The classical yeast two-hybrid system

The classical yeast two-hybrid system, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique.

Historical perspective

The yeast two-hybrid (Y2H) technique was originally developed by Stanley Fields and Ok-Kyu Song in 1989 to detect protein-protein interactions using the Saccharomyces cerevisiae yeast transcription activator GAL4. Until that time, interactions between two proteins had been studied mainly using biochemical techniques.

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Molecular analysis of eukaryotic transcription factors prompted the creation of this entirely new analytical method. Though this technique has been known for more than 25 years, it still represents one of the simplest and cheapest ways to identify and analyze protein-protein interactions, saving time, and being straightforward. This method can also be easily used to classify interacting proteins for a given protein, check interactions between two known proteins, or map interacting domains. The yeast two-hybrid (Y2H) method is the in vivo technique used to study PPIs.

Principle of Yeast two-hybrid method

The yeast two-hybrid is dependent upon the reconstitution of a functional transcription factor (TF) when two important proteins or polypeptides interact. It happens in genetically engineered yeast strains, in which a reporter gene transcription contributes to a specific phenotype, generally developing on a selective medium or changing the colour of the yeast colonies. In a colourimetric assay, the most popular reporter genes are HIS3 to select yeast on a medium that lacks histidine, and LacZ to screen yeast.

How does it work?

The yeast two-hybrid system is an effective and widely used genetic technique for investigating interactions within the nucleus of yeast between artificial fusion proteins. The Y2H assay involves two protein domains which will have two different functions: 

(i) a DNA binding domain (DBD) which helps bind to DNA, and

(ii) an activation domain (AD) responsible for activating DNA transcription. 

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Both domains are essential to transcribe a reporter gene. The basic concept was to fuse the two proteins of interest X and Y respectively to DBD and AD of Gal4 so that the interaction between X and Y would reconstitute a functional transcription factor that could then guide the expression of reporter genes.

The bacterial protein LexA in combination with Gal4 AD is also frequently used as a DBD. Gal4 protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. If the two proteins of interest interact physically, an active Gal4 transcription factor will be produced which will drive the expression of reporter genes under the influence of the GAL promoter. 

Interaction between two proteins, known as bait and prey, enables reporter genes to develop on different media or a colour reaction. During this experiment, the DNA-binding domain and activation domain of Gal4 is fused to two proteins of interest.

The protein of interest X (e.g. the glucose-sensor SNF1) was fused to the N-terminal part of GAL4 containing the DBD (GAL4DBD) in the first construct called bait. In the second construct, the prey, protein Y (e.g. the regulatory protein SNF4) was fused to the C-terminal part of Gal4 that contains the AD (GAL4AD).

The expression of both yeast fusion proteins and the interaction between bait and prey actually reconstituted a functional transcription factor for Gal4 from the two separate polypeptides. The interaction between the bait and prey protein brings GAL4’s BD and AD close enough to enable downstream reporter genes to be transcribed, which subsequently allows selective media production.

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Gal4 then recruited RNA polymerase II, resulting in a transcription of the fusion gene GAL1-lacZ. This reporter gene encodes the beta-galactosidase enzyme that identifies the yeast cell when a colourimetric substrate is being used.

The classical yeast two-hybrid system
Figure 1: Yeast two-hybrid system for protein-protein interactions

Advantages

  1. The yeast two-hybrid (Y2H) method is the in vivo technique used to study PPIs.
  2. It has a fine resolution, enabling interaction mapping within proteins.
  3. This technique detects even interactions that are transient and unstable.
  4. It is independent of endogenous protein expression.
  5. It does not require any previous knowledge of the proteins to be tested and can be performed once the corresponding genes are known, thus being suitable for large-scale applications.
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Drawbacks

  1. One drawback is that while this screen occurs inside yeast cells and thus occurs under native cellular conditions, both the bait and the prey protein must be capable of translocating to the nucleus to allow reporter gene transcription. Therefore, it is difficult to detect interactions between the bait protein and membrane-bound, integral membrane and/or proteins located to subcellular compartments and produce what is called a false negative. In addition, the bait and prey protein will not be able to autoactivate the reporter gene contributing to the presence of false positives.
  2. False positives are one of the Y2H screen’s biggest disadvantages as the presence of false positives will occur for several causes, including the one already described or non-specific interactions.
  3. Low throughput approach.
  4. It only detects binary interactions i.e. only pairwise interactions can be measured.
  5. This technique can not be used to analyze certain kinds of proteins, such as transcription factors, since their hybrids may trigger the transcription even in the absence of interaction.
  6. Extensive use of artificially produced hybrids may lead to bait and prey protein conformation changes thus preventing transcriptional activation. This is one of the possible causes of false-negative interactions.
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Applications

 This method has varieties of applications:

  1. Find new protein and new functions of the protein.
  2. Determination of sequences crucial for interaction.
  3. Study the interaction between antigen and antibody in vivo.
  4. Screening the functional site of new drugs and the effection of the drug on the interaction of the target protein.
  5. Establish a genomic protein linkage map.

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References

  1. T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, “A comprehensive two-hybrid analysis to explore the yeast protein interactome,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 8, pp. 4569–4574, 2001.
  2. Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989;340:245–246.
  3. Keegan L, Gill G, Ptashne M. Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science. 1986;231:699–704.
  4. P. Uetz, L. Glot, G. Cagney et al., “A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae,” Nature, vol. 403, no. 6770, pp. 623–627, 2000.
  5. Chien, C.-T., P. L. Bartel, R. Sternglanz, and S. Fields. 1991. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. USA 88:9578–9582.

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