Journal
SCIENTIFIC REPORTS
Volume 7, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/srep40756
Keywords
-
Categories
Funding
- Consolider Program [BFU2014-57703-REDC]
- UPStream consortium (ITN program, EU) [PITN-GA-2011-290257]
- COST (European Cooperation in Science and Technology)
- Novo Nordisk Foundation [NNF14CC0001]
- Novo Nordisk Foundation Center for Protein Research [PI Jesper Velgaard Olsen] Funding Source: researchfish
Ask authors/readers for more resources
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 nonspecific 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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available