Article |
Address correspondence to Philip Woodman, University of Manchester, Oxford Rd., Manchester M13 9PT, UK. Tel.: 44-161-275-7846. Fax: 44-161-275-5082. E-mail: pwoodman{at}fs1.scg.man.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: endocytosis; EGF receptor; down-regulation; ubiquitin; multivesicular
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies in yeast have identified a family of proteins important for endosomal sorting. Class E vacuolar protein-sorting (vps) mutants fail to transport newly synthesized hydrolases efficiently to the vacuole. Instead, hydrolases accumulate with endocytosed receptors in an exaggerated perivacuolar class E compartment (Raymond et al., 1992; Piper et al., 1995; Rieder et al., 1996). This compartment is unable to sort markers to internal vesicles, indicating that both receptor sorting and the inward invagination of membrane are directly or indirectly effected by class E vps proteins (Odorizzi et al., 1998). Mammalian homologues of class E vps proteins have been identified, including hepatocyte growth factor receptor substrate (Hrs; related to Vps27p) (Komada et al., 1997), tumor susceptibility gene 101 (TSG101; related to Vps23p) (Babst et al., 2000), and human vacuolar protein sorting (hVPS)28 (Bishop and Woodman, 2001). The precise roles that these proteins play in receptor sorting remain unclear.
Given the putative role for ubiquitination in mitogenic receptor sorting, we were intrigued by reports that the class E protein Vps23p (Babst et al., 2000) and its mammalian orthologue TSG101 (Li and Cohen, 1996) contain a domain resembling E2 ubiquitin-conjugating (Ubc) enzymes, albeit lacking the catalytic cysteine residue (Koonin and Abagyan, 1997; Ponting et al., 1997). Therefore, TSG101 might be a candidate for coupling the recognition of ubiquitinated moietie(s) to the sorting of receptors. This hypothesis formed the basis for the work presented in this article. Emr and colleagues have demonstrated independently that Vps23p assists the biosynthetic sorting of ubiquitinated vacuolar protease precursors (Katzmann et al., 2001).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The activities of class E vps proteins are likely to be linked, raising the possibility that other class E proteins might recognize ubiquitin. In particular, Hrs localizes to the sorting endosome (Komada et al., 1997; Raiborg et al., 2001) and interacts indirectly with a deubiquitinating enzyme (Kato et al., 2000). Indeed, Hrs binds ubiquitin (Fig. 1 A). As for TSG101, binding of cytosolic Hrs to ubiquitin was ATP independent and was reduced by inclusion of soluble ubiquitin (Fig. 1 B). Hrs binding was not mediated via the TSG101hVPS28 complex. When cytosol was depleted of TSG101 with anti-hVPS28, there was little depletion of Hrs (Fig. 1 D, left). Therefore, Hrs does not appear to interact with the TSG101 complex in solution. Additionally, depletion of TSG101 had little impact on the association of cytosolic Hrs with ubiquitin (Fig. 1 D, right). Furthermore, in vitrotranslated Hrs bound to ubiquitin-agarose specifically (Fig. 1 E). During the course of our studies, a putative ubiquitin-interacting motif (UIM) was identified within Hrs, with two such motifs in Vps27p (Hofmann and Falquet, 2001). Deletion of the UIM sharply reduced the efficiency with which Hrs bound ubiquitin (Fig. 1 E). Binding was also reduced by a double point mutation in which two charged amino acids within the UIM were substituted. Hence, the conserved UIM is mainly responsible for the interaction that we observe. A second UIM is present within Vps27p but is weakly conserved in Hrs. This may contribute to the residual ubiquitin binding we observed.
The TSG101 complex is localized to ubiquitin-enriched EGF-containing endosomes
Cells expressing reduced levels of TSG101 are impaired in mitogenic receptor down-regulation, consistent with the structural similarity between TSG101 and Vps23p (Babst et al., 2000). However, TSG101 has been implicated in several cellular processes, most likely involving its interaction with ubiquitinated substrate(s) (see Discussion). Hence, depletion of TSG101 may interfere with receptor sorting indirectly by allowing the accumulation of ubiquitinated intermediates in other cellular pathways and hence reducing the levels of available free ubiquitin and/or ubiquitin-interacting components. By analogy, it has been demonstrated by several laboratories that proteasomal inhibitors dramatically reduce cellular free ubiquitin levels, most likely by preventing the deubiquitination of proteasomal substrates (Patnaik et al., 2000; Schubert et al., 2000; Strack et al., 2000). Therefore, we looked for whether the TSG101hVPS28 complex contributes directly to receptor trafficking. As a first step, we examined whether the complex was localized to EGFR-enriched endosomes.
