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Proline-rich Sequence Recognition

II. PROTEOMICS ANALYSIS OF Tsg101 UBIQUITIN-E2-LIKE VARIANT (UEV) INTERACTIONS*
Open AccessPublished:June 20, 2009DOI:https://doi.org/10.1074/mcp.M800337-MCP200
      The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly “PTAP” interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFκB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461–2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes.
      Tsg101 is an essential protein involved in cancerogenesis (
      • Li L.
      • Cohen S.N.
      Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells.
      ), cell cycle progression (
      • Zhong Q.
      • Chen Y.
      • Jones D.
      • Lee W.H.
      Perturbation of TSG101 protein affects cell cycle progression.
      ,
      • Xie W.
      • Li L.
      • Cohen S.N.
      Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency.
      ), transcription regulation (
      • Watanabe M.
      • Yanagi Y.
      • Masuhiro Y.
      • Yano T.
      • Yoshikawa H.
      • Yanagisawa J.
      • Kato S.
      A putative tumor suppressor, TSG101, acts as a transcriptional suppressor through its coiled-coil domain.
      ), and endosomal sorting (
      • Babst M.
      • Odorizzi G.
      • Estepa E.J.
      • Emr S.D.
      Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking.
      ). It gained even more attention when its requirement for retroviral budding was demonstrated. Solvent-exposed proline-rich P(T/S)AP (“PTAP”)
      The abbreviations used are:
      PTAP
      P(T/S)AP
      ESCRT
      endosomal sorting complex required for transport
      ITC
      isothermal titration calorimetry
      SILAC
      stable isotope labeling by amino acids in cell culture
      UEV
      ubiquitin-E2-like variant
      E2
      ubiquitin carrier protein
      E3
      ubiquitin-protein isopeptide ligase
      PABP1
      poly(A)-binding protein 1
      TAL
      Tsg101-associated ligase
      wt
      wild type
      HIV-1
      human immunodeficiency virus, type 1
      HSQC
      heteronuclear single quantum correlation
      YFP
      yellow fluorescentprotein
      GFP
      green fluorescent protein
      HA
      hemagglutinin
      bis-Tris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxy-methyl)propane-1,3-diol
      ER
      endoplasmic reticulum
      COPII
      Coat protein complex II
      Tsg101
      Tumor susceptibility gene 101 protein
      1The abbreviations used are:PTAP
      P(T/S)AP
      ESCRT
      endosomal sorting complex required for transport
      ITC
      isothermal titration calorimetry
      SILAC
      stable isotope labeling by amino acids in cell culture
      UEV
      ubiquitin-E2-like variant
      E2
      ubiquitin carrier protein
      E3
      ubiquitin-protein isopeptide ligase
      PABP1
      poly(A)-binding protein 1
      TAL
      Tsg101-associated ligase
      wt
      wild type
      HIV-1
      human immunodeficiency virus, type 1
      HSQC
      heteronuclear single quantum correlation
      YFP
      yellow fluorescentprotein
      GFP
      green fluorescent protein
      HA
      hemagglutinin
      bis-Tris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxy-methyl)propane-1,3-diol
      ER
      endoplasmic reticulum
      COPII
      Coat protein complex II
      Tsg101
      Tumor susceptibility gene 101 protein
      motifs in retroviral structural proteins (
      • VerPlank L.
      • Bouamr F.
      • LaGrassa T.J.
      • Agresta B.
      • Kikonyogo A.
      • Leis J.
      • Carter C.A.
      Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag).
      ,
      • Garrus J.E.
      • von Schwedler U.K.
      • Pornillos O.W.
      • Morham S.G.
      • Zavitz K.H.
      • Wang H.E.
      • Wettstein D.A.
      • Stray K.M.
      • Côté M.
      • Rich R.L.
      • Myszka D.G.
      • Sundquist W.I.
      Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.
      ) interact with a conserved set of aromatic residues present in the ubiquitin-E2-like variant (UEV) domain of Tsg101 (
      • Pornillos O.
      • Alam S.L.
      • Rich R.L.
      • Myszka D.G.
      • Davis D.R.
      • Sundquist W.I.
      Structure and functional interactions of the Tsg101 UEV domain.
      ) and thereby hijack the endosomal sorting machinery for the last step of the budding process (
      • Freed E.O.
      Viral late domains.
      ). This finding provided the functional explanation for the importance of the late domain. It also shed light on endosomal sorting of ubiquitinated membrane receptors targeted for lysosomal degradation by macromolecular complexes that are called ESCRT complexes (
      • Williams R.L.
