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Mol Cell Proteomics. Nov 2009; 8(11): 2474–2486.
Published online Jun 20, 2009. doi:  10.1074/mcp.M800337-MCP200
PMCID: PMC2773715

Proline-rich Sequence Recognition

II. PROTEOMICS ANALYSIS OF Tsg101 UBIQUITIN-E2-LIKE VARIANT (UEV) INTERACTIONS*An external file that holds a picture, illustration, etc.
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Abstract

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 (1), cell cycle progression (2, 3), transcription regulation (4), and endosomal sorting (5). It gained even more attention when its requirement for retroviral budding was demonstrated. Solvent-exposed proline-rich P(T/S)AP (“PTAP”)1 motifs in retroviral structural proteins (6, 7) interact with a conserved set of aromatic residues present in the ubiquitin-E2-like variant (UEV) domain of Tsg101 (8) and thereby hijack the endosomal sorting machinery for the last step of the budding process (9). 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 (10, 11). 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 (12, 13) 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 (8). 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 (14, 15). Several other ESCRT proteins have been suggested to form similar interactions: the PTAP motif within Vps37B might contribute to its interaction with Tsg101 (16), and a PTAP motif located in the C-terminal part of Tsg101 itself could have an autoinhibitory function (8).

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 (18) 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 (1719).

The need for tight regulation of Tsg101 levels in the cell has already been described by Zhong et al. (2). 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 (20, 21). In contrast to TAL-induced polyubiquitination, the E3 ligase Mahogunin, which binds to Tsg101 UEV via its PSAP motif, appears to lead to monoubiquitination (22). 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 (23, 24), 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 (25) 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.

EXPERIMENTAL PROCEDURES

Cloning and Protein Expression of Tsg101 UEV Domain

The Tsg101 UEV domain was expressed as a GST fusion protein. Briefly the cDNA, encoding amino acids 1–145 (kindly provided by W. I. Sundquist, University of Utah), was cloned into pGEX4T1 using standard PCR methods resulting in restriction sites for BamHI and XhoI. The UEV mutants N45A, M95A, and N45A/M95A were introduced by oligonucleotide-directed mutagenesis using standard PCR. For protein expression, a single clone was inoculated in a small overnight culture transferred to either Luria broth (Carl Roth GmbH) or M9 minimal medium (including 1 mg/ml [15N]ammonium chloride and 2 mg/ml [13C]glucose for triple resonance NMR samples) at 37 °C. At an A600 of 0.6, protein production was induced by addition of 1 mM isopropyl β-D-thiogalactopyranoside, and cells were grown for another 12–18 h at 23 °C. Cells were harvested and resuspended in 50 mM Tris, 100 mM NaCl, 0.4 mg/ml lysozyme (Sigma-Aldrich), protease inhibitors (Complete Midi, Roche Applied Science), pH 7.5; stored on ice for 30 min; sonicated; and incubated for 30 min at room temperature after addition of 2 mM MgCl2 and Benzonase at 15 units/mg of bacteria pellet (Sigma-Aldrich). The GST-UEV fusion protein was purified from lysate supernatants using GSTrap 4B columns (Amersham Biosciences) on an Äkta Purifier (Amersham Biosciences) by elution with 20 mM glutathione. For pulldowns and spot analysis, peak fractions were concentrated in Amicon tubes (molecular weight cutoff, 10,000; Millipore) and directly gel filtrated on a HighLoad Superdex 75 column (Amersham Biosciences) in PBS to obtain purified fusion protein. For NMR studies and ITC measurements, fractions were incubated with thrombin (1 unit/ml; Calbiochem) overnight to cleave off GST. The reaction volume was concentrated in Amicon columns (molecular weight cutoff, 5000) and loaded onto Superdex 75 in 20 mM phosphate, 50 mM NaCl, pH 5.5 or PBS.

