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

Abstract 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. Curr. Protoc. Neurosci . 41:5.27.1‐5.27.41. © 2007 by John Wiley & Sons, Inc.