title: Analysis of SUMOylated Proteins in Cells and In Vivo Using the bioSUMO Strategy.
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.
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Bizkaia, 48160, Spain.
authors.forename: Lucia
authors.initials: L
authors.lastname: Pirone
authors.affiliation: Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, 62100, Mexico.
authors.forename: Wendy
authors.initials: W
authors.lastname: Xolalpa
authors.affiliation: Biokimika eta Biologia Molekularra Saila, Zientzia eta Teknologia Fakultatea, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, 48940, Spain.|Ikerbasque, Basque Foundation for Science, Bilbao, Bizkaia, 48013, Spain.
authors.forename: Ugo
authors.initials: U
authors.lastname: Mayor
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Bizkaia, 48160, Spain. rbarrio@cicbiogune.es.
authors.forename: Rosa
authors.initials: R
authors.lastname: Barrio
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Bizkaia, 48160, Spain. jsutherland@cicbiogune.es.
authors.forename: James D
authors.initials: JD
authors.lastname: Sutherland
mesh_terms: D000818:Animals; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007814:Larva / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; C557617:GAL4 protein, Drosophila; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; C490960:smt3 protein, Drosophila; D049452:Green Fluorescent Proteins; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli
keywords: Biotin; BirA; Drosophila; Mass spectrometry; SUMOylation