Yeast 2-Hybrid: Discovering Protein-Protein Interactions from Yeast to West

Thomas Detchemendy wrote this post as part of Dr. Stacy Krueger-Hadfield’s Science Communication course at the University of Alabama at Birmingham. He is a PhD student under the mentorship of Dr. Shahid Mukhtar. He is currently studying plant-microbial interactions in efforts to further elucidate bacterial-mediated redistribution of nutrients in Arabidopsis thaliana. Thomas earned his B.S. in Biology at the University of Tennessee at Chattanooga.

When they’re not being used to brew beer or bake bread, yeast can serve as a convenient model for discovering new information in molecular biology, such as protein-protein interactions among various biological systems. 

In the central dogma of molecular genetics, DNA is transcribed into RNA, which is then translated into a protein. After a protein is translated it may also interact with another protein to form a functional complex or dimer. 

This protein-protein interaction (PPI) could take place between two friendly proteins from the same cell, or between a pathogenic protein targeting another protein within its host. 

So, proteins interact, but how can we determine specifically, which proteins can bind together?

One solution could be yeast 2-hybrid! Although there is a plethora of methods to identify PPI, yeast 2-hybrid (Y2H) is a fast and reliable way to determine whether two proteins interact and all it requires is some basic cloning, yeast transformation, and observing growth or no growth of yeast colonies for a positive or negative interaction, respectively.   

How is a Y2H experiment performed? 

No matter which Y2H method is being used, the first step in designing the experiment involves cloning. In this case, we will discuss a modification to the GAL4-based Y2H system (Lopez & Mukhtar, 2017).In this procedure, the GAL4 transcription factor (TF) is split into two domains, the DNA-binding domain (DB) and the activation domain (AD), which are fused into two separate plasmids. The DNA binding domain plasmid contains a Leucine marker and the activation plasmid contains a Tryptophan marker, but how does this show protein interaction? 

Initially, we need clone each protein of interest into a DNA binding domain plasmid and an activation domain plasmid. For instance, if we want to know if protein X interacts with protein Y, we would clone protein X into the DNA binding domain (Bait) plasmid and protein Y into the activation domain (Prey) plasmid or vice versa. As long as each protein is in a separate plasmid, we can test their interaction! 

The next step is to transform our cloned plasmids into the respective yeast strains. DNA binding domain and activation domain plasmids are transformed into strains Y8930 and Y8800, respectively. 

As it was mentioned earlier, each plasmid has a specific marker: -Leucine for the DNA binding domain and -Tryptophan for the activation domain plasmid. These markers allow us to confirm whether our cloned plasmids have been successfully transformed into each yeast strain by visualizing growth of each strain on amino acid dropout media containing -Leucine or -Tryptophan for DNA binding domain and activation domain clones, respectively. Once confirmed, the strains are mated together to yield diploid yeast containing both the DNA binding domain and activation domain plasmids.

Positive and Negative Yeast two Hybrid Interaction

 If the proteins cloned into the DNA binding domain and activation domain plasmids do interact, the DNA binding domain and activation domain will bind together, resulting in the reconstitution of the GAL4 TF, and a reporter gene (His3) will be activated (Lopez & Mukhtar, 2017). This activation would result in yeast growth on -Leucine/-Tryptophan/-Histidine amino acid dropout media, which yields a positive interaction. Now, you can repeat this on as many proteins as you’d like! 

The applications of Y2H have been used in multiple studies, ranging from plants to cancer related PPI studies. For example, Y2H has been implemented to identify novel proteins in Arabidopsis thalianathat are targeted by various pathogenic effector proteins during an infection (Mukhtar et al., 2011). Likewise, a study interested in ECRG2, a protein associated with malignant esophageal carcinoma, colon cancer and brain tumor tissues, also utilized the Y2H system to identify proteins associated with the ECRG2 protein to further elucidate its role in tumor formation (Cui et al., 2003). Thus, if your project has anything to do with protein interaction, yeast 2-hybrid is a great starting point! 


Cui, Y.-P., Wang, J.-B., Zhang, X.-Y., Bi, M.-X., Guo, L.-P., & Lu, S.-H. (2003). Using yeast two-hybrid system to identify ECRG2 associated proteins and their possible interactions with ECRG2 gene. World journal of gastroenterology, 9(9), 1892-1896.

Lopez, J., & Mukhtar, M. S. (2017). Mapping Protein-Protein Interaction Using High-Throughput Yeast 2-Hybrid. In W. Busch (Ed.), Plant Genomics: Methods and Protocols (pp. 217-230). New York, NY: Springer New York.

Mukhtar, M. S., Carvunis, A.-R., Dreze, M., Epple, P., Steinbrenner, J., Moore, J., . . . Dangl, J. L. (2011). Independently Evolved Virulence Effectors Converge onto Hubs in a Plant Immune System Network.

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