One- plus Two-hybrid System, a Novel Yeast Genetic Selection for Specific Missense Mutations Disrupting Protein/Protein

To facilitate analysis of protein/protein interaction inter-faces, we devised a novel yeast genetic screening method, named the “one- plus two-hybrid system, ” for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mu-tants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions be