A yeast mating-selection scheme for detection of protein – protein interactions

Recently, a new approach to the study of protein—protein interactions has appeared. The 'two-hybrid system' developed by Fields and coworkers (1,2) can be used either to look for new interacting proteins, or to verify and characterize interactions between proteins that are likely to associate according to genetic or biochemical data. The two-hybrid system is a molecular genetic approach that takes advantage of the structural flexibility of the yeast transcription factor Gal4. The Gal4 protein contains two domains, the DNA-binding domain and the transcription activator domain. The two domains need not be part of the same protein to accomplish transcriptional activation (3). When the two domains are fused separately to two unrelated but interacting proteins, transcriptional activation can be achieved as a consequence of the protein—protein interaction. Usually, the search for new interacting proteins using the twohybrid system has been carried out by co-transforming a yeast strain containing an integrated copy of a UASQ^J-IOCZ reporter gene with two plasmids (2, 4—6). One plasmid encodes a fusion of the DNA-binding domain of Gal4 to the protein of interest, while the other plasmid (the library plasmid) encodes a fusion of the transcriptional activation domain of Gal4 to randomly generated coding regions. Thus, the DNA-binding domain fusion will bind to the UASQ^J element upstream of the reporter gene. If a protein encoded by the library fusion plasmid interacts with the protein of interest, the transcription activation domain becomes co-localized upstream of the reporter gene, resulting in activation of transcription. Effective use of the two-hybrid system requires the generation of a large number of yeast transformants. Since transformation of yeast is still four orders of magnitude less efficient than that of bacteria, transformation can be the limiting step for an exhaustive screen of a cDNA library.