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.

Signaling from cell surface receptors is attenuated by their ligand-induced endocytosis and degradation in lysosomes (1,2). The molecular machinery that diverts endocytosed membrane proteins from the default recycling pathway and sorts them into the degradative pathway is currently being elucidated. Two structurally related proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signaltransducing adaptor molecule), have attracted interest in this context (3,4). Both these proteins are tyrosine-phosphorylated upon growth factor and cytokine stimulation (5)(6)(7)(8), and there is evidence that that they serve to sort endocytosed membrane proteins to lysosomes (9).
Hrs contains multiple domains, including an N-terminal VHS domain, a FYVE domain that binds specifically to phosphatidylinositol 3-phosphate (10), a coiled-coil domain, and a clathrin-binding domain (11)(12)(13)(14). The FYVE and coiled-coil domains target Hrs specifically to early endosomes (10,15). Electron microscopy studies of Hrs mutant Drosophila larvae revealed an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs) 1 (16), suggesting that Hrs may play a crucial role in MVB formation and membrane trafficking from endosomes to degradative compartments. Ubiquitination functions as a sorting signal for the degradative endocytic pathway (17,18), and the binding of Hrs to ubiquitinated proteins via its ubiquitin-interacting motif (UIM) appears to be important for their sorting (19).
The two related proteins STAM1 and STAM2 (also known as Hbp (Hrs-binding protein)) share an N-terminal VHS domain, like Hrs. These proteins also contain a Src homology 3 domain, a coiled-coil domain, and an immunoreceptor tyrosine-based activation motif (7). They both interact with Janus kinases and appear to function downstream of Janus kinases 3 and 2 in cytokine signaling (8,20). A double knockout of STAM1 and STAM2 in mice is early embryonic lethal (21). Deletion mutants of STAM1 and STAM2 inhibit cytokine signaling in transfected lymphocytes (20), and conditional double knockout of STAM1 and STAM2 in mouse T-lymphocytes causes abnormalities in their development and survival (21). In contrast, mice with single knockouts of STAM1 or STAM2 are viable and have normal lymphocyte functions, indicating that the two proteins have redundant functions (21,22). Besides their roles in signal transduction, STAM proteins may also regulate endocytic membrane trafficking. A deletion mutant of STAM2 that is unable to bind Hrs suppresses the degradation of platelet-derived growth factor and its receptor but not the internalization of platelet-derived growth factor (23). This suggests that an interaction of STAM2 with Hrs is essential for the degradative sorting of platelet-derived growth factor and its receptor.
STAM1 and STAM2 form a tight interaction with Hrs via the same coiled-coil domain that targets Hrs to the early endosomal membrane (6,8,23). This has raised three possible models for the membrane recruitment of Hrs and STAM proteins. 1) STAM proteins may recruit Hrs to endosomes. 2) Hrs may recruit STAM proteins to endosomes. 3) Hrs-STAM complex formation and membrane association may be mutually exclusive. In this report we have tested these models and tried to elucidate the function of the STAM-Hrs complex. We have also investigated the relationship between the Hrs-STAM complex and the ubiquitin-binding protein Eps15.

MATERIALS AND METHODS
Antibodies-Rabbit antibodies against recombinant Hrs have been described before (14). Antisera against STAM1 and STAM2 were made * This work was supported by the Top Research Program, the Research Council of Norway, the Norwegian Cancer Society, the Novo-Nordisk Foundation, and the Anders Jahre's Foundation for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
by Eurogentec (Herstal, Belgium) by injecting rabbits with a synthetic peptide corresponding to residues 315-329 of STAM1 coupled to keyhole limpet hemocyanin or with recombinant GST-STAM2. The STAM2 antiserum was affinity-purified on Affi-Gel beads (Bio-Rad) containing immobilized MBP-STAM2, and the STAM1 antiserum was affinitypurified by coupling the same peptide that was used for injecting the rabbits to a SulfoLink Coupling Gel (Pierce) according to the manufacturer's instructions. Control experiments demonstrated that the antibodies against Hrs, STAM1, and STAM2 did not cross-react. Anti-Myc antibodies were from the 9E10 hybridoma (24). Mouse monoclonal antibodies against the VSV-G epitope were from Roche Molecular Biochemicals. Mouse monoclonal antibodies against conjugated mono-and polyubiquitin (FK2) or conjugated polyubiquitin only (FK1) (25) were from Affiniti Research Products (Exeter, UK). Human anti-EEA1 serum (26) was a gift from Ban-Hock Toh (Melbourne, Australia). Rabbit anti-Eps15 (C terminus) and goat anti-clathrin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Sheep anti-EGF-receptor was from Fitzgerald (Concord, MA). Rhodamine-, fluorescein isothiocyanate-, Cy2-, Cy3-, Cy5-, and horseradish peroxidase-labeled secondary antibodies were from Jackson Immunoresearch (West Grove, PA).