Peptide Phage Display

A randomized nonapeptide library (X9) fused to the pVIII protein of the phagemid vector pC89 (26) was used for phage display. Screening of the library was performed as follows. 120 μg of GST-UEV wt or M95A fusion protein bound to glutathione-Sepharose 4B beads (Amersham Biosciences) was incubated for 1 h with 1011–1012 infectious particles in PBS supplemented with 5 mg/ml BSA and 0.05% Tween 20 at room temperature in a total volume of 400 μl. After washing 10 times with PBS, 0.05% Tween 20, bound phages were eluted with 100 mM glycine/HCl (pH 2.2), and the eluate was neutralized with 2 M Tris. Escherichia coli XL-1Blue cells were infected by the eluted phage particles, and the phages were amplified using the helper phage VCSM13 (Stratagene) according to the standard protocol (see supplemental methods). To monitor enrichment in the process of panning, the ratios of the titers of eluted phage from GST-UEV wt or M95A versus GST-coated beads were determined after each panning step. After three rounds of selection and a final enrichment ratio of ~104 for GST-UEV wt, 29 peptide-encoding inserts were successfully sequenced to obtain the binding site consensus. No significant enrichment was observed for GST-UEV M95A after three rounds of panning.

SPOT Membranes

All selected peptide sequences were synthesized in a solid-phase mode onto a Whatman 50 cellulose membrane by an automated SPOT method (27). For incubation with GST-UEV fusion proteins, membranes were shortly rinsed with ethanol and several times with TBS. After a blocking step of 3 h at room temperature with TBS-based SPOTblock buffer (Sigma-Genosys) that included 1 mM DTT, spots were incubated with 5–40 μg/ml protein in reducing blocking buffer overnight at 4 °C. To detect binding, membranes were treated for 1 h with rabbit-derived anti-GST primary antibody Z5 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and, after extensive washing with TBS for 1 h, with goat anti-rabbit secondary antibody (1:10,000, horseradish peroxidase-coupled; Rockland Immunochemicals), each of the antibodies diluted in blocking buffer devoid of DTT. An enhanced chemiluminescence substrate (Western Lightning, Enhanced Luminol, Pierce) was used for detection on a LumiImager™ (Boehringer Ingelheim).

Peptide Synthesis

All peptides used for SILAC, NMR, and ITC measurements were synthesized in house by a standard solid-phase method based on Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Peptides were cleaved off the reaction matrix with trifluoroacetic acid, purified by preparative reverse-phase HPLC, and lyophilized, resulting in acetylated and amidated sequences. All peptides were of >96% purity as confirmed by analytical HPLC and MS analysis. For TAL-derived peptides, purity was determined as >99%. Peptides in this study are termed as follows: HIV-1-NL43-p6-(5–13) (Ac-PEPTAPPEE-NH2), Caprin-(261–269) (Ac-EAASAPAVE-NH2), CPSF7-(311–319) (Ac-MKASAPYNH-NH2), TAL-(645–668)wt (Ac-VVTPTAPQEPPESVRPSAPPAE-NH2), TAL-(645–668)mut1 (Ac-VVAAAAQEPPESVRPSAPPAE-NH2), and TAL-(645–668)mut2 (Ac-VVTPTAPQEPPESVRAAAAPAE-NH2).

Cell Culture, Isotope Labeling (SILAC), and Lysate Preparation

SILAC was performed as described by Blagoev and Mann (28). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% dialyzed FCS and 1% penicillin and streptomycin (Biochrom and Invitrogen AG, respectively). For SILAC, cells were transferred into SILAC Dulbecco’s modified Eagle’s medium (Invitrogen; according to the manufacturer’s protocol) supplemented with either 12C6- or 13C6-labeled arginine and lysine (isotopes from Cambridge Isotope Laboratories, Andover, MA) and split six times before harvesting. Cells were trypsinized, collected, and washed with PBS three times before lysis was performed in PBS containing 1% nonyl phenoxylpolyethoxylethanol (Nonidet P-40), 1 mM PMSF, 1 mM DTT, 5 mM EDTA, 1 μM pepstatin A, and protease inhibitors (Complete Midi, Roche Applied Science) for 30 min on ice. The cell lysate was centrifuged for 20 min at 16,000 × g and aliquoted prior to quick freezing at −80 °C.

Pulldowns with HeLa Cell Lysate

For pulldowns, 25 μl of glutathione-Sepharose 4B beads (Amersham Biosciences) were washed four times with PBS and incubated for 3 h at 4 °C with at least 1.5 mg of GST-UEV wt, mutants, or GST alone to saturate the matrix. Unbound protein was washed off with PBS in a stepwise manner, and the matrix was incubated with HeLa lysates overnight at 4 °C in the presence of either 2.7 mg of unlabeled protein in the case of the mutant or peptide-inhibited UEV domain or 2.7 mg of 13C-labeled protein for wt UEV. GST-UEV matrices were then washed four times with PBS, mixed at a 2:1 ratio (unlabeled:labeled), resuspended in 5× SDS sample buffer (75 μl), and heated to 95 °C for 5 min. The beads were filtered, and the filtrate was loaded onto a Tris-glycine gradient 4–20% gel (Invitrogen; in different amounts for MS applications). Gels were stained with Coomassie overnight and destained for several hours.

