Versatile Screening for Binary Protein-Protein Interactions by Yeast Two-Hybrid Mating

Identification of binary protein-protein interactions is a crucial step in determining the molecular context and functional pathways of proteins. State-of-the-art proteomics techniques provide high-throughput information on the content of proteomes and protein complexes, but give little information about transient interactions, about the binary protein pairs, or about the interacting epitopes. A powerful method to reveal this information is the yeast two-hybrid system. We have employed an optimized GAL4-based yeast two-hybrid system to dissect the photoreceptor cilium-associated protein complex around the retinitis pigmentosa GTPase regulator (RPGR) in mammalian photoreceptors. This enabled us to identify associating protein partners that, similar to RPGR, were also associated with a heterogeneous group of inherited retinal degenerations arising from ciliary defects. We describe how to generate high content pretransformed cDNA libraries, and perform an efficient yeast mating screen for protein-protein interactions with any bait protein of interest.