Using the Split-Ubiquitin Yeast Two-Hybrid System to Test Protein–Protein Interactions of Transmembrane Proteins

Proteins are responsible for many biological processes within living organisms. Many proteins have the ability to specifically interact with other proteins in order to function properly. The identification of protein–protein interactions (PPIs) can provide useful information about the function of a protein of interest. Historically, the properties of transmembrane proteins have caused difficulty in analyzing PPIs among transmembrane proteins. The development of an assay that is capable of analyzing PPIs involving transmembrane proteins, the split-ubiquitin yeast two-hybrid (SU-Y2H) assay, has provided a method to probe pairwise PPIs between two proteins of interest or to screen a single protein of interest for interaction partners. The following protocol explains how to use the SU-Y2H assay, which is compatible with the use of transmembrane proteins, to investigate PPIs between two proteins of interest and also briefly describes how to adjust the system to be used as a high-throughput screen for interaction partners of a particular protein of interest.