title: A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation.
abstract: Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: Lucia
authors.initials: L
authors.lastname: Pirone
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: Wendy
authors.initials: W
authors.lastname: Xolalpa
authors.affiliation: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark.
authors.forename: Jón Otti
authors.initials: JO
authors.lastname: Sigurðsson
authors.affiliation: Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain.
authors.forename: Juanma
authors.initials: J
authors.lastname: Ramirez
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: Coralia
authors.initials: C
authors.lastname: Pérez
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: Monika
authors.initials: M
authors.lastname: González
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: Ainara Ruiz
authors.initials: AR
authors.lastname: de Sabando
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: Félix
authors.initials: F
authors.lastname: Elortza
authors.affiliation: ITAV, IPBS, Université de Toulouse, CNRS, UPS, 1 Place Pierre Potier Oncopole entrée B, BP 50624, 31106 Toulouse Cedex 1, France.
authors.forename: Manuel S
authors.initials: MS
authors.lastname: Rodriguez
authors.affiliation: Biochemistry and Molecular Biology Department, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain.|Ikerbasque, Basque Foundation for Science, Alameda Urquijo, 36-5 Plaza Bizkaia, 48011 Bilbao, Spain.
authors.forename: Ugo
authors.initials: U
authors.lastname: Mayor
authors.affiliation: Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200 Copenhagen, Denmark.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Barrio
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
authors.forename: James D
authors.initials: JD
authors.lastname: Sutherland
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019921:Biotinylation; D002460:Cell Line; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011499:Protein Processing, Post-Translational; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins; D058207:Sumoylation; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D012097:Repressor Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins; C490960:smt3 protein, Drosophila