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, NFkappaB2, 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.
Highlights
The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis
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 ubiquitin-E2-like variant (UEV) domain [20, 21]
This definition was slightly expanded by the finding that the UEV-Hrs interaction might involve PSMP and PSGP motifs, the structure of the UEV-HIV-1-p6 5PEPTAPPEE13 complex indicates that the 9AP10 dipeptide is crucial to the interaction [31]
Summary
The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. Tsg101 is an essential protein involved in cancerogenesis [1], cell cycle progression [2, 3], transcription regulation [4], From the ‡Protein Engineering Group, Leibniz Institute for Molecular Pharmacology and Freie Universitat Berlin, Robert-Rossle-Strasse 10, 13125 Berlin, Germany, and ¶Mass Spectrometry and §Biophysics of Membrane Proteins Groups, Leibniz Institute for Molecular Pharmacology, Robert-Rossle-Strasse 10, 13125 Berlin, Germany 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. 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]. Direct interactions of these two proteins enhance mRNA translation by facilitating 5Ј- to 3Ј-end communication of mRNA, and Tsg101 conceivably modulates translation by marking these two proteins for intracellular transport
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