Ubiquitylation is a versatile posttranslational protein modification catalyzed through the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). These enzymes form transient complexes with each other and their modification substrates and determine the nature of the ubiquitin signals attached to their substrates. One challenge in the field of protein ubiquitylation is thus to identify the E2-E3 pairs that function in the cell. In this chapter, we describe the use of bimolecular fluorescence complementation to assay E2-E3 interactions in living cells, using budding yeast as a model organism.
Keywords: BiFC; Linear unmixing; Living cell; Microscopy; Protein-fragment complementation assay; Protein–protein interactions; Saccharomyces cerevisiae; Ubiquitin; Ubiquitin ligase; Ubiquitin-conjugating enzyme.