A431 cells have a large complement of EGFR. At least some of these were ubiquitinated upon ligand binding, since EGFR immunoprecipitates isolated from cells shortly after EGF internalization but not from untreated cells were labeled with a monoclonal antibody against proteinubiquitin conjugates, FK2 (Fig. 2 A). The intensity of ubiquitin staining fell sharply between 10 and 30 min after EGF internalization (though residual labeling was still detected). In addition, the appearance of antiubiquitin staining was not accompanied by any noticeable shift in mobility of the EGFR, suggesting that only a very small proportion of the receptor was ubiquitinated at any time. These data are consistent with previous reports that ligand-induced ubiquitination of the EGFR is rapid and transient (Levkowitz et al., 1998), though they might additionally indicate that receptor ubiquitination is substoichiometric. Immunofluorescence staining confirmed that EGF-rich endosomes contained ubiquitinated proteins (Fig. 2 B). Surprisingly, endosomes were strongly labeled for ubiquitinated proteins at later times when the amount of ubiquitinated EGFR had fallen. These data are consistent with ubiquitin functioning late on the endocytic pathway and with endosomally associated protein(s) distinct from the EGFR being ubiquitinated as a consequence of receptor internalization.
|
To further investigate links between ubiquitination of endosomal proteins and receptor degradation, we used the proteasome inhibitor, MG132, which has been demonstrated previously to block mitogenic receptor degradation (Rocca et al., 2001; van Kerkhof et al., 2001). Although it remains unclear whether proteasomes are directly involved in receptor degradation, it has been reported in the case of the interleukin 2 receptor ß chain that inhibition of proteasome function is accompanied by defects in receptor sorting and the elevation of levels of ubiquitinreceptor conjugate (Rocca et al., 2001). Proteasome inhibition leads to a depletion of free ubiquitin (Patnaik et al., 2000; Schubert et al., 2000; Strack et al., 2000). This may arise from the inability of deubiquitinating enzymes to function in the context of stalled proteasomes, though other reasons are also possible. As expected, preincubation of A431 cells with MG132 for 3 h before EGF binding and during EGF internalization abolished EGF degradation (Fig. 3 A, left). EGF degradation was also substantially reduced upon incubating cells with another proteasome inhibitor, lactacystin (unpublished data). The small extent of EGF-dependent EGFR ubiquitination observed in control cells was abolished by prior incubation with MG132 so that ubiquitination of the receptor could not be detected over background levels found in untreated cells (Fig. 3 B, lane 1 compared with lane 2). Fluorescently labeled EGF accumulated in small endosomes that were only weakly labeled for ubiquitin (Fig. 3 C, top) and VPS28 (unpublished data). In contrast, when MG132 was added only after EGF binding internalized EGF (whose degradation was substantially impaired) (Fig. 3 A left) entered enlarged ubiquitin-enriched endosomes as in control cells (Fig. 3 C, middle). Much EGF remained associated with ubiquitin-enriched endosomes even after a 5-h internalization (unpublished data). However, interestingly, EGFR ubiquitination and deubiquitination was unaffected by MG132 (Fig. 3 B).
|
Impairing class E protein complexes retards EGF degradation and causes ubiquitinated proteins to accumulate on endosomes
To provide functional evidence that the TSG101hVPS28 complex acts directly in receptor trafficking, we examined the fate of EGF in cells microinjected with anti-hVPS28. Anti-hVPS28 did not impair EGF internalization, since EGF labeling was similar after a 30-min internalization in cells injected with either anti-hVPS28 or control antibody (Fig. 4). In HeLa cells, degradation of intracellular EGF is essentially complete after 3 h. However, cells that had been microinjected with anti-hVPS28 retained substantial EGF labeling at this time (Fig. 4). At 3 h, the maximal intensity of EGF staining in anti-hVPS28 injected cells (n = 35) was 467 ± 31% compared with that in control injected cells (n = 31).
