TSG101/Mammalian VPS23 and Mammalian VPS28 Interact Directly and Are Recruited to VPS4-induced Endosomes

Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting. Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting. Cell surface receptors are internalized in endocytic vesicles and enter the early endosome, where they are sorted. Many receptors return to the cell surface to engage in further rounds of ligand uptake, whereas other receptors are targeted via the multivesicular body (MVB)1/late endosome for degradation in the lysosome (1Mellman I. Ann. Rev. Cell Dev. Biol... 1996; 12: 575-625Google Scholar). The molecular basis for selection of these receptors and their transfer to the MVB remains poorly characterized. Studies in yeast have, however, identified a number of gene products that are required for post-endosomal sorting. vps (vacuolar proteinsorting) mutants are defective in delivering newly synthesized hydrolases to the vacuole (the yeast lysosome) (2Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev... 1998; 62: 230-247Google Scholar). Among these, class E vps mutants are characterized by the formation of an exaggerated endocytic compartment (3Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell.. 1992; 3: 1389-1402Google Scholar). At least 15 class E vps mutants have been identified in yeast. It has been proposed that they act at a common step in endosomal sorting, and this has been borne out by several studies demonstrating physical and/or genetic interactions between them. The class E compartment contains endocytosed markers, as well as precursors of vacuolar hydrolases and markers normally associated with the trans Golgi (4Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol... 1995; 131: 603-617Google Scholar, 5Babst M. Sato T.K. Banta L.M. Emr S.D. EMBO J... 1997; 16: 1820-1831Google Scholar, 6Rieder S.E. Banta L.M. Köhrer K. McCaffery J.M. Emr S.D. Mol. Biol. Cell.. 1996; 7: 985-999Google Scholar, 7Munn A.L. Riezman H. J. Cell Biol... 1994; 127: 373-386Google Scholar, 8Nothwehr S.F. Bryant N.J. Stevens T.H. Mol. Cell. Biol... 1996; 16: 2700-2707Google Scholar). Because proteins are retained in the yeast trans Golgi by a mechanism that involves their transport to and retrieval from the endocytic pathway (8Nothwehr S.F. Bryant N.J. Stevens T.H. Mol. Cell. Biol... 1996; 16: 2700-2707Google Scholar), class E proteins appear to be important for sorting material bound for the vacuole away from proteins that cycle through the endocytic system. Indeed, recent data are consistent with class E proteins participating in the formation of MVB (9Odorizzi G. Babst M. Emr S.D. Cell.. 1998; 95: 847-858Google Scholar). To further our understanding of the function of class E proteins on the mammalian endocytic pathway, we have generated specific reagents for a number of close structural mammalian homologues of yeast class E vps proteins. We (10Bishop N. Woodman P.G. Mol. Biol. Cell.. 2000; 11: 227-239Google Scholar), and others (11Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell.. 2000; 11: 747-763Google Scholar), have shown that mammalian orthologues of the AAA (ATPases Associated with cellular Activities)-type ATPase, Vps4p, are required to maintain the morphological and functional organization of the endocytic pathway. Like yeast Vps4p (12Babst M. Wendland B. Estapa E.J. Emr S.D. EMBO J... 1998; 17: 2982-2993Google Scholar), mammalian (m)VPS4 (and the closely related SKD1 (13Périer F. Coulter K.L. Liang H. Radeke C.M. Gaber R.F. Vandenberg C.A. FEBS Lett... 1994; 351: 286-290Google Scholar)) couples its ATPase activity to a cycle of binding to and release from endosomal membranes (10Bishop N. Woodman P.G. Mol. Biol. Cell.. 2000; 11: 227-239Google Scholar, 11Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell.. 2000; 11: 747-763Google Scholar). Although the immediate substrates for mVPS4 have yet to be identified, our data are consistent with mVPS4 acting to release other soluble class E components from endosome-associated multiprotein complex(es) and thus recycle them for further rounds of sorting. In this study we have examined the interactions of TSG101, previously identified as the mammalian orthologue of Vps23p/Stp22p (14Li Y. Kane T. Tipper C. Spatrick P. Jenness D.D. Mol. Cell. Biol... 1999; 19: 3588-3599Google Scholar, 15Babst M. Odorizzi G. Estepa E.J. Emr S.D. Traffic.. 