Preparation of MS Samples and Mass Spectrometry

For MS analysis, Coomassie-stained gel bands were cut into slices (resulting in ~40–50 samples per pulldown), and proteins were in-gel digested as described previously (29). Identification and quantitation of proteins were performed on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-Tof Ultima, Micromass, Manchester, UK). A Micromass CapLC liquid chromatography system (Waters GmbH) was used to deliver the peptide solution to the electrospray source. 5 μl of the sample were injected and concentrated on a precolumn (PepMap C18, 5 μm, 100 Å, 5 mm × 300-μm inner diameter; Dionex, Idstein, Germany). LC separations were performed on a capillary column (Atlantis dC18, 3 μm, 100 Å, 150 mm × 75-μm inner diameter; Waters GmbH) at an eluent flow rate of 200 nl/min using a linear gradient of 3–64% mobile phase B in 100 min. Mobile phase A was 0.1% formic acid (v/v) in acetonitrile-water (3:97, v/v), and mobile phase B was 0.1% formic acid in acetonitrile-water (8:2, v/v). MS/MS data were acquired in a data-dependent mode (survey scanning) using one MS scan followed by MS/MS scans of the most abundant peak. The MS survey range was m/z 300–1550, and the MS/MS range was m/z 100–1990. Data analysis was performed by use of MassLynx version 4.0 software (Micromass-Waters).

Identification of Proteins

The processed MS/MS spectra and Mascot server (version 2.0; Matrix Science Ltd., London, UK) were used to search in house against the UniProt/Swiss-Prot database (release 50.0 of September 21, 2006, containing 234,112 sequence entries, comprising 85,963,701 amino acids). A maximum of two missed cleavages was allowed, and the mass tolerance of precursor and sequence ions was set to 100 ppm and 0.05 Da, respectively. Acrylamide modification of cysteine, methionine oxidation, [13C6]arginine, and [13C6]lysine were considered as possible modifications. A protein was accepted as identified if the total Mascot score was greater than the significance threshold and at least two peptides appeared the first time in the report and were the first ranking peptides.

Quantification of Proteins

Quantification was carried out by MSQuant (Peter Mortensen and Matthias Mann) open source software and was based on calculations of isotope intensity ratios of at least two arginine- or lysine-containing tryptic peptides with individual Mascot score indicating identity. Additional criteria were that no interfering mass peaks were observed and that peptides contained neither missed cleavage sites nor methionine oxidation. Relative protein ratios were calculated by averaging over all peptides. Proteins displaying 13C enrichment factors >2 were defined as hits. Standard deviations of the quantification for individual proteins were obtained from MSQuant. Analytical reproducibility was determined by multiple LC-MS measurements of selected samples showing that the experiment-to-experiment deviations of 13C:12C ratios are less than 25%.

NMR Sample Preparation and Spectra Recording

The assignment and NMR structure of Tsg101 UEV was reported previously (8) and was kindly made available by W. I. Sundquist (University of Utah). The observation that the construct we used showed partial ambiguities and additional resonances deriving from two additional amino acids as a result of thrombin cleavage required the reassignment of the free protein. A 15N- and 13C-labeled 1.2 mM sample of UEV containing 10% D2O was measured on a Bruker AV750 instrument equipped with a triple resonance probe head. 15N-1H HSQC, CBCACONNH, CBCANNH, HNCO, and HNCACO spectra were processed using TopSpin (Bruker, Rheinstetten, Germany), and assignments were obtained with Collaborative Computing Project for NMR software (Cambridge, UK). For peptide binding studies 15N-1H HSQC spectra were recorded from a 200–300 μM sample in 20 mM phosphate, 50 mM sodium chloride, pH 5.5, 10% D2O either without ligand or containing 2 mM peptides on a Bruker AV750 or DRX600 instrument (equipped with triple resonance cryoprobe heads). Spectra were processed using TopSpin or XwinNMR (Bruker), and shifts were measured with Sparky (University of California, San Francisco, CA). Binding constants could not be derived from the NMR experiments because binding was in the slow exchange regime. The integration of peak volumes for the UEV binding to TAL-derived peptides was performed on four sufficiently separately located resonances using the sum-over-box method in Sparky. Chemical shift experiments for the binding of human ubiquitin (Asla Biotech) were carried out in the buffer as mentioned above. The 15N-1H HSQC spectra of 150 μM Tsg101 UEV samples either in the absence or presence of 1 mM HIV-1-p6 peptide were recorded in the presence of increasing amounts of ubiquitin (0–3.8 mM).