      • Urbé S.
      The emerging shape of the ESCRT machinery.
      ,
      • Hurley J.H.
      ESCRT complexes and the biogenesis of multivesicular bodies.
      ). Although ESCRT-0 is involved in the initial selection of cargo, the further channeling of cargo into the lysosomal pathways requires ESCRT-I–III complexes. Tsg101 has been characterized as part of the ESCRT-I complex and through its interaction with Hrs links ESCRT-I to ESCRT-0. The interaction with Hrs, a protein that binds to ubiquitinated receptors committed to degradation, is mediated by the UEV domain of Tsg101 (
      • Bache K.G.
      • Brech A.
      • Mehlum A.
      • Stenmark H.
      Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes.
      ,
      • Katzmann D.J.
      • Stefan C.J.
      • Babst M.
      • Emr S.D.
      Vps27 recruits ESCRT machinery to endosomes during MVB sorting.
      ) and involves PTAP binding sites. A separate surface of the UEV domain conveys weak binding to ubiquitin, and this dual mode recognition is thought to mediate the seamless integration of ubiquitinated cargo into the growing ESCRT-I complex (
      • Pornillos O.
      • Alam S.L.
      • Rich R.L.
      • Myszka D.G.
      • Davis D.R.
      • Sundquist W.I.
      Structure and functional interactions of the Tsg101 UEV domain.
      ). In addition to the ESCRT-0-ESCRT-I link, binding of Tsg101 UEV to a PSAP motif within the Alix protein appears to function as a shortcut between ESCRT-I and ESCRT-III (
      • Martin-Serrano J.
      • Yarovoy A.
      • Perez-Caballero D.
      • Bieniasz P.D.
      • Yaravoy A.
      Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins.
      ,
      • von Schwedler U.K.
      • Stuchell M.
      • Müller B.
      • Ward D.M.
      • Chung H.Y.
      • Morita E.
      • Wang H.E.
      • Davis T.
      • He G.P.
      • Cimbora D.M.
      • Scott A.
      • Kräusslich H.G.
      • Kaplan J.
      • Morham S.G.
      • Sundquist W.I.
      The protein network of HIV budding.
      ). Several other ESCRT proteins have been suggested to form similar interactions: the PTAP motif within Vps37B might contribute to its interaction with Tsg101 (
      • Stuchell M.D.
      • Garrus J.E.
      • Müller B.
      • Stray K.M.
      • Ghaffarian S.
      • McKinnon R.
      • Kräusslich H.G.
      • Morham S.G.
      • Sundquist W.I.
      The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding.
      ), and a PTAP motif located in the C-terminal part of Tsg101 itself could have an autoinhibitory function (
      • Pornillos O.
      • Alam S.L.
      • Rich R.L.
      • Myszka D.G.
      • Davis D.R.
      • Sundquist W.I.
      Structure and functional interactions of the Tsg101 UEV domain.
      ).
      Additionally another protein, TOM1L1, with a modular structure similar to that of Hrs has been shown to interact with Tsg101 via its P(T/S)AP motifs (
      • Morita E.
      • Sandrin V.
      • Chung H.Y.
      • Morham S.G.
      • Gygi S.P.
      • Rodesch C.K.
      • Sundquist W.I.
      Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis.
      ) Recently both Tsg101 and TOM1L1 have been shown to localize to the midbody during cytokinesis where the final abscission step of the thin midbody membrane is mechanistically similar to the formation of multivesicular bodies or virus budding (
      • Carlton J.G.
      • Martin-Serrano J.
      Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery.
      ,
      • Morita E.
      • Sandrin V.
      • Chung H.Y.
      • Morham S.G.
      • Gygi S.P.
      • Rodesch C.K.
      • Sundquist W.I.
      Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis.
      ,
      • Yanagida-Ishizaki Y.
      • Takei T.
      • Ishizaki R.
      • Imakagura H.
      • Takahashi S.
      • Shin H.W.
      • Katoh Y.
      • Nakayama K.
      Recruitment of Tom1L1/Srcasm to endosomes and the midbody by Tsg101.
      ).
      The need for tight regulation of Tsg101 levels in the cell has already been described by Zhong et al. (
      • Zhong Q.
      • Chen Y.
      • Jones D.
      • Lee W.H.
      Perturbation of TSG101 protein affects cell cycle progression.
      ). Only recently has it been shown that Tsg101 levels are regulated by the E3 ubiquitin ligase TAL (Tsg101-associated ligase), which contains a tandem PTAP motif that mediates binding to the Tsg101 UEV domain (
      • Amit I.
