A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

TY - JOUR

T1 - A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

AU - Pirone, Lucia

AU - Xolalpa, Wendy

AU - Sigurdsson, Jón Otti

AU - Ramirez, Juanma

AU - Pérez, Coralia

AU - González, Monika

AU - de Sabando, Ainara Ruiz

AU - Elortza, Félix

AU - Rodriguez, Manuel S

AU - Mayor, Ugo

AU - Olsen, Jesper V

AU - Barrio, Rosa

AU - Sutherland, James D

PY - 2017/1/18

Y1 - 2017/1/18

N2 - 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.

AB - 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.

U2 - 10.1038/srep40756

DO - 10.1038/srep40756

M3 - Journal article

C2 - 28098257

SN - 2045-2322

VL - 7

JO - Scientific Reports

JF - Scientific Reports

M1 - 40756

ER -