Isothermal Titration Calorimetry

ITC measurements were performed on a VP-ITC device (Originlab, Northampton, MA) using freshly gel-filtrated protein at concentrations of 20–40 μM in PBS, pH 7.4 at 25 °C. All peptides were dialyzed against the reaction buffer and injected in 30 steps of 10 μl each from 0.75–1.5 mM stock solutions in 5-min intervals. Peptides were also injected into dialysis buffer to measure the heat of dilution. Power peaks were integrated, and the resulting reaction heats were plotted against the molar peptide:protein ratio and fitted using the “One Set of Sites” model using Origin 5.0 (MicroCal software), yielding the dissociation constant, KD.

Database Searches

Human ESCRT proteins were searched for (A/P)(T/S)XP motifs with the program ScanProsite or “3 of 5 complex pattern search”. Potential binary interactions between proteins identified as hits in SILAC experiments were determined using the program APID (Agile Protein Interaction DataAnalyzer).

Design and Transfection of Constructs for Immunoprecipitation

Full-length human Tsg101 in pcDNA3.1 was a kind gift of Reiner Mailer (Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany). The human PABP1 construct was kindly provided by Stephen Lee (University of Ottawa, Ottawa, Canada), and human eIF4b was contributed by Nahum Sonenberg (McGill University, Montreal, Canada). Tsg101 cDNA was N-terminally tagged with YFP by subcloning via EcoRI and XhoI restriction sites. The fusion vector (pcDNA3.1- based) was obtained from Michael Schäfer (University of Leipzig, Leipzig, Germany). Furthermore Tsg101 was N-terminally elongated by an HA tag using standard restriction-free cloning procedures (30). HeLa cells were transiently transfected with different combinations of constructs using the FuGENE HD or FuGENE 6 transfection kits according to the manufacturer’s instructions (Roche Applied Science). Cells were grown for 24 h post-transfection, harvested, and lysed as described above.

Immunoprecipitation, Ubiquitin Pulldowns, and Western Blots

Cell lysates with overexpressed proteins were incubated with the primary antibody against GFP/YFP (rabbit serum; a gift from Ralph Schülein, Leibniz Institute for Molecular Pharmacology, Berlin, Germany) overnight at 4 °C at gentle shaking. Formed complexes were precipitated with 20 μl of Protein G-Sepharose (Sigma-Aldrich), extensively washed with PBS, and boiled in SDS sample buffer. High affinity precoupled matrices were used for HA (Roche Applied Science) and FLAG (M2, Sigma-Aldrich). Samples were loaded onto NuPAGE 4–12% bis-Tris or 3–20% Tris-glycine gels (Invitrogen), separated in MES- or Tris-glycine-based running buffer, and subsequently transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 4% BSA, and proteins were detected with antibodies as described above. Additional antibodies were used for Tsg101 (clone 4A10, Santa Cruz Biotechnology), FLAG tag (F3165, Sigma-Aldrich), GFP/YFP (mouse; Clontech), and Alix (56932, Abcam Inc., Cambridge, MA). Primary antibodies were probed with the secondary fluorescence-coupled antibodies IRDye mouse 680, IRDye rabbit 800, and IRDye goat 800 (all obtained from LI-COR, Lincoln, NE) using the Odyssey Imager (LI-COR).

