Abstract
SUMOylation is a widely used protein posttranslational mechanism capable of regulating substrates localization, stability, and/or activity. Identification and characterization of bona fide SUMO substrates is a laborious task but its discovery can shed light to exquisite and crucial regulatory signaling events occurring within the cell. Experiments performed in the SUMOylation field often demand a good understanding of the putative substrate’s function and necessitate a solid knowledge regarding both in vitro and in vivo approaches. This contribution offers a simplified view into some of the most common experiments performed in biochemical and cell biological research of the SUMO pathway in mammalian systems. It also summarizes and updates well established protocols and tricks in order to improve the likelihood to obtain reliable and reproducible results.
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References
Hay RT (2013) Decoding the SUMO signal. Biochem Soc Trans 41:463–473
Tang Z, Hecker CM, Scheschonka A et al (2008) Protein interactions in the sumoylation cascade—lessons from X-ray structures. FEBS J 275:3003–3015
Wang J, Taherbhoy AM, Hunt HW et al (2010) Crystal structure of UBA2ufd-Ubc9: insights into E1-E2 interactions in sumo pathways. PLoS One 5:e15805
O’Brien SP, DeLisa MP (2012) Functional reconstitution of a tunable E3-dependent sumoylation pathway in Escherichia coli. PLoS One 7:e38671
Hickey CM, Wilson NR, Hochstrasser M (2012) Function and regulation of SUMO proteases. Nat Rev Mol Cell Biol 13:755–766
Song J, Durrin LK, Wilkinson TA et al (2004) Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc Natl Acad Sci U S A 101:14373–14378
Namanja AT, Li YJ, Su Y et al (2012) Insights into high affinity small ubiquitin-like modifier (SUMO) recognition by SUMO-interacting motifs (SIMs) revealed by a combination of NMR and peptide array analysis. J Biol Chem 287:3231–3240
Kerscher O (2007) SUMO junction-what’s your function? New insights through SUMO-interacting motifs. EMBO Rep 8:550–555
Da Silva-Ferrada E, Lopitz-Otsoa F, Lang V et al (2012) Strategies to identify recognition signals and targets of SUMOylation. Biochem Res Int 2012:875148
Mencía M, De Lorenzo V (2004) Functional transplantation of the sumoylation machinery into Escherichia coli. Protein Expr Purif 37:409–418
Da Silva-Ferrada E, Xolalpa W, Lang V et al (2013) Analysis of SUMOylated proteins using SUMO-traps. Sci Rep 3:1690
Uchimura Y, Nakao M, Saitoh H (2004) Generation of SUMO-1 modified proteins in E. coli: towards understanding the biochemistry/structural biology of the SUMO-1 pathway. FEBS Lett 564:85–90
Okada S, Nagabuchi M, Takamura Y et al (2009) Reconstitution of Arabidopsis thaliana SUMO pathways in E. coli: functional evaluation of SUMO machinery proteins and mapping of SUMOylation sites by mass spectrometry. Plant Cell Physiol 50:1049–1061
Acknowledgements
Research performed at the laboratory of Pathophysiological Cell Signaling is funded by the following bodies: FWO (G0C7514N grant), BELSPO (IAP-VII/07 program), VUB Research Council (new PI grant), and Innoviris (Brains Back to Brussels program to GJG). CC thanks financial support from the European Union’s Seventh Framework Program (FP7) 2007–2013 under grant agreement no. 264257. The authors would also like to acknowledge networking support by the Proteostasis COST Action (BM1307).
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Cedeño, C., La Monaca, E., Esposito, M., Gutierrez, G.J. (2016). Detection and Analysis of SUMOylation Substrates In Vitro and In Vivo. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_16
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DOI: https://doi.org/10.1007/978-1-4939-3756-1_16
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