|
Given that the TSG101hVPS28 complex binds to ubiquitin and contributes to endosomal sorting, we examined whether interfering with its function affected the release of ubiquitin from endosomally associated ubiquitinprotein conjugates that had formed as a consequence of receptor internalization. Indeed, microinjection of cells with anti-hVPS28 caused a dramatic increase in the level of FK2 labeling of EGF-positive endosomes (Fig. 5). The accumulation of ubiquitinprotein conjugates was largely dependent on EGFR internalization; the maximum intensity of FK2 labeling associated with cytoplasmic structures was 508 ± 23% in microinjected cells to which EGF had been bound and internalized (n = 21) compared with that observed in microinjected cells that had been mock treated (n = 22). In addition, little increase in endosomal FK2 labeling was observed in uninjected cells that had internalized EGF while being treated with the lysosomal protease inhibitor leupeptin or with primaquine, which blocks endosomal sorting by collapsing the pH gradient (Fig. 5). Hence, the accumulation of ubiquitinated moieties was not simply a consequence of preventing the degradation of receptorligand complexes.
|
|
These data are consistent with impairment of TSG101VPS28 preventing the release of ubiquitin from endosomal ubiquitinprotein conjugates. Likewise, impairing Hrs also blocked receptor sorting and release of ubiquitin. Consistent with previous reports (Komada et al., 1997), exogenously expressed Hrs localized to enlarged compartments (Fig. 7) that stained for early endosomal markers (unpublished data). These compartments partially colocalized with markers for the late endocytic pathway (unpublished data), implying that overexpression of Hrs causes defective endocytic sorting. To assess this in more detail, we examined the internalization and degradation of EGF. Hrs overexpression did not impair the internalization of EGF, since transfected cells were similarly labeled with EGF as neighboring untransfected cells after short periods of internalization (Fig. 7). At later times, internalized EGF reached compartments enriched in expressed Hrs (though not all Hrs-enriched compartments were labeled). A significant portion of EGF remained in these compartments even after 3 h internalization, in contrast to neighboring cells where the majority of EGF was degraded.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells with reduced levels of TSG101 are impaired in mitogenic receptor down-regulation and show enhanced EGF recycling (Babst et al., 2000). Our data using siRNA to deplete cellular TSG101 largely confirm these observations, though they show that a significant amount of internalized EGF is retained in a nondegradative endocytic compartment. In contrast to these earlier studies, we find that TSG101 depletion induces aberrant morphology in multiple endocytic compartments. The discrepancy between these findings may perhaps be accounted for by differences in the level to which TSG101 is depleted in each experimental system. Depletion experiments provide limited evidence that TSG101 acts directly to promote receptor trafficking, particularly since this event would be sensitive to changes in the levels of either ubiquitin or ubiquitin-interacting components that might be influenced by loss of TSG101. However, we have demonstrated that TSG101 and VPS28 are localized to ubiquitin-rich endosomes during ligand-induced EGFR internalization. Additionally, antibodies to VPS28 specifically impair EGF degradation without affecting other endosome-associated functions, such as delivery of receptors to the sorting endosome and transferrin receptor recycling. Importantly, though our data support a direct role for TSG101VPS28 in receptor trafficking they do not preclude the complex from fulfilling other cellular roles involving ubiquitin recognition (Li and Cohen, 1996; Hittelman et al., 1999).
In further support of a direct role of class E vps proteins in ubiquitin-dependent receptor trafficking, we have shown that disruption of class E vps function by three independent means causes dramatic relocalization of cellular ubiquitin to endocytic compartments. The full cause of this relocalization remains unclear. Possibly, impairment of TSG101VPS28 function prevents recruitment of further cytosolic components required for receptor sorting and that disassembly of the full complex is coupled to removal of ubiquitin chains. Consistent with this, expression of dominant negative mammalian VPS4, which may link the disassembly of such a complex to ATP hydrolysis (Babst et al., 1998; Bishop and Woodman, 2000), also causes dramatic endosomal accumulation of ubiquitinated proteins (unpublished data). Hrs might play a more direct role in releasing ubiquitin, since an Hrs-binding protein interacts with the deubiquitinating enzyme UBPY (Kato et al., 2000). Cytosolic Hrs is part of a 500-kD complex (unpublished data). We speculate that exogenously expressed Hrs, which binds to endosomes via its FYVE and coiled coil domains (Raiborg et al., 2001) (and which presumably interacts with ubiquitinated moieties on the endosome surface), would displace endogenous Hrs complexes and thus prevent recruitment of UBPY. We have been unable to analyze any further the functional significance of Hrs binding to ubiquitin since, as expected, the UIM is not solely necessary for endosomal localization of Hrs or the impairment of EGF trafficking by Hrs overexpression.
