Abstract
Covalent modification of proteins by small ubiquitin-like modifier (SUMO) regulates diverse cellular processes. While many SUMO substrates are identified through individual efforts, affinity-based approaches followed by mass spectrometry are also used to identify in vivo SUMO substrates in yeast and in mammals. Because of low steady-state levels of sumoylation and biases towards abundant targets, identifying sumoylated proteins in vivo can be challenging. The in vitro expression cloning (IVEC) method for SUMO target identification circumvents these challenges and complements the affinity-based approaches. IVEC allows for immediate validation and analysis of substrates through in vitro reconstitution. Furthermore, this method can be easily adapted to identify substrates of specific SUMO ligases.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Johnson, E. S. (2004) Protein modification by SUMO. Annu. Rev. Biochem. 73, 355–382.
Seeler, J. S., and Dejean, A. (2003) Nuclear and unclear functions of SUMO. Nat. Rev. Mol. Cell Biol. 4, 690–699.
Melchior, F., Schergaut, M., and Pichler, A. (2003) SUMO: ligases, isopeptidases and nuclear pores. Trends Biochem. Sci. 28, 612–618.
Muller, S., Ledl, A., and Schmidt, D. (2004) SUMO: a regulator of gene expression and genome integrity. Oncogene 23, 1998–2008.
Li, T., Evdokimov, E., Shen, R. F., Chao, C. C., Tekle, E., Wang, T., Stadtman, E. R., Yang, D. C., and Chock, P. B. (2004) Sumoylation of heterogeneous nuclear ribo-nucleoproteins, zinc finger proteins, and nuclear pore complex proteins: A proteomic analysis. Proc. Natl. Acad. Sci. USA101, 8551–8556.
Panse, V. G., Hardeland, U., Werner, T., Kuster, B., and Hurt, E. (2004) A Proteome-wide Approach Identifies Sumoylated Substrate Proteins in Yeast. J. Biol. Chem.279, 41346–41351.
Vertegaal, A. C., Ogg, S. C., Jaffray, E., Rodriguez, M. S., Hay, R. T., Andersen, J. S., Mann, M., and Lamond, A. I. (2004) A pro-teomic study of SUMO-2 target proteins. J. Biol. Chem. 279, 33791–33798.
Wohlschlegel, J. A., Johnson, E. S., Reed, S. I., and Yates, J. R., 3rd (2004) Global analysis of protein sumoylation in Saccharo-myces cerevisiae. J. Biol. Chem. 279, 45662–45668.
Zhao, Y., Kwon, S. W., Anselmo, A., Kaur, K., and White, M. A. (2004) Broad spectrum identification of cellular small ubiquitin- related modifier (SUMO) substrate proins. J. Biol. Chem. 279, 20999–21002.
Zhou, W., Ryan, J. J., and Zhou, H. (2004) Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. J. Biol. Chem. 279, 32262–32268.
Lustig, K. D., Stukenberg, P. T., McGarry, T. J., King, R. W., Cryns, V. L., Mead, P. E., Zon, L. I., Yuan, J., and Kirschner, M. W. (1997) Small pool expression screening: identification of genes involved in cell cycle control, apoptosis, and early development. Methods Enzymol. 283, 83–99.
Stukenberg, P. T., Lustig, K. D., McGarry, T. J., King, R. W., Kuang, J., and Kirschner, M. W. (1997) Systematic identification of mitotic phosphoproteins. Curr. Biol. 7, 338–348.
McGarry, T. J., and Kirschner, M. W. (1998) Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043–1053.
Cryns, V. L., Byun, Y., Rana, A., Mellor, H., Lustig, K. D., Ghanem, L., Parker, P. J., Kirschner, M. W., and Yuan, J. (1997) Specific proteolysis of the kinase protein kinase C-related kinase 2 by caspase-3 during apop-tosis. Identification by a novel, small pool expression cloning strategy. J. Biol. Chem. 272, 29449–29453.
Gocke, C., Yu, H., and Kang, J. (2004) Systematic identification and analysis of mammalian small ubiquitin-like modifier substrates. J. Biol. Chem. 280, 5004–5012.
Sampson, D. A., Wang, M., and Matunis, M. J. (2001) The small ubiquitin-like modi-fier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J. Biol. Chem. 276, 21664–21669.
Acknowledgments
We thank Dr. Jungseog Kang for assistance in developing the IVEC SUMO screen and Joshua Bembenek for the purification of SUMO enzymes. We also thank Frauke Melchior, Kim Orth, and Stefan Muller for providing expression constructs and purification protocols of SUMO enzymes.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gocke, C.B., Yu, H. (2009). Identification of SUMO Targets Through In Vitro Expression Cloning. 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_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-566-4_4
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-80-0
Online ISBN: 978-1-59745-566-4
eBook Packages: Springer Protocols