Unveiling Protein Interactions: The Yeast Two-Hybrid (Y2H) Assay

Kaushik Desai

1 year ago

Introduction:

In the vast world of molecular biology, understanding protein-protein interactions is crucial for deciphering the intricate workings of biological systems. The Yeast Two-Hybrid (Y2H) assay has emerged as a powerful tool to explore and uncover these interactions. This blog post dives into the principle, procedure, variations, and applications of the Y2H assay, shedding light on its significance in modern scientific research.

Principle of the Yeast Two-Hybrid Assay:

The Y2H assay utilizes the fundamental molecular properties of transcriptional activation and protein-protein interactions within the context of yeast cells. The assay is based on the modular nature of transcription factors, where the DNA-binding domain (DBD) and the activation domain (AD) can be split and reconstituted to investigate protein interactions.

The principle behind the Y2H assay involves the construction of two key plasmids: the DNA-binding domain (DBD) fusion library and the activation domain (AD) fusion library. The DBD fusion library contains a DNA-binding domain fused to a protein of interest, while the AD fusion library consists of an activation domain fused to a potential interacting protein.

When the DBD and AD fusion libraries are co-transformed into yeast cells, a protein-protein interaction between the two fused proteins leads to reconstitution of the transcription factor. This reconstituted transcription factor then activates the transcription of reporter genes, resulting in a detectable phenotype, such as growth on selective media.

Procedure of the Yeast Two-Hybrid Assay:

The Y2H assay involves several sequential steps, as outlined below:

Construction of bait and prey libraries: The bait library is created by fusing the protein of interest with the DNA-binding domain (DBD), while the prey library consists of potential interacting proteins fused with the activation domain (AD).
Co-transformation: The bait and prey libraries are co-transformed into a suitable yeast strain lacking the endogenous transcription factor.
Selection of positive interactions: The transformed yeast cells are plated on selective media lacking certain nutrients or containing specific toxins. Growth on selective media indicates potential protein-protein interactions.
Verification of interactions: Positive interactions are further confirmed through additional tests, such as β-galactosidase activity or fluorescent reporter assays. These tests provide quantitative measurements of the strength of protein interactions.
Isolation and identification of interacting proteins: The interacting proteins are isolated from yeast cells and subjected to further analysis, such as mass spectrometry, to identify the specific interacting partners.

Variations of the Yeast Two-Hybrid Assay:

Over the years, researchers have developed variations of the Y2H assay to overcome certain limitations and explore specific aspects of protein-protein interactions. Some notable variations include:

Reverse Yeast Two-Hybrid (RMYTH): In RMYTH, the bait and prey fusions are reversed compared to the conventional Y2H assay. This approach allows the study of protein interactions in the context of different cellular compartments.
Split-Ubiquitin System: The split-ubiquitin system employs a different reporter system based on the reconstitution of ubiquitin. Interaction between the bait and prey proteins leads to the reconstitution of a functional ubiquitin, resulting in the release of a reporter protein or the rescue of a selectable marker.
Membrane Yeast Two-Hybrid (MYTH): MYTH enables the investigation of protein interactions occurring at cellular membranes. It employs a modified Y2H system where the bait protein is anchored to the yeast plasma membrane.

Applications of the Yeast Two-Hybrid Assay:

The Y2H assay has found diverse applications in various areas of biological research, some of which include:

Mapping Protein-Protein Interactions: The Y2H assay allows researchers to map the intricate network of protein interactions within a cell. By identifying protein partners and their interactions, this assay contributes to the understanding of complex cellular processes and signaling pathways.
Drug Discovery: Identifying protein interactions that are crucial for disease progression can provide potential targets for drug development. The Y2H assay has been used to screen small molecules or candidate drugs for their ability to disrupt or modulate protein-protein interactions involved in diseases like cancer.
Studying Signaling Pathways: Investigating protein interactions involved in cellular signaling pathways helps unravel the mechanisms underlying normal and pathological processes. The Y2H assay enables the exploration of signaling cascades and regulatory networks by studying protein interactions in a controlled environment.
Functional Genomics: The Y2H assay can be employed to investigate the functions of uncharacterized proteins by identifying their interaction partners. By understanding the network of protein interactions, researchers gain insights into the roles and potential functions of these proteins.

Conclusion:

The Yeast Two-Hybrid (Y2H) assay has revolutionized the study of protein-protein interactions, providing a powerful tool for molecular biologists to explore the intricate molecular machinery of cells. By unraveling protein interactions, this assay contributes to our understanding of biological systems and aids in various fields, including drug discovery and functional genomics. As researchers continue to refine and expand the Y2H assay, its applications will undoubtedly uncover new insights into the complex world of protein interactions.

Test You Knowledge

What is the principle of the Yeast Two-Hybrid (Y2H) assay?

The Y2H assay is based on the modular nature of transcription factors and utilizes protein-protein interactions within yeast cells to reconstitute a functional transcription factor.

What are the two key plasmids used in the Y2H assay?

The Y2H assay involves the construction of the DNA-binding domain (DBD) fusion library and the activation domain (AD) fusion library.

How is positive interaction detected in the Y2H assay?

Positive interactions are detected through a phenotype, such as growth on selective media, resulting from the reconstitution of a functional transcription factor and subsequent activation of reporter genes.

What are some variations of the Y2H assay?

Some variations include Reverse Yeast Two-Hybrid (RMYTH), Split-Ubiquitin System, and Membrane Yeast Two-Hybrid (MYTH).

What is the significance of the Y2H assay in drug discovery?

The Y2H assay helps identify protein interactions that are important for disease progression, providing potential targets for drug development.

How does the Y2H assay contribute to studying signaling pathways?

By investigating protein interactions, the Y2H assay allows researchers to unravel the mechanisms and regulatory networks of cellular signaling pathways.

What is the role of the Y2H assay in functional genomics?

The Y2H assay helps identify interaction partners of uncharacterized proteins, providing insights into their functions and potential roles.

How does the Y2H assay aid in mapping protein-protein interactions?

The Y2H assay enables the identification of protein partners and their interactions, helping researchers understand the complex network of protein interactions within cells.

What additional tests can be used to verify protein interactions in the Y2H assay?

Additional tests, such as β-galactosidase activity or fluorescent reporter assays, can be used to quantitatively measure the strength of protein interactions.

How are interacting proteins identified in the Y2H assay?

Interacting proteins can be isolated from yeast cells and subjected to further analysis, such as mass spectrometry, to identify their specific interacting partners.

References:

Fields, S., & Song, O. (1989). A novel genetic system to detect protein–protein interactions. Nature, 340(6230), 245-246.

Bartel, P. L., & Fields, S. (1995). Analyzing protein–protein interactions using two-hybrid system. Methods in enzymology, 254, 241-263.

Uetz, P., et al. (2000). A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature, 403(6770), 623-627.

Ito, T., et al. (2001). A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proceedings of the National Academy of Sciences, 98(8), 4569-4574.

Giot, L., et al. (2003). A protein interaction map of Drosophila melanogaster. Science, 302(5651), 1727-1736.

Keywords:

Yeast Two-Hybrid assay, protein-protein interactions, principle, procedure, variations, applications