How the Yeast Two-Hybrid (Y2H) Assay Works

Introduction to Yeast Two-Hybrid (Y2H) Assay

The yeast two-hybrid (Y2H) assay represents a pivotal technology for detecting binary protein-protein interactions (PPIs) in living cells. This method emerged in 1989 and exploited the modular nature of transcription factors. It provided a simple yet powerful approach to uncovering direct molecular interactions. The assay relies on Saccharomyces cerevisiae as a cellular host. Yeast cells can be engineered to express hybrid proteins that reconstitute a transcription factor only when they interact. The resultant transcription drives reporter gene expression. This arrangement yields a clear phenotypic readout. Researchers can thus screen large libraries of potential interacting partners. The Y2H assay accelerated functional genomics in the era of whole-genome sequencing. It enabled the mapping of extensive protein interaction networks. The assay also supported drug discovery efforts by identifying novel targets. It remains an essential platform in proteomics.

Fundamental Principles of Y2H Assay

GAL4 Transcription Factor: DNA-Binding and Activation Domains

The classical Y2H system leverages the GAL4 transcription factor from yeast. GAL4 comprises two independent modules: a DNA-Binding Domain (DBD) and an Activation Domain (AD). The DBD binds specifically to upstream activating sequences (UAS) in reporter gene promoters. The AD recruits the transcriptional machinery, including RNA polymerase II. In its native form, GAL4 binds to UAS and activates the transcription of target genes in the presence of galactose. When the DBD and AD are separated, each module cannot activate transcription alone. However, when brought into proximity, they reconstitute a functional transcription factor. This principle underlies the Y2H assay.

Fusion Protein Strategy

The Y2H assay uses two hybrid proteins: a "bait" and a "prey." The bait consists of a protein of interest fused to GAL4-DBD. The prey consists of a candidate interactor fused to GAL4-AD. Yeast cells are co-transformed with vectors encoding these fusions. If the bait and prey physically interact, the GAL4-DBD and AD modules converge. This event reconstitutes the transcription factor. The reconstituted factor binds to UAS upstream of reporter genes. Recruitment of RNA polymerase II follows. The process yields reporter gene expression. Absent interaction, the DBD and AD remain separate. No transcription occurs. This binary configuration enables facile detection of interactions.

Reporter Gene Selection for Y2H Screening

Reporter genes provide a visible or selectable phenotype. Common reporter genes include:

• lacZ: Encodes β-galactosidase. Its activity can be measured via a colorimetric substrate.
• HIS3: Encodes imidazole glycerol phosphate dehydratase. Its expression allows growth in histidine-deficient media.
• ADE2: Encodes phosphoribosylaminoimidazole carboxylase. Growth on adenine-deficient media indicates interaction.
• URA3: Encodes orotidine 5'-phosphate decarboxylase. Its expression can be detected through uracil auxotrophy and 5-fluoroorotic acid sensitivity.
• LYS2: Enables growth in lysine-deficient media.

Figure 1. Y2H methods and their applications to detect protein-protein or protein-small-molecule interactions and to discover small-molecule inhibitors (Hamdi A, et al., 2012).

Experimental Design and Vector Construction

Selection Criteria for Bait and Prey Proteins

The bait protein must not self-activate reporter genes when fused to GAL4-DBD. It must fold properly in yeast and localize to the nucleus. Similarly, prey proteins should not inherently activate transcription when fused to GAL4-AD. Large or membrane-associated proteins may require truncated constructs. Such truncations should preserve relevant interaction domains. Researchers may include linkers to ensure proper folding. Proteins requiring posttranslational modifications may need coexpression of modifying enzymes. Before library screening, pilot experiments often test individual bait-prey pairs to assess background activation and expression levels.

Construction of Bait Vectors

Bait vectors contain the GAL4-DBD coding sequence under a constitutive yeast promoter (e.g., ADH1 promoter). The gene encoding the bait protein is cloned in-frame downstream of GAL4-DBD. Restriction sites flanking bait inserts facilitate subcloning. Researchers often include epitope tags (e.g., HA or c-Myc) to monitor expression by Western blot. Vectors may also carry selection markers such as TRP1 or URA3. Transformation into yeast allows selection on synthetic defined media lacking the corresponding amino acid or nucleotide.

Construction of Prey Vectors

Prey vectors mirror bait vectors but replace the GAL4-DBD with GAL4-AD. Prey libraries derive from cDNA or genomic DNA. Frameshift mutations must be minimized. Directional cloning strategies (e.g., using att sites for Gateway® cloning) improve library quality. Prey vectors often carry a complementary selection marker (e.g., LEU2) to allow co-transformation with bait vectors. Researchers can generate arrayed libraries of individual prey clones or pooled libraries in which preys are expressed as a heterogeneous mixture.

