SUMO Protocols pp 201-210 | Cite as

Preparation of Sumoylated Substrates for Biochemical Analysis

  • Puck Knipscheer
  • Helene Klug
  • Titia K. Sixma
  • Andrea Pichler
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 497)


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.

Key words

SUMO E2-25K in vitro sumoylation assay with recombinant enzymes protein purification 



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.


  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    Hay, R. T. (2005) SUMO: a history of modification. Mol. Cell 18, 1–12.PubMedCrossRefGoogle Scholar
  3. 3.
    Johnson, E. S. (2004) Protein modification by SUMO. Annu. Rev. Biochem. 73, 355–382.PubMedCrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    Reverter, D., and Lima, C. D. (2005) Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex. Nature 435, 687–692.PubMedCrossRefGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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.PubMedCrossRefGoogle Scholar
  17. 17.
    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.PubMedCrossRefGoogle Scholar
  18. 18.
    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.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    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.PubMedCrossRefGoogle Scholar
  21. 21.
    Pichler A. (2008) Posttranslational modification of proteins — Analysis of Sumoylation. Methods in Molecular Biology. 446, 131–8.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Puck Knipscheer
    • 1
  • Helene Klug
    • 2
  • Titia K. Sixma
    • 3
  • Andrea Pichler
  1. 1.Department of Biological Chemisty and Molecular PharmacologyHarvard Medical SchoolBostonUSA
  2. 2.Max F. Perutz LaboratoriesMedical University of ViennaAustria
  3. 3.The Netherlands Cancer Institute and Center for Biomedical GeneticsAmsterdamThe Netherlands

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