Two‐Hybrid Systems to Measure Protein–Protein Interactions


To understand how proteins function to control cellular processes, their interactions with other proteins must be identified and characterised. The yeast two‐hybrid system is a simple and efficient assay for protein interactions. In a yeast two‐hybrid assay, the two proteins to be tested are expressed in a yeast nucleus with each protein fused to one‐half of a transcription activator. If the two‐hybrid proteins interact, the transcription activator is reconstituted and turns on reporter genes that can be easily detected. This assay has been used to identify tens of thousands of protein interactions, to map protein interaction domains and to characterise mutant variants of proteins. A variety of related assays have been developed, all based on the ability of two interacting hybrid proteins to activate a reporter system. These assays along with the original yeast two‐hybrid assay contribute to the characterisation of the protein interactions – or protein interactome – for humans and a wide range of other organisms.

Key Concepts

  • The function of most proteins involves interacting with one or more other proteins.
  • A binary interaction is a direct physical interaction between two proteins.
  • Understanding a protein's function requires charting its binary interactions.
  • The interactome is all of the protein interactions for a particular cell or an entire organism.
  • Two‐hybrid assays detect binary protein interactions by expressing the two test proteins in cells as hybrids fused to protein moieties that when brought into proximity via the protein interaction produce a detectable signal.
  • In a yeast two‐hybrid assay, the two proteins to be tested for interaction are fused to the two halves of a transcription factor in yeast.
  • Two‐hybrid assays, like all protein interaction assays, can produce false positives, which are interactions that are detected in the assay even though they do not occur under normal conditions in vivo.
  • Two‐hybrid and other protein interaction assays can also result in missed interactions or false negatives.
  • Use of multiple different protein interaction assays can reduce the number of false negatives and provide cross‐validation to rule out false positives.

Keywords: two‐hybrid; yeast; protein interaction; interactome; networks; complementation

Figure 1. Two‐hybrid assays for protein–protein interactions. In a two‐hybrid assay, each protein to be tested is expressed fused to a tag, A or B. In this example, X interacts with Y (right) but not with Y′ (left). When A and B are brought near each other through the X–Y interaction, they activate a signal that can be detected (right). The signal may result from direct interaction between A and B, for example, if together A and B form an active enzyme. Alternatively, the signal may be generated when one tag becomes localised to a particular subcellular location as a result of the interaction, as in transcription‐based two‐hybrid systems.
Figure 2. In the transcription‐based yeast two‐hybrid system, one tag is a DNA‐binding domain (DBD) that binds to specific sites in a reporter gene. The other tag is a transcription‐activation domain (AD). An interaction between X and Y localises the AD to the reporter, where it activates transcription.


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Further Reading

Bartel PL and Field S (1997) The two‐hybrid system: a personal view. In: Bartel PL and Fields S (eds) The Yeast Two‐Hybrid System, pp. 3–7. Oxford, UK: Oxford University Press.

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Parrish JR, Gulyas KD and Finley RL Jr (2006) Yeast two‐hybrid contributions to interactome mapping. Current Opinion in Biotechnology 17 (4): 387–393.

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Suter B, Kittanakom S and Stagljar I (2008) Two‐hybrid technologies in proteomics research. Current Opinion in Biotechnology 19 (4): 316–323.

Vidal M, Cusick ME and Barabasi AL (2011) Interactome networks and human disease. Cell 144 (6): 986–998.

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Finley, Russell L, and Mairiang, Dumrong(Mar 2018) Two‐Hybrid Systems to Measure Protein–Protein Interactions. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0005980.pub3]