RESULTS

Tsg101 UEV Binding Motif

The function of the Tsg101 UEV domain was discovered in the context of viral late domains, and mutational analysis defined the binding motif as P(T/S)AP. This definition was slightly expanded by the finding that the UEV-Hrs interaction might also involve PSMP and PSGP motifs, although the structure of the UEV-HIV-1-p6 5PEPTAPPEE13 complex indicates that the 9AP10 dipeptide is crucial to the interaction (31). Fig. 1A shows the UEV-PTAP interface determined by Pornillos et al. (31) and highlights residues that are significantly affected upon binding as indicated by NMR chemical shift perturbation data. Evidently the interaction is largely stabilized by Pro10, which forms crucial hydrophobic contacts with tyrosines 63 and 68. In contrast, Pro7 is less tightly packed against the domain surface, and mutational data indicate that a P7A mutation within the HIV-1-p6 PTAP motif reduces binding to the UEV domain only 3-fold (7). Given these uncertainties, we investigated the general requirements of UEV interaction motifs. To start with, we performed a mutational screen of the original p6 peptide on a peptide SPOT membrane (Fig. 1B). In agreement with previous results (7, 31) UEV binding was abolished only when PTAP residues were mutated. These data also imply that the 9AP10 dipeptide is essential whereas position 8 has to be occupied by a threonine and serine forming critical hydrogen bonds to Ser143/Arg144 of UEV (not marked). As expected a P7A mutation was tolerated (Fig. 1B), and initial modeling results indicate that alanine can be accommodated without significant restructuring of the UEV domain itself.2

Fig. 1.
Peptide SPOT membranes and phage display. A, UEV-PTAP interface as taken from the Protein Data Bank entry 1M4P (31). The p6 peptide is shown in green sticks with only the central core motif 7PTAP10. The Tsg101 UEV domain is displayed in gray ribbon. The ...

As SPOT analysis gives local information on the effect of individual amino acid substitutions, we used phage display as a method to assess additive or cooperative effects for the peptide as a whole. For this purpose, we utilized a gene-3-fused X9 phage library. After three rounds of panning, we observed specific enrichment of phages compared with the GST control, whereas the GST-UEV M95A mutant showed no specific enrichment. 29 phage sequences were analyzed for GST-UEV (Fig. 1C) and showed a preference for PSAP (12 occurrences) over PTAP (seven occurrences), ASAP (six occurrences), and ATAP (three occurrences) motifs. Aside from this strict consensus, phage display-derived peptides often show an additional proline immediately C-terminal to the (P/A)(T/S)AP motif, and proline is the most abundant residue at this position in different HIV isolates. Because this additional proline is unlikely to contact the domain directly, it might indirectly stabilize the polyproline type II helical conformation of the ligand. The residue immediately N-terminal to the motif is often a glutamate in viral sequences, and this amino acid is also found in the Alix motif. In confirmation, we observed a bias toward overall negatively charged peptides in our phage display screen. However, one has to take keep in mind that a 9-mer sequence might be too short for accounting for expanded consensus motifs that are modulated by residues further away from the core motif. For the search of potential further interaction partners of the UEV domain, we thus focused on the consensus sequence (P/A)(T/S)AP.

Motif Hits within ESCRT Proteins

Functionally relevant interactions of Tsg101 UEV with PTAP motifs in proteins of the ESCRT machinery have been described only for Hrs (recruitment of ESCRT-I to the endosomal membrane) and Alix (links ESCRT-I and ESCRT-III). Additionally an autoinhibitory interaction of the N-terminal UEV domain of Tsg101 with its own C-terminal PTAP motif has been suggested (8). Binding to the PTAP motif within Vps37B might contribute to the Tsg101-Vps37B interaction, which is mainly mediated by coiled coil domains (16). Additional (P/A)(T/S)AP motifs are present within ESCRT proteins, and PSMP and PSGP motifs and Hrs residues 560–573 were suggested to contribute to the UEV-Hrs interaction (32). We therefore analyzed a peptide SPOT membrane with all 20-mer peptides containing (P/A)(T/S)XP motifs derived from ESCRT proteins and Hrs sequence 560–573 (Fig. 1D and supplemental Table S1). Consistent with the results described above, only (P/A)(T/S)AP spots were positive. All the previously described interactions between UEV and Hrs (PSAP), Alix (PSAP), Tsg101 (PTAP), and Vps37B (PTAP) were confirmed. Additionally peptides containing the Mvb12A ASAP and Alix ATAP motifs displayed very weak signals.

