ReLo is a simple and quick colocalization assay to identify and characterize direct protein-protein interactions

The characterization of protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, the screening and investigation of PPIs involving large or poorly soluble proteins remain challenging. As a result, we developed ReLo, a simple, rapid, and versatile cell culture-based method for detecting and investigating interactions in a cellular context. Importantly, our data strongly suggest that with ReLo specifically direct binary PPIs are detected. By applying additional bridging experiments ReLo can also be used to determine the binding topology of subunits within multiprotein complexes. Moreover, ReLo has the potential to identify protein domains that mediate complex formation, screen for interfering point mutations, study interactions that depend on conformation or protein arginine methylation, and it is sensitive to drugs that mediate or interfere with an interaction. Taken together, ReLo is a simple and quick alternative for the study of PPIs particularly when established methods fail.