Plasmid Constructs and Small Interfering RNA (siRNA)-The Hrs constructs indicated were generated by PCR with mouse Hrs (5) as the template. The STAM constructs used were generated by PCR with human STAM1 (7) and mouse STAM2 (Hbp) (23) as the templates. For use in the two-hybrid system, constructs were cloned into pLexA/ pBTM116 (27) as bait and pGAD GH (Clontech, Palo Alto, CA) as prey. For expression in mammalian cells with the T7 RNA polymerase vaccinia virus system, constructs were cloned behind the Myc epitope of pGEM-Myc4 (28). Mouse Skd1 E235Q /Vps4 E235Q was generated by PCR amplification with pTKS-mVps4 E235Q (kindly provided by Markus Babst and Scott Emr, University of California, San Diego, La Jolla, CA) as a template and cloned behind the VSV-G epitope of pGEM-VSVG (28). For bacterial expression of His 6 -tagged Eps15, the Eps15 sequence from pEGFP-Eps15 (29) (kindly provided by Alexandre Benmerah) was cloned into the expression vector pHAT (30). For siRNA experiments, 21-nucleotide RNA duplexes with 2-nucleotide 3Ј-(2-deoxy)thymidine overhangs were synthesized by Dharmacon Research, Inc. (Lafayette, CO). RNA sequences were as follows: sense, 5Ј-CGA CAA GAA CCC ACA CGU CdTdT; antisense, 5Ј-GAC GUG UGG GUU CUU GUC GdTdT. Control sequence was as follows: sense, 5Ј-GGG GCG AGG CAG CGG CAC CdTdT.
Cell Culture and Transfection-BHK-21, HEp-2, and HeLa cell cultures were maintained as recommended by ATCC. The pGEM-VSVG-STAM and pGEM-Myc-Hrs constructs and pGEM-Myc-mVps4 E235Q were expressed in BHK-21 cells using modified Ankara T7 RNA polymerase recombinant vaccinia virus (31) and lipofection as described (32). Transfection of HeLa cells with siRNA was performed as described (33). The cells were first transfected with siRNA for 3 days, and then the cells were replated and the transfection was repeated for another 3 days.
Confocal Immunofluorescence Microscopy-BHK cells grown on coverslips were fixed with 3% paraformaldehyde after transfections and stained for fluorescence microscopy as described (28). When indicated, the cells were permeabilized with 0.05% saponin prior to fixation. Coverslips were examined using a Leica TCS NT confocal microscope equipped with a krypton/argon laser and a PL Fluotar ϫ100/1.3 oil immersion objective or a Zeiss LSM 510 META microscope equipped with a Neo-Fluar ϫ100/1.45 oil immersion objective.
Protein Expression and Purification-GST fusion proteins of STAM1 and STAM2 was produced in E. coli BL21 (DE3) cells transformed with the respective pGEX constructs as described previously (14). The recombinant protein was purified on glutathione-Sepharose 4B (Amersham Biosciences) after lysis of the bacteria in B-PER TM reagent (Pierce) according to the instructions from the manufacturers. Expression of MBP-Hrs has been described previously (14). His 6 -Eps15 was expressed in Escherichia coli BL21 (DE3) cells transformed with pHAT-Eps15.
Pull-down Assays-For measurements of ubiquitin binding, aliquots (10 l) of protein A-agarose (Sigma) or ubiquitin-agarose (Sigma) were washed three times with assay buffer A (25 mM HEPES, pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 5 mM EGTA, and 1 mM dithiothreitol) before incubation with 0.02 nmol of GST-STAM2 in 300 l of assay buffer A containing 10% fetal calf serum and 0.1% Triton X-100 for 1 h at room temperature. Finally, the beads were washed four times in assay buffer and resuspended in SDS-PAGE sample buffer. STAM2 associated with the beads was detected by SDS-PAGE followed by immunoblotting with the rabbit anti-STAM2 antibody. For measurements of ternary complex formation, bacterially expressed GST, GST-STAM1 or GST-STAM2 (30 pmol) were mixed in 1 ml of assay buffer B (20 mM Hepes, pH 7.2, 140 mM NaCl, 1 mM dithiothreitol) with glutathione-Sepharose beads (10 l) and recombinant His 6 -Eps15 (30 pmol) in the absence or presence of recombinant MBP-Hrs (30 pmol). After 1 h at rotation at 4°C, the beads were washed extensively with assay buffer B and analyzed by SDS-PAGE and Western blotting with anti-Eps15.