The cellular ubiquitinated substrates for TSG101 and Hrs remain to be identified, and we have been hampered in this respect by our inability to stabilize receptor-dependent ubiquitination events in cell extracts. Candidates include the mitogenic receptors themselves. By analogy, the yeast Vps23p-containing ESCRT-1 complex binds directly to ubiquitinated carboxypeptidase S precursor (Katzmann et al., 2001). Indeed, we have detected a weak coimmunoprecipitation of the EGFR with the TSG101 complex, though this is independent of receptor ubiquitination (unpublished data). However, we anticipate that recruitment of mammalian TSG101 may be more complex. Endocytic compartments within TSG101-depleted cells accumulate striking levels of ubiquitinated proteins, yet we find no evidence that the EGFR is hyperubiquitinated. In addition, we have shown using a proteasome inhibitor that accumulation of ubiquitinated proteins within EGFR-enriched endosomes can be experimentally uncoupled from ubiquitination of the EGFR itself. The failure to increase levels of ubiquitinated EGFR under conditions where deubiquitination of endosomal proteins is apparently so diminished raises the possibility that ubiquitinated proteins other than the receptor are important for endosomal sorting and hence might act as the principle substrates for Hrs and TSG101 binding. These data are also completely consistent with recent findings that endosomal sorting of a truncated growth hormone receptor is critically dependent on a ubiquitin-recruiting motif within the receptor but does not require receptor ubiquitination (van Kerkhof et al., 2001). Furthermore, TSG101 interacts via its Ubc-like domain with a highly conserved peptide motif within the Pr55Gag protein of HIV type 1 (Garrus et al., 2001; Martin-Serrano et al., 2001; VerPlank et al., 2001), which is absent from mitogenic receptors, including EGFR. Interestingly, artificial covalent attachment of ubiquitin to HIV Pr55Gag enhances TSG101 Ubc domain binding (Garrus et al., 2001).
Our data place mammalian class E vps proteins at the center of ubiquitination-dependent receptor sorting. They extend the finding that defects in yeast Doa4p, a deubiquitinating enzyme, are suppressed by class E vps mutants (Amerik et al., 2000). At present, we cannot discriminate between models that favor ubiquitin recruiting a specific endosomal sorting machinery and those that imply that proteasome function is important for endosomal sorting. However, our finding that the TSG101 complex contributes directly to mitogenic receptor sorting supports recent evidence that the yeast ESCRT-1 complex of Vps23/28/37p promotes the ubiquitin-dependent biosynthetic sorting of vacuolar precursors (Katzmann et al., 2001). In yeast, interfering with Vps23p blocks either directly or indirectly the localization of receptors to the internal vesicles of multivascular body. We now aim to address if this specific stage is blocked when mammalian class E vps protein function is impaired.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies and reagents
Sheep were immunized with GST-mHrs purified from bacterial inclusion bodies. For fluorescence microscopy, cells were stained with an IgG fraction at a concentration that detected expressed but not endogenous Hrs. The following monoclonal antibodies were against ubiquitinated proteins (FK1, which recognizes only polyubiquitinprotein conjugates, and FK2, which recognizes both mono- and polyubiquitinated conjugates; Affiniti BioRegeants, Inc.), EEA1 (Transduction Laboratories), EGFR (clone 29.1.1; Sigma-Aldrich), 20S proteasome (HP810; Affinity BioReagents, Inc.), tubulin (TAT1; from K. Gull, University of Manchester, Manchester), TSG101 (GeneTex), CD63 (a gift from Fedor Berditchevski, University of Birmingham, Birmingham, UK). Also used were rabbit anti-Ubc1 (Affiniti BioRegeants, Inc.) and affinity purified anti-hVPS28 (Bishop and Woodman, 2001). HRP-conjugated antibodies (Dako) were used for ECL blotting. MG132 was purchased from Calbiochem and stored at 20°C in DMSO.
