Split‐Ubiquitin System for Identifying Protein‐Protein Interactions in Membrane and Full‐Length Proteins

Protein-protein interactions play a fundamental role in the regulation of almost all cellular processes. Thus, the identification of interacting proteins can help to elucidate their function. The mating-based split-ubiquitin system (mbSUS) uses yeast as a test organism to identify potential interactions between full-length membrane proteins or between a full-length membrane protein and a soluble protein. The mbSUS can also be used to provide further evidence for protein-protein interactions detected with other methods and to map the interaction domains of selected proteins. The mbSUS is optimized for systematic screening approaches employing a mating-based approach, as typically used to determine protein interactions on a genomic scale. Construction of bait and prey fusions is simplified by adapting two different cloning procedures: (i) in vivo cloning in yeast, and (ii) Gateway cloning in E. coli. Protocols for small-scale interaction tests, as well as systematic approaches using sorted bait and prey arrays, are described.