Sensitive detection of protein ubiquitylation using a protein-fragment complementation assay

Marie Le Boulch (1) , Audrey Brossard (1) , Gaëlle Le Dez (1) , Sébastien Léon (2) , Gwenaël Rabut (1)

1 IGDR - Institut de Génétique et Développement de Rennes
2 IJM (UMR_7592) - Institut Jacques Monod

Ubiquitylation is a reversible post-translational protein modification that regulates a multitude of cellular processes. Detection of ubiquitylated proteins is often challenging, because of their low abundance. Here, we present NUbiCA, a sensitive protein-fragment complementation assay to facilitate the monitoring of ubiquitylation events in cultured cells and model organisms. Using yeast as a model system, we demonstrate that NUbiCA enables to accurately monitor mono- and poly-ubiquitylation of proteins expressed at endogenous levels. We also show that it can be applied to decipher the topology of ubiquitin conjugates. Moreover, we assembled a genome wide collection of yeast strains ready to investigate the ubiquitylation of proteins with this new assay. This resource will facilitate the analysis of local or transient ubiquitylation events that are difficult to detect with current methods.

Keywords: Ubiquitin; NanoBiT; NanoLuc; PCA; Ubiquitination; UbiCREST

Domains: Life Sciences [q-bio]; Cellular Biology; Subcellular Processes [q-bio.SC]; Life Sciences [q-bio]; Biochemistry, Molecular Biology; Biochemistry [q-bio.BM]

Marie Le Boulch, Audrey Brossard, Gaëlle Le Dez, Sébastien Léon, Gwenaël Rabut. Sensitive detection of protein ubiquitylation using a protein-fragment complementation assay. Journal of Cell Science, 2020, 133 (12), pp.jcs240093. ⟨10.1242/jcs.240093⟩. ⟨hal-02635146⟩