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.
A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
L. Pirone,Wendy Xolalpa,J. O. Sigurðsson,J. Ramírez,Coralia Pérez,Monika González,Ainara Ruiz de Sabando,F. Elortza,Manuel S. Rodríguez,U. Mayor,J. Olsen,R. Barrio,J. Sutherland
Published 2017 in Scientific Reports
ABSTRACT
PUBLICATION RECORD
- Publication year
2017
- Venue
Scientific Reports
- Publication date
2017-01-18
- Fields of study
Biology, Medicine, Chemistry
- Identifiers
- External record
- Source metadata
Semantic Scholar, PubMed
CITATION MAP
EXTRACTION MAP
CLAIMS
CONCEPTS
- bioubls
A modular toolkit for studying ubiquitin-like protein modifications through biotin-based purification in Drosophila and mammalian systems.
Aliases: bioUbL system
- bira
The E. coli biotin protein ligase used as the biotinylating enzyme in the reported system.
Aliases: E. coli biotin protein ligase, biotin ligase BirA
- denaturing purification
A purification workflow that uses denaturing conditions to keep proteins unfolded during enrichment.
Aliases: purification under denaturing conditions
- drosophila cells and transgenic flies
Drosophila-based experimental systems used to express bioUbL constructs and test the method in cells and whole animals.
Aliases: fly cells and transgenic flies
- in vivo biotinylation
Biotin labeling performed inside living cells or organisms using an enzymatic tagging system.
Aliases: biotinylation in vivo
- mammalian cells
Mammalian cellular systems used to evaluate bioUbL conjugation and localization across different ubiquitin-like proteins.
Aliases: mammalian cell systems
- multicistronic expression
A gene-expression design in which multiple proteins are produced from a single vector or transcript cassette.
Aliases: multi-cistronic expression
- stringent washes
Wash steps designed to remove weakly associated contaminants and reduce non-specific binding during purification.
Aliases: high-stringency washes
- sumoylated proteins
Proteins modified by SUMO that serve as the identified substrate class in the Drosophila experiments.
Aliases: SUMO-modified proteins
- ubiquitin-like protein modifications
Post-translational modifications mediated by ubiquitin and related ubiquitin-like proteins in the systems discussed here.
Aliases: UbL modifications, UbL conjugation
- ubl conjugates
Proteins covalently modified by ubiquitin-like proteins and targeted for enrichment in the workflow.
Aliases: ubiquitin-like protein conjugates
- ufm1 substrates
Proteins proposed to be modified by UFM1 in the mammalian-cell experiments.
Aliases: UFM1 target proteins
REFERENCES
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