Preparation of Membrane and Cytosolic Fractions of HEp-2 Cells-HEp-2 cells were washed three times with ice-cold phosphate-buffered saline and homogenized in homogenization buffer (10 mM HEPES, 3 mM imidazole, pH 7.2, 250 mM sucrose, mammalian protease inhibitor mixture (Sigma)) by repeated passages through a 22-gauge needle at 4°C. Membrane particulate and cytosolic fractions were prepared from postnuclear supernatants by ultracentrifugation for 15 min at 65,000 rpm in a TLA-100 rotor, using a Beckman table top ultracentrifuge.
Coimmunoprecipitation of Hrs and Eps15 with STAM2-HEp-2 cells were starved for 4 h and stimulated or not with 100 ng/ml EGF for 8 min. The cells were lysed, and STAM2 was immunoprecipitated as described, using 25 l of affinity-purified anti-STAM2. Complexes of STAM2, Hrs, and Eps15 were eluted in sample buffer for SDS-PAGE and run on 10% gels. Proteins were transferred to 0.45-m pore size polyvinylidene difluoride membranes following SDS-PAGE and incubated with primary and secondary antibodies before detection with the SuperSignal chemiluminescence kit.
Quantitation of EGF-R in Cells Treated with siRNA against Hrs-HeLa cells were transfected twice at 72-h intervals with a siRNA duplex specific for nucleotides 161-180 of the Hrs coding sequence, with a sense RNA as a negative control. Hrs levels were analyzed by Western blotting of total cell lysates. The amount of total EGF-R was measured by immunofluorescence after a 30-min pulse of 200 ng/ml EGF and undegraded EGF-R after a 3-h chase in the presence of 10 g/ml cycloheximide to prevent synthesis of new EGF-R. The cells were permeabilized with 0.05% saponin prior to fixation and stained with anti-EGF-R. The mean intensity of the Cy2 EGF-R signal from 20 siRNAtreated and 20 control cells after 3 h of chase was quantified and was presented as the percentage of total EGF-R. The mean intensity of total EGF-R was quantified by counting 20 control and 20 siRNA cells after a 30-min EGF pulse. No significant differences were found in the amount of total EGF-R in control and siRNA cells at this time point. Error bars denote S.E.

STAM Proteins and Eps15 Are Coimmunoprecipitated with
Hrs from Cytosol and Membrane Fractions-Since the coiledcoil domain of Hrs binds STAM1/STAM2 (6, 23) and is essential for the localization of Hrs to early endosomes (15), this raises the question of whether Hrs can bind to both STAM proteins and the endosomal membrane at the same time. We investigated this by immunoprecipitating Hrs from the cytosol and membrane fractions of HEp-2 cells and studied whether STAM1 and STAM2 coprecipitated from both. As shown in Fig.  1, although Hrs was most abundant in the cytosolic fraction, STAM1 and STAM2 coprecipitated with Hrs from both the membranes and the cytosol. No STAM1/STAM2 was detected in the absence of anti-Hrs (not shown). These results indicate that the association between Hrs and STAM is not disturbed by membrane localization and that STAM proteins and Hrs are present in a complex both in the cytosol and on membranes.
Besides STAM proteins, Eps15, a protein that regulates endocytosis, has also been found to interact directly with Hrs (34). To study whether Eps15 is associated with Hrs in cytosol or on membranes, we therefore also probed the blot with an antibody to Eps15 (Fig. 1). The result showed that Eps15, like STAM proteins, coimmunoprecipitates with Hrs both from cytosol and membrane fractions. Eps15 has been shown to relocalize to the plasma membrane and subsequently to EGF-R-positive endosomes upon activation of the EGF-R kinase (35). The finding that Eps15 interacts with Hrs in membrane fractions suggests that Hrs could be important in order to determine the relocalization of Eps15 during endocytosis.
Quantitative Western blotting experiments using recombinant proteins as reference standards indicated that approximately equimolar amounts of Eps15 were coimmunoprecipitated with Hrs, whereas the amount of STAM proteins coimmunoprecipitated varied between equimolar amounts and 20% (not shown). Whereas the reason for this variation is not known, these results are consistent with the possibility that Hrs is present in stoichiometric complexes with Eps15 and STAM1/STAM2. STAM Proteins, Eps15, and Hrs Are Found in a Ternary Complex-The binding sites in Hrs for STAM2 and Eps15 are distinct, but it is not known if Hrs is capable of interacting with these proteins in a ternary complex. We therefore investigated whether both Hrs and Eps15 could be coimmunoprecipitated with STAM2. We found, by analyzing the samples on SDS-PAGE and Western blotting, that all three proteins were present in the STAM2 immunoprecipitate ( Fig. 2A). The extent of coimmunoprecipitation was independent of EGF stimulation, which is known to cause the phosphorylation and ubiquitination of Hrs, STAM proteins, and Eps15 (5, 6, 8, 23, 36 -39). This indicates that Hrs interacts with Eps15 and STAM2 in a manner that is independent of phosphorylation and ubiquitination of the individual components.