      • Yakir L.
      • Katz M.
      • Zwang Y.
      • Marmor M.D.
      • Citri A.
      • Shtiegman K.
      • Alroy I.
      • Tuvia S.
      • Reiss Y.
      • Roubini E.
      • Cohen M.
      • Wides R.
      • Bacharach E.
      • Schubert U.
      • Yarden Y.
      Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding.
      ,
      • McDonald B.
      • Martin-Serrano J.
      Regulation of Tsg101 expression by the steadiness box: a role of Tsg101-associated ligase.
      ). In contrast to TAL-induced polyubiquitination, the E3 ligase Mahogunin, which binds to Tsg101 UEV via its PSAP motif, appears to lead to monoubiquitination (
      • Kim B.Y.
      • Olzmann J.A.
      • Barsh G.S.
      • Chin L.S.
      • Li L.
      Spongiform neurodegeneration-associated E3 ligase Mahogunin ubiquitylates TSG101 and regulates endosomal trafficking.
      ). Besides its own regulation via the ubiquitin-proteasome pathway, Tsg101 probably uses its UEV domain, which is a nonfunctional E2 enzyme domain, to regulate protein levels of other proteins. In concert with the E3 ligase MDM2 it regulates protein levels of the transcription factor p53 in a feedback loop (
      • Li L.
      • Liao J.
      • Ruland J.
      • Mak T.W.
      • Cohen S.N.
      A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control.
      ,
      • Ruland J.
      • Sirard C.
      • Elia A.
      • MacPherson D.
      • Wakeham A.
      • Li L.
      • de la Pompa J.L.
      • Cohen S.N.
      • Mak T.W.
      p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101.
      ), and this function seems to be PTAP-independent. The Tsg101 UEV domain is the only known protein domain interacting with PTAP motifs, and the definition of this peptide sequence was based on the occurrence of P(T/S)AP sequences in viral late domains. However, within viruses an affinity-optimized motif seems to have evolved compared with that of cellular interaction partners, and we were therefore interested in defining sequence specificity and affinity by selecting high affinity binders from a randomized 9-mer phage display library. In conjunction with peptide SPOT experiments we refined the UEV interaction motif to (A/P)(T/S)AP. ESCRT proteins containing this motif were analyzed for binding to Tsg101 UEV by SPOT analysis. SILAC-based pulldown experiments (
      • Ong S.E.
      • Blagoev B.
      • Kratchmarova I.
      • Kristensen D.B.
      • Steen H.
      • Pandey A.
      • Mann M.
      Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
      ) with GST-UEV and mutational variants were performed, and cellular interaction partners were identified by mass spectrometry. Known and novel potential interaction partners were among the 30 isotopically enriched proteins. Alix, Hrs, and the E3 ubiquitin ligase TAL were confirmed as known Tsg101 targets. NMR and ITC analysis showed that the two (A/P)(T/S)AP motifs of TAL bind UEV with graded affinity. Importantly, two proteins involved in translational control, namely PABP1 and eIF4b, were shown to bind as full-length proteins to Tsg101 in cellular lysates. Direct interactions of these two proteins enhance mRNA translation by facilitating 5′- to 3′-end communication of mRNA, and Tsg101 conceivably modulates translation by marking these two proteins for intracellular transport.

      DISCUSSION

      The assigned function of Tsg101 in ESCRT-dependent endosomal sorting (
      • Babst M.
      • Odorizzi G.
      • Estepa E.J.
      • Emr S.D.
      Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking.
      ,
      • Williams R.L.
      • Urbé S.
      The emerging shape of the ESCRT machinery.
      ,
      • Hurley J.H.
      ESCRT complexes and the biogenesis of multivesicular bodies.
      ), viral budding (
      • Fujii K.
      • Hurley J.H.
      • Freed E.O.
      Beyond Tsg101: the role of Alix in ‘ESCRTing’ HIV-1.
      ), and cytokinesis, (
      • Carlton J.G.
      • Martin-Serrano J.
      Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery.
      ,
      • Morita E.
      • Sandrin V.
      • Chung H.Y.
      • Morham S.G.
      • Gygi S.P.
      • Rodesch C.K.
      • Sundquist W.I.
      Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis.
      ) leaves open the question of additional roles of the protein in other physiological processes. We therefore designed a proteomics screen that more fully comprises the putative targets of Tsg101. UEV domain mutants either devoid of PTAP or ubiquitin binding sites or both were compared in their ability to interact with cellular proteins by utilizing the SILAC technology (
      • Ong S.E.
