Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome

We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all ~4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About ~20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors.Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a measure of reproducibility without additional assays.The yeast two-hybrid (Y2H) system has been among the most powerful methods to identify protein-protein interactions. However, it has also been criticized for generating large numbers of false positive and false negative data. While false positives can be minimized by including controls, retesting by independent methods, and bioinformatic filtering, false negatives pose a much bigger problem. In fact, many interactions must go undetected in two-hybrid screens because of the sterical constraints the system involves: the two fusion proteins must interact with each other, their interaction interfaces must be exposed, and all component