Library Screening vs. Pairwise Interaction Testing

Pairwise Testing: Researchers test specific bait–prey combinations. This method is suited to confirm interactions suggested by other techniques (e.g., co-immunoprecipitation or computational prediction). It yields high specificity but is low throughput.

Library Screening: A single bait is screened against an entire prey library. Yeast cells are co-transformed with the bait vector and the pooled prey library. Transformants are plated on selective media. Positive colonies indicate potential interactors. These colonies are isolated, and prey plasmids are recovered and sequenced to identify the interacting partner. Library screening enables proteome-wide exploration but may generate false positives.

Yeast Strains and Transformation Protocols

Commonly Used Yeast Strains for Y2H

• AH109: Carries HIS3, ADE2, and lacZ reporters. It uses the LYS2 and TRP1 loci for selection markers.
• Y2HGold: Contains HIS3, AUR1-C, ADE2, and MEL1 reporters. It also features higher stringency to reduce background.

These strains are engineered to minimize endogenous transcription factor noise. They also lack proteases that degrade hybrid proteins. Strain choice influences assay sensitivity and background.

Yeast Transformation Techniques

Lithium Acetate Method

• Yeast cells are treated with lithium acetate to increase cell wall permeability.
• Carrier DNA (e.g., salmon sperm DNA) is added to enhance transformation efficiency.
• Plasmid DNA is mixed with cells, polyethylene glycol (PEG), and lithium acetate solution.
• A heat shock step promotes DNA uptake.
• This method is simple and cost-effective.

Electroporation

• Yeast cells are grown to mid-log phase and washed to remove salts.
• Cells are mixed with DNA and subjected to a brief electrical pulse.
• Membrane pores open briefly, allowing DNA entry.
• Requires specialized equipment (electroporator) and high-voltage pulses.
• Lithium acetate transformation suffices for most Y2H assays. Electroporation is preferred for large-scale library screens requiring maximal efficiency.

Selection Media and Growth Conditions for Y2H Assays

Y2H assays employ defined synthetic dropout media to select for plasmid uptake and reporter activation. For example:

• -Trp-Leu medium selects for retention of bait (TRP1 marker) and prey (LEU2 marker) plasmids.
• -Trp-Leu-His medium selects for HIS3 reporter activation.
• -Trp-Leu-Ade medium selects for ADE2 reporter activation.

Screening and Detection of PPIs

Qualitative Reporter Assays

Qualitative detection relies on the growth of yeast colonies on selective plates. When bait and prey interact, reporter genes enable auxotrophy rescue. For instance:

• Activation of HIS3 permits growth on histidine-deficient medium.
• Activation of ADE2 permits growth on an adenine-deficient medium.
• Activation of LYS2 permits growth on a lysine-deficient medium.

Quantitative Detection Methods

Quantitative assessment uses the β-galactosidase assay. Yeast colonies or liquid cultures are subjected to a colorimetric assay using ONPG (o-nitrophenyl-β-D-galactopyranoside). The reaction yields o-nitrophenol, which absorbs at 420 nm. Researchers measure absorbance and calculate Miller Units to quantify transcriptional activation. This value correlates with interaction strength.

Confirmation of Positive Hits

Candidate interactions must be validated through additional steps:

• Retransformation: Reintroduce purified bait and prey plasmids into fresh yeast cells. This step confirms that the plasmids, not spontaneous mutations, cause the interaction phenotype.
• Reciprocal Testing: Swap bait and prey fusions to detect orientation-specific artifacts.
• Secondary Reporter Assays: Test alternative reporters to reduce false positives.

Integration of Y2H with Complementary PPIs Techniques

Combining Y2H with Co-Immunoprecipitation (Co-IP) for Validation

Co-IP serves as an orthogonal validation method. Researchers express epitope-tagged bait and prey in yeast or mammalian cells. Cell lysates are incubated with an antibody against the bait tag. The immune complex is captured on Protein A/G beads. Western blot analysis with an antibody against the prey reveals co-precipitation. Positive Co-IP corroborates Y2H results in a different biological context. It confirms that interactions occur in native membranes and multi-protein complexes. Combining Y2H and Co-IP strengthens confidence in identified interaction pairs.

Orthogonal Confirmation via Bimolecular Fluorescence Complementation (BiFC)

BiFC exploits fragments of fluorescent proteins (e.g., split-YFP or split-GFP). Each fragment is fused to one of the interacting proteins. Upon interaction, the fragments reassemble into a functional fluorescent protein. This event emits fluorescence detectable by microscopy or flow cytometry. BiFC offers subcellular localization information. It confirms Y2H-identified interactions in mammalian cells. Moreover, BiFC can detect transient or weak interactions not captured by Y2H under stringent conditions.