2000; 1: 248-258Google Scholar), another class E vps protein. TSG101, like Vps23p/Stp22p, positively regulates the lysosomal degradation of cell surface proteins, including mitogenic receptors (14Li Y. Kane T. Tipper C. Spatrick P. Jenness D.D. Mol. Cell. Biol... 1999; 19: 3588-3599Google Scholar, 15Babst M. Odorizzi G. Estepa E.J. Emr S.D. Traffic.. 2000; 1: 248-258Google Scholar). Tsg101 was identified originally as a tumor susceptibility gene by an assay for cellular transformation upon random gene disruption (16Li L. Cohen S.N. Cell.. 1996; 85: 319-329Google Scholar), although its importance as a tumor suppressor gene is still debated. A number of studies have reported the presence of aberrant splice variants ofTSG101 in specific human tumors (16Li L. Cohen S.N. Cell.. 1996; 85: 319-329Google Scholar, 17Ferrer M. López-Borges S. Lazo P.A. Oncogene.. 1999; 18: 2253-2259Google Scholar), although the significance of these findings has been disputed (18Steiner P. Barnes D.M. Harris W.H. Weinberg R.A. Nat. Genet... 1997; 16: 332-333Google Scholar, 19Hampl M. Hempl J. Plaschke J. Fitze G. Scheckert G. Saeger H.D. Schackert H.K. Biochem. Biophys. Res. Commun... 1998; 248: 753-760Google Scholar). Although the sorting defect associated with TSG101-deficient cells could underlie the susceptibility of these cells to transformation, limited evidence is available to explain how TSG101 acts to regulate mitogenic receptor down-regulation. The TSG101 peptide sequence was first interpreted to contain two DNA-binding motifs, perhaps indicative of a transcription factor (16Li L. Cohen S.N. Cell.. 1996; 85: 319-329Google Scholar). However, the existence of the DNA-binding motifs has not been substantiated in subsequent analyses (20Ponting C.P. Cai Y.-D. Bork P. J. Mol. Med... 1997; 75: 467-469Google Scholar, 21Koonin E.V. Abagyan R.A. Nat. Genet... 1997; 16: 330-331Google Scholar). Notwithstanding this, recent reports have linked TSG101 to transcriptional regulation and have provided evidence that TSG101 can act in vitro as a transcriptional suppressor (22Watanabe M. Yanagi Y. Masuhiro Y. Yano T. Yoshikawa H. Yanagisawa J. Kato S. Biochem. Biophys. Res. Commun... 1998; 245: 900-905Google Scholar, 23Hittelman A.B. Burakov D. Iniguez-Lluhı́ J.A. Freedman L.P. Garabedian M.J. EMBO J... 1999; 18: 5380-5388Google Scholar). Therefore, TSG101 could act indirectly to aid receptor degradation by modulating the expression of specific protein(s), themselves involved in endosomal trafficking. Alternatively, TSG101 might interact with a number of other class E vps proteins to form a sorting complex on the surface of the sorting endosome. Here, it might function to recognize cargo destined for inclusion in the multivesicular body, or provide a structural component for the sorting event. As part of our studies we have identified a mammalian orthologue of a further class E vps protein, Vps28p. We now demonstrate that human VPS28 (hVPS28) interacts directly with TSG101. Moreover, both TSG101 and hVPS28 can be recruited to early endosome membranes by co-expression of a dominant-negative mutant of VPS4, consistent with a direct role for the TSG101 complex in receptor sorting. Escherichia colistrain XL1Blue (Stratagene) was used for maintenance, sequencing, and mutation of plasmids. Luria-Bertani medium was used for growth ofE. coli cells. For selection of plasmids, 100 μg/ml ampicillin or 25 μg/ml kanamycin was added to the media. Proteins tagged withmyc/His6 were detected using mouse monoclonal antibody 9E10 (Sigma) or anti-polyhistidine (Sigma). Anti-TSG101 was from AbCam, Cambridge, UK. An anti-hVPS28 antibody was raised in sheep, with His6-tagged recombinant hVPS28 used as antigen. Immunizations were performed by Diagnostics Scotland (Carluke, Lanarkshire). Antibody was affinity-purified using glutathioneS-transferase-tagged hVPS28. Standard protocols for recombinant DNA manipulation were used (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The ABI PRISM big dye terminator system was used for DNA sequencing, and mutagenesis was performed using the QuikChange (Stratagene) method according to the manufacturer's instructions, or by cassette mutagenesis (see below). Mutagenesis and subcloning constructs were confirmed by DNA sequencing. A human cDNA EST clone from a Soares senescent fibroblast (NbHSF) library (Clone ID 1331807) was obtained from the IMAGE consortium (25Lennon G.G. Auffray C. Polymeropoulos M. Soares M.B. Genomics.. 