Previous work has shown that the chick homolog of STAM2, EAST, coimmunoprecipitates with Eps15 (40). Both EAST and STAM contain an N-terminal NPF motif that, in principle, might bind to the epsin homology domains of Eps15. Although mutagenesis of the NPF motif in EAST did not abolish its coimmunoprecipitation with Eps15 (40), we could not rule out the possibility that Eps15 may interact directly with STAM1/ STAM2 and thus participate in two different complexes, one with STAM1/STAM2 and one with Hrs. To investigate this possibility, we incubated GST-STAM1 and GST-STAM2 with recombinant Eps15 in the absence or presence of recombinant Hrs. We retrieved the GST fusion proteins with glutathione-Sepharose beads and analyzed their possible content of Eps15 by Western blotting. As shown in Fig. 2B, Eps15 did not associate with beads containing GST-STAM1 or GST-STAM2. However, in the presence of Hrs, Eps15 was associated with both GST-STAM1 and GST-STAM2. This indicates that Hrs is able to bridge STAM proteins with Eps15 and that Hrs, STAM proteins, and Eps15 are present in the same complex.
STAM2 Is Recruited to Early Endosomes by Hrs but Not Vice Versa-Given that Hrs interacts with STAM1 and STAM2 and that the association to endosomal membranes does not appear to interfere with this interaction, we addressed whether Hrs recruits STAM proteins to membranes or if STAM proteins may recruit Hrs. Because our STAM2 antibody was superior to that of STAM1 for immunocytochemistry, we restricted our investigation to STAM2. By overexpressing STAM2, we found that it was mainly cytosolic (Fig. 3, A and C), and it had no effect on the endosomal distribution of endogenous Hrs (Fig. 3, B and D). When we permeabilized the cells prior to fixation, almost all of the overexpressed STAM2 was extracted (Fig. 3C), in agreement with its predominant localization to the cytosol. In contrast, when STAM2 was coexpressed together with Hrs (Fig. 3, E and F), it colocalized strongly with Hrs on clustered vesicles positively stained for the early endosomal marker EEA1 (26) (Fig. 3G). Overexpression of Hrs alone (Fig. 3J) gave the same pattern of Hrs on clustered EEA1-positive vesicles (Fig. 3K). Significantly, we observed a very strong endosomal staining of endogenous STAM2 in cells transfected with Hrs ( Fig. 3I), whereas the staining of endogenous STAM2 in untransfected cells gave a weaker signal (Fig. 3M). This is probably due to a strong recruitment of STAM2 from cytosol to endosomes in the presence of overexpressed Hrs. Even in untransfected cells, STAM2 colocalized extensively with EEA1 (Fig. 3, M-O), indicating that a portion of endogenous STAM2 is indeed associated with early endosome membranes.
The above results indicated that overexpression of Hrs causes an increased recruitment of STAM2 to endosomes. To study whether Hrs is required for the membrane recruitment of STAM2, we attempted to deplete HeLa cells of Hrs by using siRNA (41). Control cells (treated with a Hrs-specific sense RNA) and siRNA-treated cells were fractionated into cytosol and membrane fractions, and the levels of Hrs and STAM2 were analyzed by Western blotting. We loaded relatively higher amounts of membrane fractions, since Hrs is most abundant in the cytosol (see Fig. 1). As shown in Fig. 4A (upper panel), siRNA treatment caused a strong reduction in Hrs levels both in the cytosol (lanes 1 and 2) and membrane fractions (lanes 3 and 4). Importantly, the decreased level of Hrs in the membrane fraction was accompanied by a strong reduction in membrane-associated STAM2 (middle panel, lanes 3 and 4). This indicates that the recruitment of STAM2 to membranes requires Hrs.
To assess whether the reduced association of STAM2 with the membrane fraction of Hrs-depleted cells reflected an impaired recruitment to endosomes, we studied the siRNAtreated cells by confocal immunofluorescence microscopy (Fig.  4B). In control cells, STAM2 could be readily detected on EEA1-positive endosomes. By contrast, in siRNA-treated cells, a diffuse, cytosolic STAM2 staining could be observed, with only faint labeling of endosomes. Interestingly, enlarged endosomes could be observed in siRNA-treated cells, reminiscent to the phenotype observed in Hrs Ϫ/Ϫ mouse embryos (42). Taken together, these results indicate that whereas Hrs is independent of STAM2 for the binding to endosomes, Hrs is required for the recruitment of STAM2 to endosomes.
