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

ABSTRACTUbiquitylation 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 ubiquitin chain linkages. We used NUbiCA to investigate the ubiquitylation of the low abundance centromeric histone Cse4, and found that it is ubiquitylated during S-phase. Finally, 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.Summary statementWe describe a sensitive protein-fragment complementation assay to facilitate the monitoring of ubiquitylation events that take place in cultured cells or model organisms.