1996; 33: 151-152Google Scholar) in vector pT7T3D, with a modified polylinker. The EST sequence had amino acid similarity to the 5′-end of yeast VPS28. The DNA insert was subcloned into pBluescript SKII(−) for sequencing, and it was found that the cDNA encoded a human homologue of yeastVPS28, denoted hVPS28 (human VPS28; GenBank™ accession number AF316887). Multiple sequence alignments were performed using the BCM search launcher and the ClustalW 1.8 program (26Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res... 1994; 22: 4673-4680Google Scholar, 27Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol... 1996; 266: 383-402Google Scholar), and graphics were performed using the BOXSHADE server. The cDNA insert for hVPS28 was subcloned into two mammalian expression vectors pEGFPC (CLONTECH) and vectors were to hVPS28 with an protein at the of the protein or at the or were from these vectors were hVPS28 with a or and For hVPS28 was subcloned into and vectors by pBluescript SKII(−) a mouse TSG101 cDNA was obtained from C. The gene was subcloned into with the still the and was The was to expression of with an at the C-terminal An was at the of vector to with an TSG101 were performed by as in A. The DNA was using of and was using a of via the server. G. B. 1: Scholar), M.J. 1996; Scholar), J. B. J. Mol. Biol... Scholar, J. B. J. Mol. Biol... Scholar, J. B. Methods Enzymol... 1996; 266: Scholar, C. C. C. G. 2000; 16: Scholar), Y. C. P. G. 1999; Scholar), B. C. J. Mol. Biol... Scholar), D. P. 1996; Scholar), J.M. B. J. FEBS Lett... Scholar), and C. G. 1994; 7: Scholar, C. G. 1995; 11: Scholar). A human cDNA (CLONTECH) was using according to the manufacturer's instructions, using and to the of the The of products were the their as by of cycle that the of in could be directly The used to the hVPS28 gene were the and in the A in the was used to of a related of the gene The of the were and The used had been using the expression of several of the to the of hVPS28 The products were the its by products at products were in with DNA markers cells were in modified medium modified For cells were when using the method Molecular of expression vectors insert and were on were using at and using were examined using a For cells were in medium and using the cells were in and by The cells were by through a and were by for at protein of were by For were by cell at for 15 in a were to a in 100 and on a system. Proteins were using D. Scholar) and for and In were performed using and from of DNA and of were by of The of was confirmed by and For of was as previously D. M. Methods Cell Biol... Scholar) and used to in at the of 25 of J. N. S. Woodman P. Mol. Biol. Cell.. 1998; Scholar). For was to in 100 of and on for 15 of protein was and the with to For cross-linking products were at least and for using a Cambridge, were performed as For protein bound were with of for at of of were on and The were with a antibody for at were and were as for The sequence of the EST clone with accession number was at the amino acid with on the data that the cDNA encoded a human homologue of yeast Vps28p accession number and was hVPS28 with other proteins on the data that homologues of Vps28p are found in and of the VPS28 protein of the an sequence Vps28p S.E. Banta L.M. Köhrer K. McCaffery J.M. Emr S.D. Mol. Biol. Cell.. 1996; 7: 985-999Google Scholar) amino acid sequence with hVPS28 with a further an sequence similarity of VPS28 is most closely related to that of D. The in protein sequence between S. protein and that of other is an of amino in the of the protein. proteins are with to a to protein sequence motifs were by the or data A cDNA was using to the hVPS28 and The hVPS28 is a of was detected in human examined and variants were detected when of the not splice variants of hVPS28 are not To the cellular of mammalian a mammalian expression vector hVPS28 to at the was As shown in hVPS28 in cells was cytosolic. An affinity-purified antibody that hVPS28 by the hVPS28 cells with antibody that VPS28 was the the was for hVPS28 At we not the of VPS28 from that of VPS28. experiments not that VPS28 is However, it is that of this is for The VPS28 not with markers for early or not from yeast that class E vps proteins interact with other to form multiprotein To hVPS28 to other mammalian class E vps proteins, hVPS28 was in and with other class E proteins. Among for its to hVPS28 was TSG101 (16Li L. Cohen S.N. Cell.. 1996; 85: 319-329Google Scholar), the mammalian orthologue of Vps23p/Stp22p (14Li Y. Kane T. Tipper C. Spatrick P. Jenness D.D. Mol. Cell. Biol... 1999; 19: 3588-3599Google Scholar, 15Babst M. Odorizzi G. Estepa E.J. Emr S.D. Traffic.. 