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.
Highlights
Ub is the most conserved protein found in all eukaryotes
When applying the BirA/biotinylation-target peptide (Bio) tagging system to other UbLs, we modelled the initial vectors on the biotinylated Ub (bioUb)-BirA fusion, which relied on the capacity of endogenous DUBs to process the tandem bioUb-BirA precursor for the separation of the bioUb and the biotinylating enzyme[33,34]
In some cases the third ORF position was exchanged for the respective E2 conjugating enzyme for the UbL being studied, which enhanced the recovery of bioUbL conjugates
Summary
Ub is the most conserved protein found in all eukaryotes. Approximately 20 proteins have been identified that are related to Ub, known as UbLs4,5. All of them share the beta-grasp fold characteristic of Ub and all participate in processes similar to ubiquitination, suggesting a common ancestry to this family of proteins[4]. The Ub Related Modifier 1, URM1, is conserved in eukaryotes and its structure resembles that of ancient prokaryotic sulfur carriers. Given the variety of UbLs that exist, the different types of monotypic chains that they can potentially form, and that there can even exist hybrid chains containing different types of UbLs, a complex UbL code may underlie numerous biological and cellular processes This has prompted researchers to develop different systems to isolate UbL-modified proteins using cultured cells, model organisms, and clinical tissue samples. Monoclonal antibodies with mapped epitopes in SUMO1 or SUMO2/3 allow the identification of SUMOylated proteins in endogenous conditions[23]. Identification of other UbL-modifications has been performed using exogenous epitope-tagged versions or using antibodies to detect endogenous substrates[26,27,28,29,30,31]
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