Yeast 2 hybrid system

Figure 5.1. Yeast two-hybrid system. Interaction of proteins X and Y upstream of a reporter gene leads to transcriptional activation. Protein X is part of a fusion protein that binds to a site on DNA upstream of the reporter gene by means of a DNA binding domain. Protein Y is part of a fusion protein that contains a transcriptional activation domain. Interaction of proteins X and Y places the activation domain in the vicinity of the reporter gene and stimulates its transcription.

A bacterial two-hybrid system has been developed that, similar to the yeast system, functions via activation of transcription (Dove and Hochschild, 1998 Joung et al., 2000). RNA polymerase (RNAP) in E. coli consists of an enzymatic core composed of the a, (3, and (3 subunits in the stoichiometry a2(3(3, and one of several c factors that enable the enzyme to recognize specific promoters (Heilman and Chamberlin, 1988). Many bacterial transcriptional activator proteins bind the promoters they regulate and interact directly with subunits of RNAP. The most commonly observed contact is between activator proteins and the a subunit of RNAP (Ebright and Busby, 1995). The function of the a subunit is to initiate the assembly of RNAP by forming a dimer (Igarashi et al., 1991). 

The bacterial one and two-hybrid systems have potential advantages over the yeast two-hybrid system due to the higher transformation efficiency and faster growth rate of coli. To date, however, the bacterial two-hybrid system has not been used for genome-scale analysis of protein-protein interactions. 

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

Bai, C., and Elledge, S. J. (1997). Gene identification using the yeast two-hybrid system. Methods Enzymol. 283, 141-156. 

Expression of a recombinant protein using an inducible vector system would permit expression at endogenous levels to simulate physiologic levels of expression of a protein of interest. Tandem affinity purification strategies have recently been employed and facilitate the analyses of highly interactive proteins when the bait protein is expressed at endogenous levels. Immunoaffinity or immunoprecipitation followed by LC-MS/MS does not readily permit determination of the stoichiometry of interacting partners. Additionally, when compared to yeast hybrid experiments, it is difficult to determine whether interactions are binary when identified in complexes by MS/MS. 

M. C., Yu, C. H., Lin, P. I., and Wu, W. F. Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system. /. Bacteriol. 2003, 185, 2393-2401. 

The Sos recruitment yeast two-hybrid system was developed by Aronheim and colleagues (1997). It is now known as the CytoTrap yeast two-hybrid system and marketed by Stratagene (La Jolla, California, USA). The CytoTrap system differs from both the GAL4 and the LexA systems in that it is not dependent on transcription factor activation in the nucleus for the detection of protein-protein association. Instead, protein interactions are detected in the cytoplasm and involve the reconstitution of the Sos/Ras signaling pathway in conjunction with the temperature-sensitive yeast strain, cdc25H. 

Schematic diagram outlining the principle of the CytoTrap, Sos recruitment, yeast two-hybrid system. The figure shows the bait and fish fusion constructs. The bait vector encodes a Sos fusion protein. The fish vector encodes a myristylation sequence that targets the fusion protein to the cell membrane. Interaction between the fish and bait proteins targets the Sos fusion protein to the intracellular face of the cell membrane where it can interact with Ras and rescue cell growth at 37°C...

Identification of Interacting Proteins Using Transcription Factor-Based Yeast Two-Hybrid Systems... 

By far, the most widely used application of the yeast two-hybrid system as intimated in Introduction is the identification of protein partners for a test protein of either known or unknown function. Here, the DNA encoding the test protein or the domain of a test protein is cloned in frame into the bait vector. The fish vector contains cDNA and it is constructed so that there is one cDNA molecule per vector. Fish and bait vectors are cotransformed into the appropriate strain of competent yeast, and the resultant transformed yeast cells are screened for growth on SD media and for reporter gene activities. Putative positive clones are then isolated and characterized further. In the next section, each of these stages is discussed in detail. 

Autoactivation may be circumvented by modification of the bait protein usually by the construction of truncation or deletion bait proteins that do not yield autoactivation. The disadvantage is that this may result in the loss of protein-protein interaction domains. Again, the use of two different yeast two-hybrid systems in parallel would negate these potential problems. O Figure 19-4 shows an example of manipulation... 

Bartel PL, Fields S, editors. 1997. The yeast two-hybrid system. Oxford, UK Oxford University Press. 

Clontech MATCHMAKER GAL4 Two-hybrid system 3 and libraries user manual. Clontech yeast protocols handbook. 

Smith MJ, Beck M, Stephenson FA. 2003. Can the yeast two-hybrid system be used to study assembly domains of GABA-A receptor subunits British Neurosci Assoc Abstr 17 26.12. 

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