2000; 1: 248-258Google Scholar). As shown in His6-tagged hVPS28 could not be with antibody when However, of with of by evidence for an between the proteins. of with TSG101 tagged at the with an of by not To demonstrate that this was direct and was not by components within the a cross-linking was Here, was or with hVPS28 or TSG101 that had been in vitro in the presence of acid and that a cross-linking D. M. Methods Cell Biol... Scholar, P. S. Scholar, J. Cell Biol... 1996; Scholar). were with or and with molecular cross-linking were upon with and However, was with two molecular were detected upon were obtained the were confirmed to contain by with antibody The molecular were was with not was or with and these that TSG101 and hVPS28 directly to other that TSG101 and hVPS28 are not evidence for of TSG101 or hVPS28 was obtained and proteins were and with antibody not The molecular of and for the are consistent with formation of both and However, cross-linking products on that other of these data are To provide further evidence that cytosolic and hVPS28 a from cells was by and by TSG101 as a with an molecular of consistent with findings M. Odorizzi G. Estepa E.J. Emr S.D. Traffic.. 2000; 1: 248-258Google Scholar), and the of hVPS28 was with TSG101 Moreover, of the of the TSG101 complex Although of the to a consistent with a the presence of in a of TSG101 molecular between and It remains TSG101 are within the cell by or the is of the complex TSG101 was from cell with anti-hVPS28 antibody that not cellular TSG101 is to hVPS28. It is that the to complex contains components involved in endosomal sorting, we have found evidence that TSG101 or hVPS28 can The gene was originally as a a of and a (16Li L. Cohen S.N. Cell.. 1996; 85: 319-329Google Scholar). the presence of an with to (20Ponting C.P. Cai Y.-D. Bork P. J. Mol. Med... 1997; 75: 467-469Google Scholar, 21Koonin E.V. Abagyan R.A. Nat. Genet... 1997; 16: 330-331Google Scholar). The interacts with proteins (22Watanabe M. Yanagi Y. Masuhiro Y. Yano T. Yoshikawa H. Yanagisawa J. Kato S. Biochem. Biophys. Res. Commun... 1998; 245: 900-905Google Scholar, 23Hittelman A.B. Burakov D. Iniguez-Lluhı́ J.A. Freedman L.P. Garabedian M.J. EMBO J... 1999; 18: 5380-5388Google Scholar) and is to interact with the cytosolic protein A. Biochem. 16: Scholar, A. A. S. 1995; Scholar). the yeast was found to be to human and M. Odorizzi G. Estepa E.J. Emr S.D. Traffic.. 2000; 1: 248-258Google Scholar), demonstrating that is analyses of the protein sequence of mammalian TSG101 have identified further of this protein. In to orthologues in and S. we have found orthologues in C. and A. although the of the S. gene has not been The between of these proteins was using the ClustalW program (26Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res... 1994; 22: 4673-4680Google Scholar), and was performed to an The that human and mouse TSG101 are most closely related to other and are most to C. TSG101 A protein closely related to TSG101 is found in D. number protein the first part of the (see The portion of the is to a conserved in proteins, and the first of the E.V. Abagyan R.A. Nat. Genet... 1997; 16: 330-331Google Scholar). the this protein the to TSG101 with a a and a C-terminal The functional of this protein to TSG101 is The C-terminal portion of TSG101 has the to form an (see as using a of via the server. The C-terminal is the most conserved within TSG101 M. Odorizzi G. Estepa E.J. Emr S.D. Traffic.. 2000; 1: 248-258Google Scholar). We have found that the of mammalian TSG101 has the to form a first for a acid with at the first and TSG101 has a of at as well as have been identified, including within the protein that has at and H. M. 1999; Scholar). The in a are with and in TSG101 as found in other J.M. J. Biol. 1996; P. F. G. EMBO J... 1997; 16: Scholar). The of this was by of the program A. M. J. Scholar). As well as in mammalian TSG101, the is conserved in C. S. S. and A. whereas it is in S. and C. To of TSG101 required for its with mutants of TSG101 were generated by into its of in TSG101 were and with As shown in TSG101 the or with hVPS28 as well as TSG101. In TSG101 the C-terminal was to whereas TSG101 the bound to hVPS28 with The C-terminal of TSG101 were to for its binding to a the and C-terminal bound hVPS28 to the as TSG101 Indeed, the C-terminal of TSG101 bound to hVPS28 as as TSG101. the most conserved within TSG101 is for its with perhaps that the with VPS28 a important role in TSG101 the C-terminal is conserved between TSG101 and other proteins an (20Ponting C.P. Cai Y.