Summary
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 involved in regulation of protein function, nuclear targeting, and the formation of subcellular structures. Because SUMO is involved in such a plethora of functions and modifies numerous proteins it is important to identify proteins that are sumoylated in order to increase our understanding of how this modification affects protein function and localization. This overview describes techniques utilized for the detection of sumoylated proteins. The techniques covered include immunoprecipitation, an in vitro sumoylation assay, and gel shift mobility assays that have been used to identify SUMO-modified proteins.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Mahajan, R., Delphin, C., Guan, T., Gerace, L., and Melchior, F. (1997) A small ubiquitinrelated polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97–107.
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.
Verger, A., Perdomo, J., and Crossley, M. (2003) Modification with SUMO. EMBO Rep. 4, 137–42.
Kim, K. I., Baek, S. H., and Chung, C. H. (2002) Versatile protein tag, SUMO: its enzymology and biological function. J. Cell Physiol. 191, 257–268.
Sarge, K. D., Murphy, S. P., and Morimoto, R. I. (1993) Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress. Mol. Cell Biol. 13, 1392–1407.
Matunis, M. J., Coutavas, E., and Blobel, G. (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J. Cell Biol. 135, 1457–1470.
Desterro, J. M., Thomson, J., and Hay, R. T. (1997) Ubch9 conjugates SUMO but not ubiquitin. FEBS Lett. 417, 297–300.
Sambrook, J. and Russell, D. (2001) Molecular Cloning, A Laboratory Manual, 3rd edit. 3 vols, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Jaffray, E., Wood, K. M., and Hay, R. T. (1995) Domain organization of I kappa B alpha and sites of interaction with NF-kappa B p65. Mol. Cell Biol. 15, 2166–2172.
Tatham, M. H., Jaffray, E., Vaughan, O. A., et al. (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.
Hong, Y., Rogers, R., Matunis, M. J., et al. (2001) Regulation of heat shock transcription factor 1 by stress-induced SUMO-1 modification. J. Biol. Chem. 276, 40263–40267.
Desterro, J. M., Rodriguez, M. S., and Hay, R. T. (1998) SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. Mol. Cell 2, 233–239.
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 (HSF2), a promyelocytic leukemia nuclear body associated transcription factor. J. Biol. Chem. 276, 18,513–18,518.
Acknowledgments
The authors are very grateful to Mike Matunis (Johns Hopkins) for many reagents and advice on the in vitro sumoylation assay, to Chris Lima (Weill Medical College of Cornell University) for his generous gift of constructs encoding recombinant E1 heterodimers, and to the Journal of Biological Chemistry for allowing us to reprint figures from two of our papers (11,13). This work was supported by NIH grants GM61053 and GM64606 to K.D.S.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Hilgarth, R.S., Sarge, K.D. (2005). Detection of Sumoylated Proteins. In: Patterson, C., Cyr, D.M. (eds) Ubiquitin-Proteasome Protocols. Methods in Molecular Biology™, vol 301. Humana Press. https://doi.org/10.1385/1-59259-895-1:329
Download citation
DOI: https://doi.org/10.1385/1-59259-895-1:329
Publisher Name: Humana Press
Print ISBN: 978-1-58829-252-0
Online ISBN: 978-1-59259-895-3
eBook Packages: Springer Protocols