Incorporation of Mass Spectrometry and Proteomics Platforms

Affinity Purification-Mass Spectrometry (AP/MS) complements Y2H by identifying multi-protein complexes. Researchers generate a cell line stably expressing an epitope-tagged bait. The bait and its interactors are isolated via affinity purification. Mass spectrometry then identifies co-purified proteins. AP/MS captures direct and indirect interactors within a native complex. Networking Y2H and AP/MS data provides both binary interactions and multi-component assemblies. Integrating Y2H with quantitative proteomics (e.g., SILAC, TMT labeling) yields dynamic interaction maps under various conditions. This integrated approach advances systems biology by constructing robust interactomes with temporal and spatial resolution.

Applications of Y2H in Research

Mapping Protein Interaction Networks in Yeast and Mammalian Systems

In Saccharomyces cerevisiae, systematic array-based screening of 6,000 open reading frames (ORFs) yielded over 5,600 interactions covering 70 % of the proteome. Similar efforts in Caenorhabditis elegans, Drosophila melanogaster, and human cells have revealed thousands of interactions. These interactomes serve as blueprints for cellular wiring diagrams. They facilitate identification of network hubs, modules, and pathways. The data also support computational models of cellular function.

Identification of Novel Drug Targets Using Y2H Assays

Y2H assays have identified interactors of disease-associated proteins. For example, the interaction between p53 and MDM2 was confirmed in Y2H. This discovery highlighted MDM2 as a therapeutic target. Screening against oncogenic Ras uncovered the Ras-Raf interaction. This knowledge spurred the development of Raf inhibitors. Library-based Y2H screens of viral proteins have identified host-pathogen interactions. Such screens help pinpoint human proteins that serve as potential antiviral targets.

Use of Y2H in Functional Genomics and Proteomics

Integration of Y2H data with gene expression profiles, genetic screens, and phenotypic assays creates a multi-omic view of cellular processes. In proteomics, Y2H data complement mass spectrometry-based methods. They provide binary interaction validation for complexes identified by affinity purification. This synergy refines PPI networks and reduces false positives.

Case Study

A reference map of the human binary protein interactome

Journal: Nature

Published: 2020

DOI: 10.1038/s41586-020-2188-x

Background

Comprehensive maps of PPIs are essential to understand how genotype gives rise to phenotype. Existing datasets were either limited to small-scale, hypothesis-driven studies or skewed by protein expression biases. Y2H remains the only binary PPI assay scalable to proteome-wide screening, providing uniform coverage with minimal ascertainment bias

Purpose

To generate a systematic, "all-by-all" reference map of human binary PPIs—termed HuRI—covering most protein-coding genes, thereby enabling functional exploration of cellular networks across diverse physiological and pathological contexts

Methods

• ORFeome Expansion: Updated to encompass ~90 % of the protein-coding genome (17,408 genes).
• Y2H Screening: Employed three complementary Y2H assay versions across nine independent screens of the ~150 million possible pairwise search space.
• Verification: Candidate interactions underwent quadruplicate retesting, sequence confirmation, and orthogonal validation using MAPPIT and GPCA assays.
• Stringency Controls: Benchmarked against gold-standard positive (Lit-BM) and random reference sets (RRS), with each assay version exhibiting high sensitivity and low false-positive rates

Results

• HuRI Dataset (HI-III-20): 52,569 high-confidence binary PPIs involving 8,275 unique proteins.
• Coverage Expansion: Four-fold more PPIs than prior systematic maps (HI-II-14).
• Direct Contacts: ~90 % of HuRI PPIs correspond to direct biophysical contacts, outperforming literature-curated datasets (81 %)
• Assay Complementarity: Switching between Y2H versions yielded incremental PPI discoveries, highlighting the prevalence of weaker or transient interactions.
• Biological Insights: Integration with genomic, transcriptomic, and proteomic data revealed tissue-specific network architectures and suggested molecular mechanisms underlying Mendelian disease phenotypes

Figure 2. Generation of a reference interactome map using a panel of binary assays.

Conclusion

HuRI represents the most comprehensive, systematically derived reference map of the human binary interactome to date. Its high coverage and rigorous validation establish a foundational resource linking genomic variation to cellular function and disease, and it will catalyze discoveries in systems biology, functional genomics, and therapeutic target identification.

References

• Hamdi A, Colas P. Yeast two-hybrid methods and their applications in drug discovery. Trends in pharmacological sciences, 2012, 33(2): 109-118. DOI: 10.1016/j.tips.2011.10.008
• Brückner A, et al. Yeast two-hybrid, a powerful tool for systems biology. International journal of molecular sciences, 2009, 10(6): 2763-2788. DOI: 10.3390/ijms10062763
• Lopez J, Mukhtar M S. Mapping protein-protein interaction using high-throughput yeast 2-hybrid. Plant Genomics: Methods and Protocols, 2017: 217-230. DOI: 10.1007/978-1-4939-7003-2_14