Abstract
Posttranslational regulation of proteins by conjugation of ubiquitin- and ubiquitin-like molecules is a common theme in almost every known biological pathway. SUMO (small ubiquitin-related modifier) is dynamically added and deleted from many cellular substrates to control activity, localization, and recruitment of other SUMO-recognizing protein complexes. The dynamic nature of this modification and its low abundance in resting cells make it challenging to study, with susceptibility to deSUMOylases further complicating its analysis. Here we describe bioSUMO, a general method to isolate and analyze SUMOylated proteins from cultured cells, using Drosophila as a highlighted example. The method also has been validated in transgenic flies, as well as human cells. SUMOylated substrates are labeled by in vivo biotinylation, which facilitates their subsequent purification using streptavidin-based affinity chromatography under stringent conditions and with very low background. The bioSUMO approach can be used to validate whether a specific protein is modified, or used to analyze an entire SUMO subproteome. If coupled to quantitative proteomics methods, it may reveal how the SUMO landscape changes with different stimuli, or in diverse cell or tissue types. This technique offers a complementary approach to study SUMO biology and we expect that the strategy can be extended to other ubiquitin-like proteins.
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Acknowledgments
We would like to acknowledge other colleagues who initiated us in the biotin-based protocols: Maribel Franco, Juanma Ramirez, Aitor Martinez, Benoit Lectez, and So Young Lee. We would also like to thank the excellent MS support received from Jesper Olsen, Jón Otti Sigurðsson (University of Copenhagen, DK) and Félix Elortza (CIC bioGUNE, Bizkaia, Spain). This chapter is based upon work from COST Action (PROTEOSTASIS BM1307), supported by COST (European Cooperation in Science and Technology). R.B. acknowledges the Spanish Ministry of Economy and Competitiveness grant (BFU2014-52282-P), the Consolider network (BFU2014-57703- REDC), the Department of Industry of the Basque Government, and the Bizkaia County. L.P. and R.B. acknowledge the UPStream ITN network (PITN-GA-2011-290257). U.M. acknowledges the Spanish Ministry of Economy and Competitiveness grant (SAF2013-44782-P).
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Pirone, L., Xolalpa, W., Mayor, U., Barrio, R., Sutherland, J.D. (2016). Analysis of SUMOylated Proteins in Cells and In Vivo Using the bioSUMO Strategy. In: Rodriguez, M. (eds) SUMO. Methods in Molecular Biology, vol 1475. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6358-4_12
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DOI: https://doi.org/10.1007/978-1-4939-6358-4_12
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