-D. Bork P. J. Mol. Med... 1997; 75: 467-469Google Scholar). data that TSG101 regulates the transport of receptors to endocytic M. Odorizzi G. Estepa E.J. Emr S.D. Traffic.. 2000; 1: 248-258Google Scholar), the mechanism by this is is TSG101 interact directly with an endosome-associated receptor sorting Alternatively, evidence that TSG101 is a transcriptional (22Watanabe M. Yanagi Y. Masuhiro Y. Yano T. Yoshikawa H. Yanagisawa J. Kato S. Biochem. Biophys. Res. Commun... 1998; 245: 900-905Google Scholar, 23Hittelman A.B. Burakov D. Iniguez-Lluhı́ J.A. Freedman L.P. Garabedian M.J. EMBO J... 1999; 18: 5380-5388Google Scholar), it might its indirectly by of expression of specific gene To provide evidence for a direct role for TSG101 and its interacting in receptor sorting, we examined could to endosomal reports have shown that TSG101 is a predominantly cytosolic protein at Y. D. Res... 1998; Scholar, L. Cohen S.N. S. 1998; 95: Scholar), although it to the Y. D. Res... 1998; Scholar). To demonstrate TSG101 and hVPS28 might at least to we for that might their Studies in yeast that the endosome of several class E vps proteins are (4Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol... 1995; 131: 603-617Google Scholar, M. Wendland B. Estapa E.J. Emr S.D. EMBO J... 1998; 17: 2982-2993Google Scholar). We examined modulating the activity of another mammalian orthologue of a class E protein could the of TSG101 and hVPS28. Vps4p is a class E protein that is a of the AAA of proteins M. Sato T.K. Banta L.M. Emr S.D. EMBO J... 1997; 16: 1820-1831Google Scholar), of appear to in molecular F. M. 1995; 17: Scholar, K. A. T. 1999; Scholar, S. M. Cell Biol... 1998; Scholar). this basis it has been proposed that Vps4p acts to interactions between other components of the vacuolar sorting pathway, including other class E proteins. Indeed, expression of Vps4p that is defective in to the of and on the surface of aberrant class E in yeast (12Babst M. Wendland B. Estapa E.J. Emr S.D. EMBO J... 1998; 17: 2982-2993Google Scholar), that a in Vps4p function might soluble components required for endocytic sorting from from the endosomal We have demonstrated that expression of human to aberrant that are and defective in sorting of (10Bishop N. Woodman P.G. Mol. Biol. Cell.. 2000; 11: 227-239Google Scholar). with this, mutants of the closely related mouse SKD1 growth factor receptor degradation (11Yoshimori T. Yamagata F. Yamamoto A. Mizushima N. Kabeya Y. Nara A. Miwako I. Ohashi M. Ohsumi M. Ohsumi Y. Mol. Biol. Cell.. 2000; 11: 747-763Google Scholar). The of both TSG101 and hVPS28 were examined in cells with or expression of a dominant-negative mutant of might the complex from from aberrant As demonstrated in TSG101 is cytosolic in although the is is to that for VPS28 (see expression of a portion of both TSG101 and hVPS28 associated with endosomal were with TSG101 or however, consistent with the of in studies (10Bishop N. Woodman P.G. Mol. Biol. Cell.. 2000; 11: 227-239Google Scholar). At we not the mechanism mutant of on the surface of Although it is that interacts directly with the TSG101 it is that a number of components are and that of function a in the of of these, a portion of both TSG101 and hVPS28 to TSG101 with the endosome in the first is however, the presence of a conserved the that TSG101 interacts with is evidence that of receptors and their associated proteins a role in the endocytic pathway J. Cell 1999; Scholar), including post-endosomal sorting to the MVB and lysosome L. H. M. I. S. K. Y. A. S. Y. Mol. Cell.. 1999; Scholar). findings provide evidence that TSG101 and its interacting components with the endocytic pathway and are to a direct role in mitogenic receptor and role they in gene It is important to that our data not reports TSG101 function as a transcriptional Indeed, is evidence of between the regulation of the endocytic pathway, and gene regulation (see Cell Biol... 1999; 11: for J. H. P. J. Cell Biol... 2000; We for DNA sequencing. multivesicular body protein vacuolar protein sorting tumor suppressor gene acid associated with cellular sequence