Hrs and STAM2 Accumulate on Aberrant Endosomes Induced by Expression of an ATPase-deficient Form of mVps4 -The yeast homologue of Hrs, Vps27p, was identified in a screen for vps mutants defective in vacuolar protein sorting (43)(44)(45)(46). vps27 belongs to the class E vps mutants, which accumulate vacuolar, endocytic, and trans-Golgi markers in an aberrant multilamellar structure, the class E compartment (47)(48)(49). An AAA ATPase, Vps4p, may serve to modulate interactions between other class E proteins, and it has been demonstrated that Vps4p influences binding of the class E proteins Vps2p, Vps20p, Vps23p, Vps24p, and Vps32p/Snf7p to endosomal membranes (18,50,51). In addition, it has been shown that expression of ATPase-defective mammalian Vps4 (mVps4), mVps4 E235Q , causes a portion of TSG101 (the mammalian homologue of Vps23p) and human VPS28 to become associated with aberrant endosomes (52). To study whether the membrane localization of Hrs and STAM2 is regulated in a similar manner, we transfected cells transiently with mVps4 E235Q (Fig.  5, A and D). Confocal microscopy showed a distinct staining of both Hrs and STAM2 on the aberrant endosomal structures induced (53,54). Compared with untransfected cells, in which a speckled, cytosolic staining pattern was seen, the transfected cells contained little cytosolic Hrs and STAM2 and showed instead a strong, vesicular staining (Fig. 5, B and E). This staining showed strong colocalization with EEA1 (Fig. 5, C and  F), which is consistent with the idea that mVps4 regulates the morphology and the transport functions of endosomes (54). Based on these findings, we suggest that mVps4 is involved in the regulation of a membrane complex containing Hrs, STAM, and additional proteins and that ATP hydrolysis is an important step of this regulation.
STAM2 Binds Ubiquitin-Sequence analyses show that Hrs, Eps15, STAM1, and STAM2 contain UIMs (55). It has recently been demonstrated that Hrs binds ubiquitin (16,56) and that the binding occurs directly via the UIM (16,19,38,56). Furthermore, Hrs binds ubiquitinated receptors and colocalizes with ubiquitinated proteins in endosomal microdomains (19). The Hrs/ubiquitin interaction appears to be involved in the sorting of receptors from the recycling to the degradative pathway by retaining ubiquitinated receptors in a clathrin-coated area of the sorting endosome that is not involved in recycling (19). Due to the low affinity of Hrs to monoubiquitin (K D ϭ 300 M), it is possible that other proteins in complex with Hrs may also interact with neighboring ubiquitin molecules and thereby increase the avidity. Since also Eps15 has been found to bind ubiquitin (38), we investigated whether STAM2 is capable of binding ubiquitin. For this purpose, we incubated recombinant STAM2 in the presence of agarose beads to which ubiquitin had been covalently bound. By SDS-PAGE and Western blotting, we found that STAM2 was pelleted with the ubiquitin beads (Fig. 6, lane 2) but not with control beads containing protein A instead of ubiquitin (Fig. 6, lane 1). This indicates that STAM2, like Hrs and Eps15, interacts directly with ubiquitin.
