Y2H

Y2H stands for yeast two-hybrid, a molecular biology method used to detect protein–protein interactions in vivo in Saccharomyces cerevisiae. Developed by Fields and Song in 1989, the system relies on reconstituting a functional transcription factor from two separate domains. A protein of interest is fused to a DNA-binding domain (DBD), while a second protein is fused to an activation domain (AD). If the two proteins interact, the DBD and AD are brought together, activating transcription of reporter genes in the yeast nucleus.

In a typical workflow, the bait protein is fused to the DBD in a bait vector and the prey protein to the AD in a prey vector. These constructs are co-transformed into a yeast strain that carries reporters such as HIS3, ADE2, or lacZ. Interaction leads to growth on selective media lacking certain nutrients or to a colorimetric readout, enabling identification of interacting pairs. Positive results are usually confirmed with reciprocal assays and appropriate controls to rule out autoactivation by either fusion.

Limitations and scope: Y2H detects direct binary interactions but is susceptible to false positives from nonspecific binding or autoactivation, and false negatives from issues such as improper folding, lack of relevant post-translational modifications, or dependence on a cellular context not present in yeast. Membrane proteins often require specialized variants, such as split-ubiquitin systems. Consequently, Y2H findings are typically followed by secondary validation using methods like co-immunoprecipitation, pull-down assays, or fluorescence-based interaction assays. Despite limitations, Y2H remains a foundational approach for mapping protein interaction networks.