Isolating Interaction-Null/Impaired Mutants Using the Yeast Two-Hybrid Assay

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Interactions between biomolecules are the most basic part of biological phenomena. Protein-protein interactions constitute a significant part of such interactions. Therefore, identification of the interaction partner(s) of a protein of interest is critical to further elucidate the function of the protein/gene of interest. The yeast two-hybrid (Y2H) method is a popular technique to identify protein-protein interactions in vivo¹. In this system, two proteins (X and Y) whose interaction is to be tested are fused to the DNA-binding (DB) domain and transcriptional activation domain (AD), respectively. The DB-X fusion protein binds to a reco....

1. Construction of the mutant library

1. Set up the polymerase chain reaction (PCR) (an example is shown below). Generally, prepare 50 µL of the reaction, which is divided into ten aliquots of 5 µL, and amplify the target gene fragment in a PCR machine⁴.
   NOTE: Users should select an appropriate polymerase to efficiently introduce mutations. If the DNA fragment to be amplified is short, then a polymerase with a higher error rate, such as Taq polymerase, wh.......

Recently, it was found that the C-terminal half of Pol2 protein (Pol2-C) interacts with Mcm10. Both proteins are essential for the initiation of DNA replication and, hence, for cell growth in the budding yeast Saccharomyces cerevisiae^13,14,15. To help understand the biological significance of this interaction, Pol2-C mutants that have no/diminished interaction with Mcm10 were isolated using the method described here.

This article describes how to isolate interaction-null/impaired mutants using the Y2H assay. Such mutants are powerful tools to analyze the function of a protein of interest. To isolate such mutants, rY2H assays were developed previously by modifying the Y2H host strain^2,3. However, they have not greatly reduced the amount of labor. By contrast, mutants can be isolated with this method without a significant amount of labor. In this method, modification of the Y2H.......

Y. Tanaka performed the technical improvement of Y2H. This work is supported by JSPS KAKENHI Grant Number JP22K06336 and the Institute for Fermentation, Osaka.