Interaction Analysis of Tsg101 UEV Domain

To define those PTAP and ubiquitin interactions that are of physiological relevance and possibly determine new interaction partners of the UEV domain, we performed a SILAC-based approach as schematized in Fig. 2 and described in detail in the accompanying article (48). We were also interested in whether the binding of PTAP sequences and ubiquitin would influence each other. Although we did not observe a change in the UEV-p6 interaction in the presence of ubiquitin (supplemental Fig. S1) it is still conceivable that avidity-driven interactions arise from simultaneous binding of PTAP and ubiquitin moieties within target proteins. Wild type GST-UEV was used for pulldown experiments with lysates of HeLa cells stably labeled with [13C]lysine and [13C]arginine. A corresponding unlabeled lysate was applied to a GST-UEV variant that contained either (i) a dysfunctional PTAP site (M95A), (ii) a dysfunctional ubiquitin binding epitope (N45A), or (iii) dysfunctional PTAP and ubiquitin interaction sites (M95A/N45A). Upon mixing the eluates of the two samples in a ratio of 2:1 (unlabeled:labeled), the combined sample was boiled and separated by one-dimensional SDS-PAGE. Subsequent MS analysis will detect peptides binding equally to both wild type and mutant proteins in a ratio of 2:1, whereas this ratio will be shifted significantly in cases where the mutation affects the affinity of a cellular binding partner. As an alternative way to block the PTAP binding site, the HIV-1-p6 peptide was added to the lysate prior to precipitation with wild type GST-UEV. Proteins identified as potential UEV binding partners interacting either directly via their (A/P)(T/S)AP motifs or as part of a complex are shown in Table I. The quantification of the ratio of 13C to 12C in the MS spectra of several peptides (13C enrichment) of these proteins is shown in supplemental Table S2, and lists of all quantified proteins are found in supplemental Tables S3–S6. By thoroughly quantifying 13C:12C ratios, 29 proteins (plus ubiquitin) were reliably identified as hits characterized by 13C enrichment factors >2 in at least two of the three experiments blocking the PTAP binding site (competition with PTAP peptide and comparison with the mutant UEV M95A and the double mutant UEV M95A/N45A). All of the 29 “hits” also fulfill the criteria of the accompanying article (48) (supplemental Table 2). Of these proteins 18 contain (A/P)(T/S)AP motifs allowing for potential direct UEV binding, whereas the remaining proteins are likely to be part of an interacting complex. No significantly enriched proteins were present in the ubiquitin-binding mutant (N45A), indicating that all of the proteins interacting with wild type UEV can also bind to the mutant domain with sufficient affinity not to detect a difference in binding in our experimental setup (Table I). As expected, the pulldown with the double mutant contains a very similar set of enriched proteins when compared with the PTAP blocking experiments. Interestingly the experiment with the ubiquitin-binding mutant showed less enrichment of ubiquitin compared with the PTAP and the double mutant, suggesting that ubiquitin enrichment depends on PTAP target recognition (supplemental Table S2).

Fig. 2.
Principle of the SILAC experiment as it was performed in our laboratory and as it is described in detail in the accompanying GYF domain article (48). Mutant or peptide-inhibited UEV was incubated with unlabeled lysate, whereas wild type UEV was incubated ...
Table I
List of all proteins defined as potential interaction partners based on 13C enrichment factors >2 in at least two of the experiments using a block of PTAP binding

We identified three proteins known to interact with Tsg101 UEV in all three pulldown experiments: the ESCRT proteins Alix (PDC6I) and Hrs (HGS) as well as the ubiquitin ligase TAL (LSRM1). Thus, we identified those ESCRT proteins with an assigned functional interaction with Tsg101 UEV, whereas we did not find Vps37B or Tsg101 itself. Apart from the known interaction partners, we found a set of potentially new interaction partners (Table I). One functional annotation class comprises proteins involved in mRNA processing/turnover, as for example Caprin-1 and PABP1, whereas other proteins found in our screen are annotated in the context of NFκB signaling or COPII-mediated transport.

To further confirm the finding that Tsg101 UEV interacts with A(T/S)AP as well as with P(T/S)AP motifs, we chose two proteins identified by the SILAC approach that contain ASAP motifs and belong to the largest functional cluster of proteins associated with mRNA regulation. 9-mer peptides of Caprin-1 and CPSF7 spanning the motif were analyzed for binding to Tsg101 UEV by NMR. Fig. 3A shows that both proteins bound essentially to the same interface as the HIV-1-p6 peptide. In contrast, binding of these peptides to the UEV M95A mutant was almost completely abolished (supplemental Fig. S2).

Fig. 3.
Chemical shift perturbations of the Tsg101 UEV domain upon binding peptide sequences from selected hits. A shows an overlay of 1H-15N HSQC spectra recorded from the Tsg101 UEV domain alone (cyan) and with one of the peptides, HIV-1-p6-(5–13) ( ...