Cells
Cells were grown in DME (GIBCO BRL) containing 10% FCS. Cells were transfected with DNA constructs using FuGene (Roche) and analyzed after 1624 h. For siRNA experiments, 21 nucleotide RNA duplexes corresponding to the Tsg101 coding nucleotides AACGATGGCAGTTCCAGGGAA with symmetric 2 nucleotide 3' (2'-deoxy) thymidine overhangs (sense, 5' CGAUGGCAGUUCCAGGGAAdTdT; antisense, 5' UUCCCUGGAACUGCCAUCGdTdT) were synthesized and annealed (Dharmacon Research). HeLa cells were transfected with 6 µl 20 µM siRNA duplex per well of a 12-well dish using oligofectamine (Invitrogen) and standard procedures (Elbashir et al., 2001). Knock-down of cellular TSG101 was routinely observed after 48 h. For Western blot analysis of cell extracts, cells were solubilized in buffer A containing 1% Triton X-100, and proteins were precipitated immediately in chloroform/methanol before SDS PAGE. For immunoprecipitations, cells were lysed in 0.4 ml boiling buffer A (100 mM NaCl, 1 mM MgCl2, 20 mM Hepes-NaOH, pH 7.4) containing 50 mM NaF, 1 mM Na3VO4, and 1% SDS. Cleared samples were diluted into 5 vol of buffer A containing 2% Triton X-100, incubated at 4°C overnight with antibody and 10 µl protein A-sepharose, and then washed four times before preparation for SDS-PAGE. To measure degradation of 125I EGF, iodinated EGF (50 µCi/µg) was bound (50 ng/ml) to A431 cells for 1 h at 4°C with CO2-independent medium (GIBCO BRL) containing 2 mg/ml BSA (BM). Cells were washed and incubated in 1 ml BM. As indicated, medium was removed and TCA added to 10%. After 12 h on ice, samples were centrifuged, and the supernatant was counted for radioactivity.
Fluorescence microscopy
Cells were fixed in 3.5% (wt/vol) PFA with a 0.05% saponin pretreatment as indicated or with methanol. Secondary antibodies were from Jackson ImmunoResearch Laboratories. For EGF uptake, cells were washed in PBS and incubated in BM containing 0.4 µg/ml Oregon green or Texas red EGF (Molecular Probes), washed, and then incubated at 37°C. For transferrin uptake, 20 µg/ml Texas red transferrin (Molecular Probes) was bound and internalized in medium containing 0.1 mg/ml transferrin and 50 µM desferroxamine (Ciba). Cells were examined using a Leica NT confocal microscope or an inverted Olympus IX-70 linked to a Delta Vision system (Applied Precision, Inc.). Quantitation was performed using an Olympus BX60 microscope linked to a cooled slow-scan CCD camera (Roper Scientific) driven by Metamorph software (Universal Imaging Corp.). Images were processed using Adobe Photoshop® 5.0.
Microinjection
Antibodies were dialyzed against 48 mM K2HPO4, 4.5 mM KH2PO4, 14 mM NaH2PO4, pH 7.2, and concentrated. Antibody solutions (0.5 mg/ml) containing 25 µg/ml DAPI were injected using an Eppendorf 5171 micromanipulator and microinjector. Cells were normally incubated for 1 h at 37°C between microinjection and the start of internalization assays.
Ubiquitin binding
In vitro translation (Promega) products (5 µl) were diluted in 250 µl buffer A containing 1% Triton X-100 and incubated for 2 h at 4°C with 10 µl prewashed ubiquitin- (Affinity BioReagents, Inc.) or GSH-agarose beads, washed four times in buffer A, and boiled for SDS PAGE. Gels were analyzed by phosphorimaging. For native proteins, HeLa cytosols were incubated for 15 min with 50 IU/ml hexokinase and 5 mM glucose final to deplete ATP and desalted into buffer A. Samples (1 mg total protein) were incubated with 20 µl ubiquitin- or GSH-agarose beads as above and then analyzed by Western blotting.
![]() |
Footnotes |
---|
* Abbreviations used in this paper: EEA1, early endosome-associated antigen 1; EGFR, EGF receptor; GSH, glutathione; Hrs, hepatocyte growth factor receptor substrate; hVPS, human VPS; mHrs, mouse Hrs; siRNA, small interfering RNA; TSG101, tumor susceptibility gene 101; Ubc, ubiquitin-conjugating; UIM, ubiquitin-interacting motif; VPS, vacuolar protein sorting.
![]() |
Acknowledgments |
---|
N. Bishop is funded by a Wellcome Trust Career Development Fellowship (061045/Z/00/Z/CH/TH/lc), and A. Horman is funded by a Biotechnology and Biological Sciences Research Council studentship (99/A1/C/05291). This work is supported by the Medical Research Council (G117/153, G9722026, and G0001128).