      • Blagoev B.
      • Kratchmarova I.
      • Kristensen D.B.
      • Steen H.
      • Pandey A.
      • Mann M.
      Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
      ). 13C enrichment of peptides was used as a measure to classify proteins that bind to UEV in a PTAP-dependent manner. Destruction of the PTAP site was either achieved by including an excess of HIV-1-p6 peptide in the pulldown experiment or by a mutation in the center of the binding site (M95A). Interestingly a few more hits were found when the pulldown was performed with peptide competition as compared with the PTAP mutant (M95A). This might indicate that despite the 52-fold reduction in binding affinity (
      • Pornillos O.
      • Alam S.L.
      • Rich R.L.
      • Myszka D.G.
      • Davis D.R.
      • Sundquist W.I.
      Structure and functional interactions of the Tsg101 UEV domain.
      ) the mutant still allows weak interactions with certain proteins, whereas competition with external peptide completely blocks all interactions. Strikingly largely overlapping sets of proteins were found for the peptide competition experiment and the experiments utilizing the PTAP mutant or the mutant devoid of PTAP and ubiquitin binding. In contrast, the mutant only devoid of ubiquitin binding did not have an influence on the interaction network mediated by UEV. Target selection therefore depends predominantly on the PTAP sequence, and ubiquitin binding occurs preferably within the PTAP-selected cargos.
      The validity of our approach was demonstrated by the finding of three functionally known interaction partners: Alix and Hrs as part of the ESCRT machinery and TAL, which controls Tsg101 levels in living cells. The latter protein was investigated in more detail by NMR and ITC to unravel the function of its double PTAP-PSAP motif, which had been shown to mediate its interaction with Tsg101. We found a strong preference for the PSAP motif as reflected by the roughly 6-fold higher affinity. Conceivably the second motif, which is in close proximity to the RING domain mediating ubiquitination, has evolved as the primary target site. Like many high affinity ligands the PSAP motif is C-terminally flanked by an additional proline, which is consistent with our phage display results (Fig. 1C).
      Among the highly enriched proteins we found the transcription factor eIF4b as a Tsg101 UEV interaction partner in our pulldown experiment. eIF4b is part of the translation initiation complex that binds to the cap structure at the 5′-end of mRNA (
      • van Heugten H.A.
      • Thomas A.A.
      • Voorma H.O.
      Interaction of protein synthesis initiation factors with the mRNA cap structure.
      ) and contributes to cell cycle- or stress-dependent regulation of translation (
      • Yamasaki S.
      • Anderson P.
      Reprogramming mRNA translation during stress.
      ). Recently eIF4b has also been shown to be regulated by 14-3-3σ (
      • Wilker E.W.
      • van Vugt M.A.
      • Artim S.A.
      • Huang P.H.
      • Petersen C.P.
      • Reinhardt H.C.
      • Feng Y.
      • Sharp P.A.
      • Sonenberg N.
      • White F.M.
      • Yaffe M.B.
      14-3-3sigma controls mitotic translation to facilitate cytokinesis.
      ), a p53-induced gene that functions in mitotic exit and cytokinesis. Another protein identified in our experiments was PABP1, which has been described to stabilize mRNAs at the 5′-end by interacting with the poly(A) tail (
      • Mangus D.A.
      • Evans M.C.
      • Jacobson A.
      Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression.
      ). eIF4b and PABP1 have been shown to interact with each other (
      • Le H.
      • Tanguay R.L.
      • Balasta M.L.
      • Wei C.C.
      • Browning K.S.
      • Metz A.M.
      • Goss D.J.
      • Gallie D.R.
      Translation initiation factors eIF-iso4G and eIF-4B interact with the poly(A)-binding protein and increase its RNA binding activity.
      ), and reduced levels of these two proteins lead to stress granule formation in HeLa cells (
      • Mokas S.
      • Mills J.R.
      • Garreau C.
      • Fournier M.J.
      • Robert F.
      • Arya P.
      • Kaufman R.J.
      • Pelletier J.
      • Mazroui R.
      Uncoupling stress granule assembly and translation initiation inhibition.
      ). Our demonstration of a direct interaction of Tsg101 with both proteins suggests an exciting link between the tumor maintenance protein Tsg101 and cellular stress factors. Another scenario comes from the observation that Tsg101 partakes in cytokinesis (
      • Carlton J.G.
      • Martin-Serrano J.
      Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery.
      ,
      • Morita E.
      • Sandrin V.
      • Chung H.Y.
      • Morham S.G.
      • Gygi S.P.
      • Rodesch C.K.
      • Sundquist W.I.
      Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis.
      ) where its main function seems to be the recruitment of ESCRT components during the final steps of abscission. During cell division, the enhancement of cap-independent translation correlates with the partial inhibition of cap-dependent translation conferred by binding of 14-3-3σ to eIF4b (
      • Wilker E.W.
      • van Vugt M.A.
      • Artim S.A.
      • Huang P.H.
      • Petersen C.P.
      • Reinhardt H.C.
      • Feng Y.
      • Sharp P.A.
      • Sonenberg N.
      • White F.M.
      • Yaffe M.B.
      14-3-3sigma controls mitotic translation to facilitate cytokinesis.
      ). Possibly Tsg101 contributes to the relative abundance of eIF4b by transport of the initiation factor away from ribosomal complexes. Clearly microscopic investigations of eIF4b and PABP1 localization and of cap-dependent mRNA translation in the presence and absence of Tsg101 have to be performed to more vigorously demonstrate such a functional interplay.
      We defined a set of PTAP-containing proteins that connect molecular complexes involved in ER trafficking, mRNA surveillance, and transcriptional control to the UEV domain (Fig. 6). The link between mRNA surveillance and the heat-shock ubiquitin-proteasome pathway has been established (
      • Laroia G.
      • Cuesta R.
      • Brewer G.
      • Schneider R.J.
      Control of mRNA decay by heat shock-ubiquitin-proteasome pathway.
      ), and it appears likely that Tsg101 functions in this context by regulating ubiquitination reactions via its non-functional UEV domain. Tsg101 could either stabilize its interaction partners by using its catalytically inactive UEV domain to prevent their ubiquitination as has been suggested for the function of Tsg101 in the MDM2/p53 feedback loop (
      • Li L.
      • Liao J.
      • Ruland J.
      • Mak T.W.
      • Cohen S.N.
      A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control.
      ). Alternatively it might work in concert with functional E2 enzymes to direct Lys63-linked polyubiquitination to defined cargos as was shown for two other human UEV proteins, UEV1a and hMms2 (
      • Andersen P.L.
      • Zhou H.
      • Pastushok L.
      • Moraes T.
      • McKenna S.
      • Ziola B.
      • Ellison M.J.
      • Dixit V.M.
      • Xiao W.
      Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.
      ). The Ubc13-UEV1a complex uses this pathway for NFκB activation. The fact that we identified NFκB2 as a potential Tsg101 UEV interaction partner suggests that a similar mechanism could link NFκB activation to endosomal sorting processes, and such a link had been suggested by Rodriguez et al. (
      • Rodriguez P.L.
      • Sahay S.
      • Olabisi O.O.
      • Whitehead I.P.
      ROCK I-mediated activation of NF-kappaB by RhoB.
      ). A role of Tsg101 as a general modifier of target protein ubiquitination, however, remains speculative. For example, we did not observe major ubiquitinated species when probing PABP1 or eIF4b in our pulldown experiments (data not shown), and further experiments are needed to see whether Tsg101 has no influence or a protective or enhancing effect on the concentration and localization of these two proteins in the cell.
      Figure thumbnail gr6
      Fig. 6Functional clusters of proteins identified as potential interaction partners of Tsg101 UEV (the crystal structure shown refers to the Protein Data Bank entry 2F0R (
      • Palencia A.
      • Martinez J.C.
      • Mateo P.L.
      • Luque I.
      • Camara-Artigas A.
      Structure of human TSG101 UEV domain.
      ). Proteins containing (P/A)(T/S)AP motifs are colored red, whereas others are kept in gray (see key). Proteins forming a binary interaction according to the program APID (Agile Protein Interaction DataAnalyzer) are connected by arrows. VASP, vasodilator-stimulated phosphoprotein; Ub, ubiquitin; NONO, non-POU domain-containing octamer-binding protein; SPFQ, splicing factor, proline- and glutamine-rich; TFG, Trk-fused gene protein; EWS, Ewing sarcoma breakpoint region 1 protein.
      In the context of COPII coat proteins Tsg101 UEV might exhibit additional ESCRT-related functions. SEC16A and S23IP, which interact with the main COPII coat component Sec23/Sec24, were shown to localize to ER exit sites (
      • Watson P.
      • Townley A.K.
      • Koka P.
      • Palmer K.J.
      • Stephens D.J.
      Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells.
      ,
      • Shimoi W.
      • Ezawa I.
      • Nakamoto K.
      • Uesaki S.
      • Gabreski G.
      • Aridor M.
      • Yamamoto A.
      • Nagahama M.
      • Tagaya M.
      • Tani K.
      p125 is localized in endoplasmic reticulum exit sites and involved in their organization.
      ). No direct link between COPII-mediated trafficking from the endoplasmic reticulum to the trans-Golgi network and the endosomal sorting pathway has been described. However, the retromer connects the endosome to the trans-Golgi network (
      • Bonifacino J.S.
      • Hurley J.H.
      Retromer.
      ), and a more direct link between endosomes and the ER is conceivable in cases of shared cargo.
      The simple approach we describe for interaction mapping of proline-rich recognition domains represents an important step in deciphering their contribution in a cellular context. Of course, many questions remain open. For example, it is still unclear whether the Tsg101 UEV domain is regulated by an intramolecular autoinhibitory interaction in the respective full-length protein. Because Tsg101 itself could not be identified in our UEV pulldown experiments despite the presence of a binding motif at its C terminus, intramolecular masking of its own PTAP motif offers a reasonable explanation. Alternatively the levels of free Tsg101 in the cell may be so low that it escapes identification by our method. Similarly PTAP motifs of other cellular proteins might be shielded or occupied by interaction partners in tightly packed protein complexes and are consequently not captured by GST-UEV. This might be one reason why only a small fraction of PTAP-containing proteins is found in our pulldown experiments. Alternatively expression levels might be very low, or target proteins might be localized to membranes, two conditions that would probably prevent identification by our approach. When comparing with the yeast two-hybrid database (part of the Human Protein Reference Database) four of seven proteins harboring a (P/A)(S/T)AP motif were also found in at least one of our pulldowns, indicating that observation of certain interactions depends on the cellular environment or experimental setup. Although certain interaction partners might not be captured by the reasons given above we argue that we still identified non-stoichiometric and spurious interaction partners because we used a large excess of UEV domain in our pulldown experiments. Using tandem affinity purification tag technology for mildly overexpressed proteins might seem to be more appropriate for capturing cognate interactors, but this approach also requires larger efforts to establish the corresponding cell lines and purification protocols (
      • Bürckstümmer T.
      • Bennett K.L.
      • Preradovic A.
      • Schütze G.
      • Hantschel O.
      • Superti-Furga G.
      • Bauch A.
      An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.
      ). In addition, a significant number of unspecific binders is still expected, and analysis might be hampered by low protein amounts. We argue that our approach captures “moonlighting” functions of an individual domain, some of which might not be physiologically relevant. On the other side, low affinity encounters as captured by our approach might still contribute to the formation of more stable complexes as they are detected by the tandem affinity purification tag methodology. Our approach also ignores the compartmentalization of proteins that might prevent the direct encounter of potential interaction partners in the cell. However, many proteins and protein complexes shuttle between compartments, and their transport might depend on developmental status or extracellular cues. For example, when nuclear pore membranes are broken down during cell division or when intracellular compartments are formed, protein complex composition and distribution are conceivably quite different from those of the steady-state level of asynchronously growing cells. Therefore, we believe that our approach, when carefully interpreted, represents a robust and rapid method to encounter the full proteomic potential of an individual domain or an individual interaction epitope.
      In conclusion and complimentary to the results obtained in the accompanying article (
      • Kofler M.
      • Schuemann M.
      • Merz C.
      • Kosslick D.
      • Schlundt A.
      • Tannert A.
      • Schaefer M.
      • Lührmann R.
      • Krause E.
      • Freund C.
      Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes.
      ) on GYF and WW domains, we have shown that domain-based pulldown experiments in combination with site-specific inhibition and SILAC/MS analysis allows one to deconvolute the contributions of individual epitopes to protein complex formation. All three domain families investigated here belong to the superfamily of proline-rich binding domains. Although GYF domains seem to operate in a setting similar to that of WW domains, namely the formation of the early spliceosome, the UEV domain participates in protein assemblies that form during vesicle transport, mRNA surveillance, and NFκB signaling.

      Acknowledgments

      We are thankful to Dr. Michael Beyermann for synthesis of UEV-interacting peptides and to Angelika Ehrlich for synthesis of UEV SPOT membranes. We also thank Dr. Peter Schmieder for help with the NMR spectrometers and Sandra Bittman and Gesa Albert for technical support and optimization of transfection, respectively.

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