The third identified protein known to interact with Tsg101 UEV in a PTAP-dependent fashion (20) is the ubiquitin ligase TAL. It has also been shown that TAL polyubiquitinates Tsg101 itself, thereby maintaining low Tsg101 levels when not associated with the ESCRT machinery (21). This requires a tight association of the two proteins, and we were interested in whether the affinities of the two proteins are in the range that allows TAL to monitor low levels of free Tsg101. NMR and ITC measurements were performed to address these questions more thoroughly.

Tsg101-TAL Interaction

The 15N-labeled UEV domain was used in NMR titration experiments with the long TAL peptide containing both putative interaction motifs (Fig. 3, B and C). For comparison, peptides with each of the motifs mutated to alanine were used. Comparison of the respective spectra allowed us to assign the shifts of individual resonances with regard to one of the individual motifs. Interestingly when adding wild type peptide, individual populations of UEV-bound peptide could be identified because chemical exchange is slow in relation to the NMR time scale (Fig. 3B). Peak intensities were averaged for several resonances and showed that ~75% of UEV is bound to the second motif, whereas 25% interacts with the first PTAP sequence.

ITC was performed to determine the thermodynamic parameters that drive complex formation. Fig. 4 shows the corresponding titrations from which the dissociation constants for the single motifs and an estimation of the affinity of the double motif were derived. The peptide containing the second PSAP motif bound with a KD of 62 μM, whereas the first motif (PTAP) had a considerably higher value of 344 μM. The peptide containing both motifs revealed an apparent dissociation constant of 72 μM (data not shown). On the basis of the NMR results that showed two clearly distinguishable interaction sites, this value represents the composite binding of the UEV domain to the individual motifs.

Fig. 4.
ITC of the UEV-TAL complex at 25 °C. Tsg101 UEV was titrated with either of the two TAL single motif mutants: TAL-(645–668)mut2 (A) or TAL-(645–668)mut1 (B). Shown is the differential heating power, Δp, versus time, t. ...

Interaction of Tsg101 with PABP1 and eIF4b

To confirm the results of the UEV domain-based pulldown experiments in the context of full-length Tsg101 we probed two of the novel and functionally interesting interaction partners by immunoprecipitation. Specifically PABP1 and eIF4b, two proteins involved in mRNA translational control, were detected in pulldown experiments with HA-tagged Tsg101 (Fig. 5A). Clearly for the M95A mutant levels of PABP1 and eIF4b were reduced to background levels that are comparable to the amount of the well known interaction partner Alix. Subsequently YFP-Tsg101 and FLAG-eIF4b (Fig. 5B) or HA-Tsg101 and GFP-PABP1 (Fig. 5C) were co-transfected into HeLa cells. We observed co-precipitation when using either of the two respective proteins as bait, whereas the M95A mutant of Tsg101 showed almost no binding. Taken together these results define PABP1 and eIF4b as two new interaction partners for Tsg101.

Fig. 5.
Immunoprecipitations and co-immunoprecipitations of Tsg101 with PABP1 and eIF4b. A, Western blot of HeLa cells that were transiently transfected with HA-Tsg101 (wt or the M95A mutant). Membranes were incubated with the indicated antibodies to probe for ...

DISCUSSION

The assigned function of Tsg101 in ESCRT-dependent endosomal sorting (5, 10, 11), viral budding (33), and cytokinesis, (17, 18) 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 (25). 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 (8) 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 (34) and contributes to cell cycle- or stress-dependent regulation of translation (35). Recently eIF4b has also been shown to be regulated by 14-3-3σ (36), 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 (37). eIF4b and PABP1 have been shown to interact with each other (38), and reduced levels of these two proteins lead to stress granule formation in HeLa cells (39). 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 (17, 18) 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 (36). 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 (40), 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 (23). 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 (41). 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. (42). 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.

Fig. 6.
Functional clusters of proteins identified as potential interaction partners of Tsg101 UEV (the crystal structure shown refers to the Protein Data Bank entry 2F0R (47). Proteins containing (P/A)(T/S)AP motifs are colored red, whereas others are kept in ...

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 (43, 44). 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 (45), 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 (46). 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 (48) 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.

Supplementary Material

[Supplemental Data]

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.

Footnotes

* This work was supported by Deutsche Forschungsgemeinschaft Grants FG806, SFB740, and SFB765.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpg The on-line version of this article (available at http://www.mcponline.org) contains supplemental Figs. S1 and S2, Tables S1–S6, and methods.

2 R. Kuehne, personal communication

1 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 fluorescent protein
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

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