Ubiquitinated Proteins Localize to the Same Microdomains as STAM2 on Endosomes-If STAM2 is involved in the sorting of ubiquitinated proteins together with Hrs, we would expect to find STAM2 and ubiquitinated proteins in the same microdomains of the endosomal membrane. When cells are transfected with a GTPase-defective mutant of Rab5, Rab5 Q79L , enlarged early endosomes are formed due to increased fusion (57), and the detection of endosomal microdomains by confocal immunofluorescence microscopy is facilitated (14). We therefore transfected BHK cells with Rab5 Q79L and stained the fixed cells with antibodies that recognize endogenous STAM2 and conjugated FIG. 4. Endosomal localization of STAM2 depends on Hrs. A, HeLa cells treated with control RNA (Ϫ) or with siRNA against Hrs (ϩ) were fractionated into membrane and cytosolic fractions as described under "Materials and Methods." Hrs left in the cytosol and on membranes after siRNA treatment as described under "Materials and Methods" was analyzed by SDS-PAGE (upper panel), and the corresponding levels of STAM2 were shown by sequential blotting of the same membrane with anti-STAM2 (middle panel). Because Hrs is most abundant in the cytosol fraction (see Fig. 1), the loaded amount of membrane fraction was 5-fold higher than that of the cytosol fraction. To visualize transferred proteins, the blot was stained with Ponceau S (lower panel) prior to detection of Hrs and STAM2. B, HeLa cells treated with control RNA (upper panels) or with siRNA against Hrs (lower panels) were fixed on coverslips and stained with antibodies against STAM2 and EEA1. Merged images are shown to the right, with yellow indicating colocalization. Bar, 5 m. ubiquitin (FK2) as well as EEA1. We found that in contrast to EEA1, which is found in microdomains distinct from STAM2 (Fig. 7, D-F), ubiquitinated proteins colocalized with STAM2 on enlarged endosomes (Fig. 7, A-C). This is in agreement with a previous finding that Hrs colocalizes with ubiquitinated proteins in endosomal microdomains (19). Due to the finding that Hrs is monoubiquitinated (38), we included an antibody (FK1) that specifically recognizes polyubiquitinated proteins. Polyubiquitinated proteins also showed colocalization with both STAM2 (Fig. 7, G-I) and Hrs (Fig. 7, J-L). This indicates that the STAM2/Hrs microdomains contain ubiquitinated proteins other than Hrs itself. A general colocalization between STAM2 and ubiquitin-conjugated proteins on early endosomes was also seen in untransfected cells (not shown). These results support the idea that STAM2 not only binds ubiquitin but also colocalizes with ubiquitinated proteins on microdomains of early endosomes and thereby may take part in their sorting together with Hrs.
Clathrin and Eps15 Accumulate on Aberrant Endosomes Induced by Expression of mVps4 E235Q -Previous work has shown that Hrs interacts with clathrin (14) and that Hrs localizes to endosomal microdomains characterized by a flat bilayered clathrin coat (14,58). Given that mVps4 regulates the membrane association of Hrs, we therefore reasoned that mVps4 may regulate the association of clathrin with endosomes. To investigate this, we transfected cells with mVps4 E235Q and stained the fixed cells with antibodies against Hrs and clathrin. Interestingly, the enlarged endosomes caused by mVps4 E235Q were stained strongly for clathrin (Fig. 8, A-C), indicating that mVps4, probably through its regulation of Hrs, is able to control the recruitment of clathrin to endosome membranes.
Because Eps15 interacts with Hrs (34) and coimmunoprecipitates with Hrs from membranes (Fig. 1), we also investigated whether the endosomal localization of Eps15 is controlled by mVps4. As shown in Fig. 8, D-F, Eps15 accumulates strongly on EEA1-positive large endosomes induced by mVps4 E235Q . These results suggest that ATP hydrolysis by mVps4 regulates the membrane localization of an entire complex consisting of Hrs and a number of associated proteins, in agreement with the proposed function of Vps4p in yeast (50).
Eps15 Localizes to the Same Microdomains as Ubiquitinated Proteins and Clathrin-Due to the ubiquitin interacting properties of Eps15, we asked whether Eps15, like STAM2, is present in the same microdomains on early endosomes as Hrs and ubiquitinated proteins. We transfected BHK cells with the Rab5 Q79L mutant as described above and stained with antibodies against Eps15, EEA1, clathrin, and polyubiquitinated proteins (FK1). Clathrin has been shown to localize to the same microdomains as Hrs (14), and because our antibodies against Hrs and Eps15 are both raised in rabbit, we used a goat anti-clathrin antibody to study whether Eps15 localizes to the Hrs/clathrin microdomains. We found that, like Hrs and STAM2, Eps15 did not colocalize with EEA1-containing microdomains (Fig. 9, A-C) but was instead found in the same areas as clathrin (Fig. 9, D-F) and polyubiquitinated proteins (Fig. 9, G-I) on enlarged early endosomes. This illustrates that Eps15, like Hrs and STAM2, localizes to the same microdomains as its potential ligands, ubiquitinated proteins.

Hrs Is Essential for Efficient Degradation of the Epidermal
Growth Factor Receptor-Given that Hrs is needed for recruitment of other ubiquitin-binding proteins to the early endosome and that this process is important for proper sorting of ubiquitinated receptors to the degradative pathway, we would expect Hrs to be important for degradation of the EGF-R, whose down-regulation is ubiquitin-dependent (59). In order to investigate this, we used siRNA against Hrs and examined degradation of EGF-R 3 h after a 30-min pulse of EGF. We found that whereas control cells had degraded 80 -90% of the internalized EGF-R, siRNA-treated cells had degraded less than 50% (Fig.  10). Some degradation was expected in siRNA-treated cells, since a small amount of Hrs was present even in these cells (Fig  10, inset). This result indicates that Hrs is required for liganddependent degradation of EGF-R. DISCUSSION In the work presented here, we show that Hrs recruits STAM2 to membranes, that Hrs can be coimmunoprecipitated with Eps15 and STAM2 from membrane fractions, and that recombinant Hrs, STAM2, and Eps15 form a ternary complex. We show that all of these proteins colocalize with ubiquitinated proteins on endosomal microdomains and that their membrane association is controlled by mVps4. Finally, we show that Hrs is required both for the recruitment of STAM2 to membranes and for the efficient degradation of endocytosed EGF-R. Our findings are consistent with the idea that Hrs, STAM proteins, and Eps15, which all bind ubiquitin, may participate in a multivalent complex that sorts ubiquitinated membrane proteins into the MVB pathway.
Given that Hrs and STAM2 are found in a complex, what might its function be? Hrs was recently shown to bind ubiquitin via its UIM with low affinity (19,38). Here we found also that STAM2 binds directly to ubiquitin and that it colocalizes with ubiquitinated proteins on microdomains of early endosomes. This suggests that STAM2 is involved in a sorting complex on early endosomes together with Hrs. It was recently reported that Hse1p, a yeast protein with sequence similarity to STAM, is associated with the yeast Hrs homolog Vps27p and requires Vps27p for its membrane targeting (9). Hse1p is thus likely to be a homolog of STAM. Interestingly, mutations in the UIM domain of Hse1p, when combined with UIM mutations in Vps27p, have been shown to lead to impaired sorting of a ubiquitinated reporter construct, indicating that Hse1p functions in the sorting of ubiquitinated proteins. It is also worth noting that a chick homologue of STAM2 (EAST) binds the activated EGF-R (40). Since the EGF-R has been shown to be ubiquitinated (59), this interaction might take place via the UIM in EAST and the ubiquitin linked to EGF-R. Further evidence for this idea comes from our finding that siRNA- mediated depletion of Hrs, which inhibits the targeting of STAM2 to endosomes, is associated with an impaired degradation of endocytosed EGF-R. However, there are several additional candidate ligands for Hrs-STAM, since multiple plasma membrane and endosomal proteins are known to be ubiquitinated (60,61). Identification of cellular ubiquitinated ligands for Hrs as well as another mammalian class E Vps protein, TSG101/mammalian Vps23p, has turned out to be difficult due to instability in the receptor-dependent ubiquitination events in cellular extracts (56). However, in yeast, such a ligand has been found by detection of an interaction between the Vps23pcontaining ESCRT-1 complex and carboxypeptidase S precursors (18).
How would such a sorting complex and its assembly and disassembly from early endosomes be regulated? In yeast, it has been shown that Vps4p, an AAA-type ATPase necessary for efficient protein transport from an endosomal compartment to the yeast vacuole/lysosome, catalyzes disassembly of an endosome-bound class E Vps protein complex and thereby functions to regulate the dynamic structure and transport activity of the prevacuolar endosome (50). The mammalian homologue of Vps4p, mVps4, displays a high degree of sequence identity to Vps4p, and these proteins have been suggested to fulfill similar functions (62). Members of the AAA ATPase family couple their ATPase cycle to the binding and release of substrate proteins (63,64). Changing one amino acid in the AAA cassette (E233Q and E235Q for Vps4p and mVps4, respectively) leads to ATPase-deficient versions of the proteins that become constitutively membrane-associated (50). Studies of cells expressing ATPase deficient mVps4 revealed induction of aberrant endosomes in which cholesterol accumulated, consistent with a mVps4-induced defect in postendosomal sorting (53). In addition, overexpression of mVps4 E235Q results in decreased amounts of transferrin receptor on the cell surface, along with increased amounts of the receptor in perinuclear compartments positively stained for mVps4 E235Q and EEA1 (54). These observations suggest that transferrin receptor accumulate in compartments belonging to the endocytic pathway in mVps4 E235Q -transfected cells and raises the possibility that the mammalian counterpart of Vps4p is involved in the function of endosomes (54).
Although we observed Hrs and STAM2 mostly in the cytosol in untransfected cells, these proteins translocated to the aberrant endosomes formed in mVps4 E235Q -transfected cells. This indicates that the membrane association of the Hrs-STAM complex is regulated by mVps4. Both the expression of Rab5 Q79L and mVps4 E235Q causes the formation of enlarged endosomes, presumably by quite distinct mechanisms. Whereas punctate Hrs-STAM-Eps15-containing microdomains can be readily distinguished on the Rab5 Q79L -induced large endosomes, the staining of Hrs-STAM-Eps15 on mVps4 E235Qinduced structures was more evenly distributed. The reason is probably that Rab5 does not regulate the membrane association of Hrs (15), whereas mVps4 E235Q causes a hyperrecruitment of Hrs and its associated proteins.
