Vps27/Hse1 complex on a membrane show how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.