Abstract
Posttranslational modification of proteins with the small ubiquitin-related modifier (SUMO) has been implicated in many important physiological functions, including the regulation of transcription and DNA repair. In most cases, only a small fraction of the total cellular amounts of a given protein is sumoylated at a certain point in time. Sensitive detection of sumoylated forms of proteins by western blotting is, therefore, an important step in the identification and/or characterization of a protein control by sumoylation. Polysumoylated proteins are recognized and targeted to the proteasome by specific ubiquitin ligases bearing SUMO interaction motifs. Sumoylation itself is reversible by the action of desumoylating enzymes. Their activities cause a rapid loss of SUMO conjugates in most standard cell extracts. To preserve SUMO–protein conjugates, therefore, a preparation of extracts under denaturing conditions is recommended. Here, we describe the application of an alkaline lysis procedure and a western blot protocol for the analysis of SUMO conjugates in yeast and human cells. In addition, we describe the application of another extraction procedure combined with immobilized metal affinity chromatography for the analysis of ubiquitin–SUMO hybrid conjugates from yeast and human cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Johnson ES (2004) Protein modification by SUMO. Annu Rev Biochem 73:355–382.
Dohmen RJ (2004) SUMO protein modification. Biochim Biophys Acta 1695:113–131.
Hay RT (2005) SUMO: a history of modification. Mol Cell 18:1–12.
Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. Nature 398:246–251.
Hay RT (2007) SUMO-specific proteases: a twist in the tail. Trends Cell Biol 17:370–376.
Mukhopadhyay D, Dasso M (2007) Modification in reverse: the SUMO proteases. Trends Biochem Sci 32:286–295.
Yeh ET (2009) SUMOylation and De-SUMOylation: wrestling with life’s processes. J Biol Chem 284:8223–8227.
Uzunova K, Gottsche K, Miteva M et al (2007) Ubiquitin-dependent proteolytic control of SUMO conjugates. J Biol Chem 282:34167–34175.
Prudden J, Pebernard S, Raffa G et al (2007) SUMO-targeted ubiquitin ligases in genome stability. EMBO J 26:4089–4101.
Sun H, Leverson JD, Hunter T (2007) Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO J 26:4102–4112.
Xie Y, Kerscher O, Kroetz MB et al (2007) The yeast HEX3-SLX8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation. J Biol Chem 282: 34176–34184.
Tatham MH, Geoffroy MC, Shen L et al (2008) RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation. Nat Cell Biol 10:538–546.
Tatham MH, Jaffray E, Vaughan OA 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.
Weisshaar SR, Keusekotten K, Krause A et al (2008) Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML. FEBS Lett 582:3174–3178.
Matic I, van Hagen M, Schimmel J et al (2008) In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy. Mol Cell Proteomics 7:132–144.
Miteva M, Keusekotten K, Hofmann K et al (2010) Sumoylation as a signal for polyubiquitylation and proteasomal degradation. Subcell Biochem 54:195–214.
Johnson ES, Blobel G (1999) Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins. J Cell Biol 147:981–994.
Bylebyl GR, Belichenko I, Johnson ES (2003) The SUMO isopeptidase Ulp2 prevents accumulation of SUMO chains in yeast. J Biol Chem 278:44112–44120.
Boutell C, Orr A, Everett RD (2003) PML residue lysine 160 is required for the degradation of PML induced by herpes simplex virus type 1 regulatory protein ICP0. J Virol 77:8686–8694.
Acknowledgments
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB635 and SPP1365) to GJKP and RJD.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Schnellhardt, M. et al. (2012). Analysis of Cellular SUMO and SUMO–Ubiquitin Hybrid Conjugates. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_4
Download citation
DOI: https://doi.org/10.1007/978-1-61779-474-2_4
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-473-5
Online ISBN: 978-1-61779-474-2
eBook Packages: Springer Protocols