Abstract
Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating a variety of different protein functional properties, including protein-protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as of nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Because sumoylation plays an important role in regulating so many important cellular processes, there has been intense interest in identifying new proteins that are targets of this modification and determining what role sumoylation plays in regulating the protein functions. This chapter presents methodologies for determining whether a particular protein is a substrate of sumoylation, and for identifying the lysine residue(s) where the modification occurs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mahajan, R., Delphin, C., Guan, T., Gerace, L., and Melchior, F. (1997) A small ubiquitin related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell. 88, 97-107
Matunis, M.J., Wu, J., 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
Bossis, G. and Melchior, F. (2006) SUMO: regulating the regulator. Cell Div. Jun 29, 1-13
Kerscher, O., Felberbaum, R., and Hochstrasser, 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., Schwienhorst, I., Dohmen, R.J., and Blobel, G. (1997) The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 16, 5509-5519
Desterro, J.M., Rodriguez, M.S., Kemp, G.D., and Hay, R.T. (1999) Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1. J. Biol. Chem. 274, 10618-10624
Gong, L., Li, B., Millas, S., and Yeh, E.T. (1999) Molecular cloning and characterization of human AOS1 and UBA2, components of the sentrin-activating enzyme complex. FEBS Lett. 448, 185-189
Okuma, T., Honda, R., Ichikawa, G., Tsumagari, N., and Yasuda, H. (1999) In vitro SUMO-1 modification requires two enzymatic steps, E1 and E2. Biochem. Biophys. Res. Commun. 254, 693-698
Desterro, J.M., Thomson, J., Hay, R.T. (1997) Ubch9 conjugates SUMO but not ubiquitin. FEBS Lett. 417, 297-300
Johnson, E.S. and Blobel, G. (1997) Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. J. Biol. Chem. 272, 26799-26802
Rodriguez, M.S., Dargemont, C., and Hay, R.T. (2001) SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276, 12654-12659
Sampson, D.A., Wang, M., and Matunis, M.J. (2001) The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J. Biol. Chem. 276, 21664-21669
Tatham, M.H., Jaffray, E., Vaughan, O.A., Desterro, J.M., Botting, C.H., Naismith, J.H., and Hay, R.T. (2001) Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J. Biol. Chem. 276, 35368-35374
Goodson, M.L., Hong, Y., Rogers, R., Matunis, M.J., Park-Sarge, O.K., and Sarge, K.D. (2001) Sumo-1 modification regulates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor. J. Biol. Chem. 276, 18513-18518
Acknowledgments
We are very grateful to Mike Matunis (Johns Hopkins), Ron Hay (University of St. Andrews), and Chris Lima (Sloan Kettering Institute) for providing constructs and reagents.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Park-Sarge, OK., Sarge, K.D. (2008). Detection of Sumoylated Proteins. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 464. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-461-6_14
Download citation
DOI: https://doi.org/10.1007/978-1-60327-461-6_14
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-460-9
Online ISBN: 978-1-60327-461-6
eBook Packages: Springer Protocols