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

• Published in: Molecular & cellular proteomics 2007-10, Vol.6 (10), p.1727-1740
• Main Authors: Kim, Ji Young, Park, Ok Gu, Lee, Jae Woon, Lee, Young Chul
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Cell Line; Clone Cells; Glutathione Transferase - metabolism; Humans; Molecular Sequence Data; Mutagenesis; Mutant Proteins - chemistry; Mutant Proteins - isolation & purification; Mutant Proteins - metabolism; Mutation, Missense - genetics; Protein Binding; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear - chemistry; Receptors, Cytoplasmic and Nuclear - isolation & purification; Receptors, Cytoplasmic and Nuclear - metabolism; Receptors, Retinoic Acid - metabolism; Repressor Proteins - chemistry; Repressor Proteins - metabolism; Reproducibility of Results; Retinoid X Receptors - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Selection, Genetic; Transcription, Genetic; Two-Hybrid System Techniques
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M700079-MCP200

To facilitate analysis of protein/protein interaction interfaces, 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 mutants 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 between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.