Abstract
Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via “loss of modification” analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a “gain in modification” analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.
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References
Kerscher, O., Felberbaum, R., and Hoch-strasser, M. (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell. Dev. Biol. 22, 159–180.
Hay, R. T. (2005) SUMO: a history of modification. Mol. Cell 18, 1–12.
Johnson, E. S. (2004) Protein modification by SUMO. Annu. Rev. Biochem. 73, 355–382.
Pichler, A., Knipscheer, P., Saitoh, H., Sixma, T. K., and Melchior, F. (2004) The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type. Nat. Struct. Mol. Biol. 11, 984–991.
Hannich, J. T., Lewis, A., Kroetz, M. B., Li, S. J., Heide, H., Emili, A., and Hoch-strasser, M. (2005) Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. J. Biol. Chem. 280, 4102–4110.
Hecker, C. M., Rabiller, M., Haglund, K., Bayer, P., and Dikic, I. (2006) Specification of SUMO1- and SUMO2-interacting motifs. J. Biol. Chem. 281, 16117–16127.
Minty, A., Dumont, X., Kaghad, M., and Caput, D. (2000) Covalent modification of p73alpha by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-inter-acting proteins and a SUMO-1 interaction motif. J. Biol. Chem. 275, 36316–36323.
Song, J., Durrin, L. K., Wilkinson, T. A., Krontiris, T. G., and Chen, Y. (2004) Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl. Acad. Sci. U S A 101, 14373–14378.
Song, J., Zhang, Z., Hu, W., and Chen, Y. (2005) Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J. Biol. Chem. 280, 40122–40129.
Mahajan, R., Delphin, C., Guan, T., Gerace, L., and Melchior, F. (1997) A small ubiqui-tin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97–107.
Mahajan, R., Gerace, L., and Melchior, F. (1998) Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association. J. Cell. Biol. 140, 259–270.
Matunis, M. J., Coutavas, E., and Blobel, G. (1996) A novel ubiquitin-like modifi- cation modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell. Biol. 135, 1457–1470.
Matunis, M. J., Wu, J., and Blobel, G. (1998) SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex. J. Cell. Biol. 140, 499–509.
Reverter, D., and Lima, C. D. (2005) Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex. Nature 435, 687–692.
Shen, T. H., Lin, H. K., Scaglioni, P. P., Yung, T. M., and Pandolfi, P. P. (2006) The mechanisms of PML-nuclear body formation. Mol. Cell 24, 331–339.
Lin, D. Y., Huang, Y. S., Jeng, J. C., Kuo, H. Y., Chang, C. C., Chao, T. T., Ho, C. C., Chen, Y. C., Lin, T. P., Fang, H. I., Hung, C. C., Suen, C. S., Hwang, M. J., Chang, K. S., Maul, G. G., and Shih, H. M. (2006) Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol. Cell 24, 341–354.
Baba, D., Maita, N., Jee, J. G., Uchimura, Y., Saitoh, H., Sugasawa, K., Hanaoka, F., Tochio, H., Hiroaki, H., and Shirakawa, M. (2005) Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature 435, 979–982.
Pichler, A., Knipscheer, P., Oberhofer, E., van Dijk, W. J., Korner, R., Olsen, J. V., Jentsch, S., Melchior, F., and Sixma, T. K. (2005) SUMO modification of the ubiq-uitin-conjugating enzyme E2–25K. Nat. Struct. Mol. Biol. 12, 264–269.
Pichler, A., Gast, A., Seeler, J. S., Dejean, A., and Melchior, F. (2002) The nucleop-orin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109–120.
Bossis, G., Chmielarska, K., Gartner, U., Pichler, A., Stieger, E., and Melchior, F. (2005) A fluorescence resonance energy transfer-based assay to study SUMO modification in solution. Methods Enzymol. 398, 20–32.
Pichler A. (2008) Posttranslational modification of proteins — Analysis of Sumoylation. Methods in Molecular Biology. 446, 131–8.
Acknowledgments
Our special thanks go to Katharina Maderböck and Neha Nigam for critical reading of the manuscript. This work was funded by the Vienna Science and Technology Fund WWTF LS05003 and FWF P18584-B12 to A.P., and EU-Rubicon, NWO-CW pionier and CBG to T.K.S.
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Knipscheer, P., Klug, H., Sixma, T.K., Pichler, A. (2009). Preparation of Sumoylated Substrates for Biochemical Analysis. In: Ulrich, H.D. (eds) SUMO Protocols. METHODS IN MOLECULAR BIOLOGY™, vol 497. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-566-4_13
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DOI: https://doi.org/10.1007/978-1-59745-566-4_13
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