title: A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation.
abstract: Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays. These assays provide a fast readout for in vitro SUMO chain formation activity of E3 ligases in a paralog-specific manner. Finally, we critically analyze the application of fluorescent SUMO proteins to study substrate modification in vitro revealing also the drawbacks of the system.
authors.affiliation: Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108, Freiburg, Germany.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Eisenhardt
authors.affiliation: Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108, Freiburg, Germany.|MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, DD1 5EH, Dundee, UK.
authors.forename: Viduth K
authors.initials: VK
authors.lastname: Chaugule
authors.affiliation: Department of Epigenetics, Max Planck Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108, Freiburg, Germany. pichler@ie-freiburg.mpg.de.
authors.forename: Andrea
authors.initials: A
authors.lastname: Pichler
mesh_terms: D001681:Biological Assay; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D050828:Protein Inhibitors of Activated STAT / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling / Q000379:methods; D058207:Sumoylation; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D005456:Fluorescent Dyes; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; C106529:PIAS1 protein, human; D050828:Protein Inhibitors of Activated STAT; D011994:Recombinant Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins; C094493:ran-binding protein 2; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
keywords: E3 ligase; In vitro sumoylation; PIAS1; RanBP2; SUMO; SUMO chains; SUMO protease; Sp100; ZNF451