Previous studies have indicated that chick EAST/STAM2 coimmunoprecipitates with Eps15 (40) and that mammalian Hrs interacts directly with Eps15 (34). However, it was not known whether STAM proteins interact with Eps15 and whether STAM proteins, Eps15, and Hrs can be found in a ternary complex. Our present studies with recombinant proteins indicate that STAM proteins do not interact directly with Eps15 but that a ternary complex between STAM proteins, Eps15, and Hrs is formed through the ability of Hrs to interact simultaneously with STAM proteins and Eps15. This raises the possibility that Hrs, Eps15, and STAM proteins are subunits of a larger ubiquitin-binding complex on endosomes. Consistent with this, we found that Eps15, Hrs, and STAM2 are found in the same microdomains as polyubiquitinylated proteins on early endosomes and recruited to endosomal membranes by mVps4 E235Q . Size exclusion gel filtration chromatography indicates that Hrs is present in two complexes of 180 and 500 -550 kDa, respectively (56,65). The larger complex could potentially contain one or more subunits of Hrs, Eps15, and a STAM protein (total M r of about 300 kDa), and it is also quite possible that it contains additional proteins such as sorting nexin 1 (65). Recent work has shown that UIM-containing proteins, including Hrs, Eps15, and STAM1, become monoubiquitinated (38,39). This intriguing finding raises the possibility that Hrs, Eps15, and STAM could strengthen their interactions by transinteracting with ubiquitin moieties of other subunits of the complex. However, given that even the recombinant, nonubiquitinated proteins appear to interact quite strongly (6,34), ubiquitination might alternatively serve to down-regulate the ability of these molecules to interact with ubiquitinated cargo because of cis-interactions between the UIMs and the ubiquitin moieties of Hrs, Eps15, and STAM (38). Ubiquitination could also function to sterically regulate interactions with other components of the endocytic sorting machinery.
In addition to its interaction with STAM proteins and Eps15, Hrs binds directly to clathrin and is found in a flat, bilayered clathrin coat of early endosomes (14,19,58). This suggests that a Hrs-containing complex could function to retain ubiquitinated proteins in clathrin-containing microdomains. The functional relevance of the Hrs-clathrin interaction is underscored by our finding that also clathrin is recruited onto mVps4 E235Qcontaining endosomes. We speculate that Hrs, STAM proteins, and Eps15 constitute a multivalent ubiquitin-interacting complex on early endosomes. Although the individual interactions between UIMs and ubiquitin are of low affinity (19,38), such a complex would have high avidity for proteins that are ubiquitinated on several lysine residues. Moreover, the restricted localization to clathrin lattices might further enhance the efficacy of this sorting mechanism. In agreement with this hypothesis, an accumulation of endosomal ubiquitinated proteins is observed in dominant negative mVps4-transfected cells (56). This suggests that both a possible sorting complex and its putative cargo are regulated by mVps4.
Hrs is an essential mammalian protein (42), but its exact functions still remain to be elucidated. We present evidence that Hrs is required both for the membrane localization of STAM2 and for degradation of the EGF-R. The EGF-R is thus a likely substrate for a Hrs-STAM2-containing sorting complex, which is consistent with the fact that Hrs mutant Drosophila displays increased EGF signaling (16). However, in mutant pupae the total levels of EGF-R are decreased, although the levels of activated receptors are increased (16). In the absence of Hrs in vivo, it is possible that compensatory mechanisms may be activated in order to counteract the sustained signaling caused by endocytosed growth factor receptors that do not enter into MVBs. Our finding that Hrs is required for EGF-R degradation is consistent with the fact that depletion of two other mammalian Vps class E proteins, hVps28 and TSG101, causes the endosomal accumulation of ubiquitinated proteins and inhibits EGF degradation (56).
Based on the present and previous results, we propose that a clathrin-associated complex containing Hrs, STAM2, and Eps15 binds ubiquitinated cargo proteins on the endosomes and excludes these from the default recycling pathway by preventing them from entering the tubular parts of the endosome. The relationship between this complex and the ubiquitin-binding ESCRT-I complex (18) is not known, but one possibility is that the two complexes act sequentially in the recognition and sorting of ubiquitinated proteins. Dissociation of the complexes, controlled by mVps4, may control clathrin disassembly and allow cargo to proceed along the MVB pathway to lysosomes. In future studies, we hope to be able to test this model.