Generation of a Yeast Two-Hybrid Strain Suitable for Competitive Protein Binding Analysis

Abstract

The yeast two-hybrid (Y2H) system is one of the most common applied genetic systems utilized for the identi-fication of protein-protein interactions in vivo (1). Over the years this system has been systematically exploited for analyzing the physiological function of proteins via the generation of huge protein interaction networks (2). It is based on the principle that different transcription factors can be split into a DNA binding and a polymerase II activation domain. When these independently functional domains are brought into close proximity, the activity of the transcription factor is restored. If an interaction of a protein fused to the DNA binding domain (bait protein) and another protein fused to the activation domain (prey protein) occurs, a functionally transcription factor is reconstructed, and transcription of certain reporter genes involved in amino acid biosynthesis is initiated. The colorimetric reporter LacZ is often exploited as well, because it allows the relative quantification of its activity and, consequently, the relative strength of protein-protein interactions in vivo (3–5).Since proteins are multifunctional and interact with many factors, it would be obviously advantageous and in particular less time-consuming to characterize the interplay between proteins in vivo by the Y2H system in comparison to existing in vitro approaches (6,7). Therefore, the Y2H technology has been broadened and applied for further characterization of protein-protein interactions. Reverse approaches have been developed allowing the identification of particular protein mutations, peptides, or agents that inhibit the protein-protein inter-action of interest (8,9). Furthermore, Y2H systems have been generated in which the interaction between proteins is enhanced or depends upon expression of a third protein or ligand (5,10,11). However, the simultaneous expression of a third protein or factor to analyze competitive protein binding in the Y2H background can be difficult, because often the auxotrophic marker genes available have been exhausted in the course of generating the strains for a particular Y2H system. Moreover, many Y2H systems are incompatible to each other because of the different promoters and the auxotrophic markers that have to be used. Thus, switching between the available Y2H systems and strategies is often impossible or very time-consuming due to required cloning efforts for the creation of different bait and prey plasmids needed.Hence, to facilitate competitive protein binding approaches in a quite frequent Y2H system, we aimed to create a strain still exploitable for Y2H screens, but additionally allowing the simultaneous expression of a third protein (Figure 1A). Given that most yeast expression vectors are based on auxothropic markers, we decided to replace the URA3 gene in the standard strain L40c [MATa his3∆200 trp1–901 leu2–3,112 ade2 lys2–801am can1 URA3::(lexAop)