The yeast two-hybrid system detects protein-protein interactions in living yeast cells by reconstituting a functional transcription factor. It is a powerful and widely used method for discovering and characterizing binary protein interactions.
Principle
The system is based on the modular nature of transcription factors, which have separable DNA-binding and activation domains. The protein of interest, or bait, is fused to the DNA-binding domain of a transcription factor such as Gal4. Potential interacting proteins, or prey, are fused to the activation domain. Interaction between bait and prey brings the activation domain into proximity with the DNA-binding domain, reconstituting a functional transcription factor that activates reporter gene expression.
Components
The bait is typically cloned into a vector that expresses the bait protein fused to the Gal4 DNA-binding domain. The prey is expressed from a library vector fused to the Gal4 activation domain. Both plasmids are transformed into a yeast reporter strain containing one or more reporter genes under the control of Gal4-responsive upstream activation sequences.
Reporter genes commonly include HIS3, which allows growth on histidine-deficient medium, ADE2 for adenine biosynthesis, and lacZ or GFP for colorimetric or fluorescent detection. Multiple reporters with different promoters reduce false positives. Counter-selectable markers such as URA3 and CYH2 can eliminate false positives by negative selection under certain conditions.
Library Screening
A typical Y2H screen begins with constructing a cDNA library in the prey vector. The bait is transformed into the yeast reporter strain, and the prey library is introduced by large-scale transformation. Transformants are plated on selective medium that requires interaction for growth. Colonies appearing after 3 to 7 days are picked, and the prey plasmids are recovered and sequenced to identify the interacting protein.
Screening requires careful optimization. The bait must be tested for autoactivation, where the bait alone activates reporter transcription. If autoactivation occurs, the bait can be truncated or alternative vectors can be used. The expression level of both bait and prey affects sensitivity. High-throughput screens using automation and pooling strategies enable whole-proteome interaction mapping.
Advantages and Limitations
Y2H detects direct, binary interactions and can identify weak or transient interactions that may be missed by co-immunoprecipitation and other biochemical protein extraction and purification methods. The system is scalable for high-throughput, cost-effective, and does not require protein-specific antibodies. Interactions are detected in a eukaryotic cellular environment.
Limitations include false positives from autoactivators, sticky proteins, and fortuitous interactions. False negatives occur when proteins fail to fold properly in yeast, require post-translational modifications absent in yeast, or are toxic when overexpressed. Membrane proteins are problematic because the nuclear localization of the system is incompatible with membrane environments. Variants such as the split-ubiquitin system address some of these limitations.
Variants
Several Y2H variants extend the basic technique. The reverse two-hybrid system detects disruption of interactions by screening for loss of reporter expression, useful for identifying mutations or drugs that disrupt specific interactions. The one-hybrid system detects protein-DNA interactions by fusing the DNA-binding domain to a known transcription factor and screening for activation. The three-hybrid system detects RNA-protein interactions using a hybrid RNA molecule that bridges the bait and prey.
Membrane yeast two-hybrid uses the split-ubiquitin approach, where bait and prey are fused to halves of ubiquitin. Interaction reconstitutes full ubiquitin, which is cleaved by ubiquitin-specific proteases, releasing a transcription factor that activates reporter genes. This allows detection of interactions involving membrane proteins in their native environment.
Applications
Y2H has been used to generate proteome-scale interaction maps for organisms including yeast, fly, worm, and human. These maps reveal functional modules, identify novel pathway components, and predict functions of uncharacterized proteins. Y2H screens have identified disease-relevant interactions, such as interactions between proteins encoded by genes linked to genetic disorders. The technology has also been applied to map host-pathogen interactions, revealing how viral and bacterial proteins subvert host cellular machinery.