Submitted: 17 December 2001
Revised: 15 February 2002
Accepted: 19 February 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amerik, A.Y., J. Nowak, S. Swaminathan, and M. Hochstrasser. 2000. The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol. Biol. Cell. 11:33653380.
Babst, M., B. Wendland, E.J. Estapa, and S.D. Emr. 1998. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17:29822993.
Bishop, N., and P.G. Woodman. 2000. ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking. Mol. Biol. Cell. 11:227239.
Bishop, N., and P. Woodman. 2001. Tsg101/mammalian vps23 and mammalian vps28 interact directly and are recruited to vps4-induced endosomes. J. Biol. Chem. 276:1173511742.
Felder, S., K. Miller, G. Moehren, A. Ullrich, J. Schlessinger, and C.R. Hopkins. 1990. Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell. 61:623624.[Medline]
Hershko, A., H. Heller, S. Elias, and A. Ciechanover. 1983. Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J. Biol. Chem. 258:82068214.
Hittelman, A.B., D. Burakov, J.A. Iniguez-Lluhí, L.P. Freedman, and M.J. Garabedian. 1999. Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins. EMBO J. 18:53805388.
Hofmann, R.M., and C.M. Pickart. 1999. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell. 96:645653.[Medline]
Joazeiro, C.A.P., S.S. Wing, H.-K. Huang, J.D. Leverson, T. Hunter, and Y.-C. Liu. 1999. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science. 286:309312.
Kato, M., K. Miyazawa, and N. Kitamura. 2000. A deubiquitinating enzyme UBPY interacts with the Src homology 3 domain of Hrs-binding protein via a novel binding motif PX(V/I)(D/N)RXXKP. J. Biol. Chem. 275:3748137487.
Komada, M., R. Masaki, A. Yamamoto, and N. Kitamura. 1997. Hrs, a tyrosine kinase substrate with a conserved double zinc finger domain, is localized to the cytoplasmic surface of early endosomes. J. Biol. Chem. 272:2053820544.
Levkowitz, G., H. Waterman, E. Zamir, Z. Kam, S. Oved, W.Y. Langdon, L. Beguinot, B. Geiger, and Y. Yarden. 1998. c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12:36633674.
Li, L., and S.N. Cohen. 1996. tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. Cell. 85:319329.[Medline]
Odorizzi, G., M. Babst, and S.D. Emr. 1998. Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell. 95:847858.[Medline]
Patnaik, A., V. Chau, and J.W. Wills. 2000. Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Sci. USA. 97:1306913074.
Piper, R.C., A.A. Cooper, H. Yang, and T.H. Stevens. 1995. VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae. J. Cell Biol. 131:603617.[Abstract]
Raiborg, C., B. Bremnes, A. Mehlum, D.J. Gillooly, A. D'Arrigo, E. Stang, and H. Stenmark. 2001. FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes. J. Cell Sci. 114:22552263.
Raymond, C.K., I. Howald-Stevenson, C.A. Vater, and T.H. Stevens. 1992. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol. Cell. 3:13891402.[Abstract]
Rieder, S.E., L.M. Banta, K. Köhrer, J.M. McCaffery, and S.D. Emr. 1996. Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Mol. Biol. Cell. 7:985999.[Abstract]
Rocca, A., C. Lamaze, A. Subtil, and A. Dautry-Varsat. 2001. Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor ß chain to late endocytic compartments. Mol. Biol. Cell. 12:12931301.
Schubert, U., D.E. Ott, E.N. Chertova, R. Welker, U. Tessmer, M.F. Princiotta, J.R. Bennink, H.-G. Kräusslich, and J.W. Yewdell. 2000. Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. USA. 97:1305713062.
Strack, B., A. Calistri, M.A. Accola, G. Palu, and H.G. Gottlinger. 2000. A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci. USA. 97:1306313068.
van Kerkhof, P., C.M. Alves dos Santos, M. Sachse, J. Klumperman, G. Bu, and G.J. Strous. 2001. Proteasome inhibitors block a late step in lysosomal transport of selected membrane but not soluble proteins. Mol. Biol. Cell. 12:25562566.
VerPlank, L., F. Bouamr, T.J. LaGrassa, B. Agresta, A. Kikonyogo, J. Leis, and C.A. Carter. 2001. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag. Proc. Natl. Acad. Sci. USA. 98:77247729.