Abstract
Protein SUMOylation is a posttranslational protein modification that is emerging as a key regulatory process in neurobiology. To date, however, SUMOylation in vivo has only been studied cursorily. Knock-in mice expressing His6-HA-SUMO1 from the Sumo1 locus allow for the highly specific localization and identification of endogenous SUMO1 substrates under physiological and pathophysiological conditions. By making use of the HA-tag and using wild-type mice for highly stringent negative control samples, SUMO1 targets can be specifically localized in and purified from cultured mouse nerve cells and mouse tissues.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Flotho A, Melchior F (2013) Sumoylation: a regulatory protein modification in health and disease. Annu Rev Biochem 82:357–385
Psakhye I, Jentsch S (2012) Protein group modification and synergy in the SUMO pathway as exemplified in DNA repair. Cell 151:807–820
Jentsch S, Psakhye I (2013) Control of nuclear activities by substrate-selective and protein-group SUMOylation. Annu Rev Genet 47:167–186
Gareau JR, Lima CD (2010) The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition. Nat Rev Mol Cell Biol 11:861–871
Barysch SV, Dittner C, Flotho A, Becker J, Melchior F (2014) Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies. Nat Protoc 9:896–909
Pichler A (2008) Analysis of sumoylation. Methods Mol Biol 446:131–138
Hendriks IA, D’Souza RC, Chang JG, Mann M, Vertegaal AC (2015) System-wide identification of wild-type SUMO-2 conjugation sites. Nat Commun 6:7289
Niedenthal R (2009) Enhanced detection of in vivo SUMO conjugation by Ubc9 fusion-dependent sumoylation (UFDS). Methods Mol Biol 497:63–79
Lang V, Aillet F, Da Silva-Ferrada E, Xolalpa W, Zabaleta L, Rivas C, Rodriguez MS (2015) Analysis of PTEN ubiquitylation and SUMOylation using molecular traps. Methods 77–78:112–118
Da Silva-Ferrada E, Xolalpa W, Lang V, Aillet F, Martin-Ruiz I, de la Cruz-Herrera CF, Lopitz-Otsoa F, Carracedo A, Goldenberg SJ, Rivas C, England P, Rodriguez MS (2013) Analysis of SUMOylated proteins using SUMO-traps. Sci Rep 3:1690
Rosas-Acosta G, Russell WK, Deyrieux A, Russell DH, Wilson VG (2005) A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers. Mol Cell Proteomics 4:56–72
Tatham MH, Matic I, Mann M, Hay RT (2011) Comparative proteomic analysis identifies a role for SUMO in protein quality control. Sci Signal 4:rs4. doi: 10.1126/scisignal.2001484
Matic I, van Hagen M, Schimmel J, Macek B, Ogg SC, Tatham MH, Hay RT, Lamond AI, Mann M, Vertegaal AC (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
Matsuzaki S, Lee L, Knock E, Srikumar T, Sakurai M, Hazrati LN, Katayama T, Staniszewski A, Raught B, Arancio O, Fraser PE (2015) SUMO1 affects synaptic function, spine density and memory. Sci Rep 5:10730
Tirard M, Hsiao HH, Nikolov M, Urlaub H, Melchior F, Brose N (2012) In vivo localization and identification of SUMOylated proteins in the brain of His6-HA-SUMO1 knock-in mice. Proc Natl Acad Sci U S A 109:21122–21127
Rossner MJ, Tirard M (2014) Thy1.2 driven expression of transgenic His(6)-SUMO2 in the brain of mice alters a restricted set of genes. Brain Res 1575:1–11
Yang W, Sheng H, Thompson JW, Zhao S, Wang L, Miao P, Liu X, Moseley MA, Paschen W (2014) Small ubiquitin-like modifier 3-modified proteome regulated by brain ischemia in novel small ubiquitin-like modifier transgenic mice: putative protective proteins/pathways. Stroke 45:1115
Wang L, Rodriguiz RM, Wetsel WC, Sheng H, Zhao S, Liu X, Paschen W, Yang W (2014) Neuron-specific Sumo1-3 knockdown in mice impairs episodic and fear memories. J Psychiatry Neurosci 39:259–266
Lee YJ, Mou Y, Maric D, Klimanis D, Auh S, Hallenbeck JM (2011) Elevated global SUMOylation in Ubc9 transgenic mice protects their brains against focal cerebral ischemic damage. PLoS One 6:e25852. doi: 10.1371/journal.pone.0025852
Zhang FP, Mikkonen L, Toppari J, Palvimo JJ, Thesleff I, Janne OA (2008) Sumo-1 function is dispensable in normal mouse development. Mol Cell Biol 28:5381–5390
Wang L, Wansleeben C, Zhao S, Miao P, Paschen W, Yang W (2014) SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development. EMBO Rep 15:878–885
Hsiao HH, Meulmeester E, Urlaub H (2012) Identification of endogenous SUMO1 accepter sites by mass spectrometry. Methods Mol Biol 893:431–441
Peng Y, Xiong WC, Mei L (2013) Culture of dissociated hippocampal neurons. Methods Mol Biol 1018:39–47
Gage GJ, Kipke DR, Shain W (2012) Whole animal perfusion fixation for rodents. J Vis Exp (65). doi: 10.3791/3564
Krumova P, Meulmeester E, Garrido M, Tirard M, Hsiao HH, Bossis G, Urlaub H, Zweckstetter M, Kugler S, Melchior F, Bahr M, Weishaupt JH (2011) Sumoylation inhibits alpha-synuclein aggregation and toxicity. J Cell Biol 194:49–60
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Tirard, M., Brose, N. (2016). Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1. 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_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6358-4_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6356-0
Online ISBN: 978-1-4939-6358-4
eBook Packages: Springer Protocols