In their article, the authors omitted to mention ubiquitin-mediated fluorescence complementation (UbFC), a method previously designed to probe the conjugation of ubiquitin or ubiquitin-like proteins (Ubls) to their substrates (Fang and Kerppola, 2004; Sung et al, 2013). UbFC and NUbiCA are similar in that both methods rely on a protein-fragment complementation assay to detect Ubl conjugation, but the reporters (fluorescent proteins for UbFC, the NanoLuc luciferase for NUbiCA) and assay conditions (live or fixed cells for UbFC, purified proteins for NUbiCA) are different. UbFC is appealing since it enables direct visualization of the subcellular localization of Ubl conjugates in living cells, which is not possible with NUbiCA. Yet, the irreversible assembly and slow maturation of fluorescent proteins (Hu et al., 2002; Kodama and Hu, 2012) precludes real-time monitoring of Ubl conjugation and deconjugation events, which may complicate the interpretation of UbFC results, especially for short-lived or dynamically modified conjugates. Other approaches based on FRET and BRET have also been devised to probe ubiquitylation of proteins in living cells, but their sensitivity has not yet been carefully assessed (Ganesan et al., 2006; Batters et al., 2010; Riching et al., 2018).
The authors apologise to readers for this omission, which does not impact the results or conclusions of the paper.