Yeast Two-hybrid System

The yeast two-hybrid system. 

a, Different ORFs are expressed as fusion proteins to either the GAL4 DNA-binding domain (GAL4-BD) or its activation domain(GAL4-AD). If the proteins encoded by the ORFs do not interact with each other, the fusion proteins are not brought into close proximity and there is no activation of transcription of the reporter gene containing the upstream GAL4-binding sites.
b, If the ORFs encode proteins that interact with each other, the fusion proteins are assembled at the GAL4-binding site of the reporter gene, which leads to activation of transcription. 
c, Library-based yeast two-hybrid screening method. In this strategy, two different yeast strains containing two different cDNA libraries are prepared. In one case, the ORFs are expressed as GAL4-BD fusions and in the other case, they are expressed as GAL4-AD fusions. The two yeast strains are then mated and diploids selected on deficient media. Thus, only the yeast cells expressing interacting proteins survive. The inserts from both the plasmids are then sequenced to obtain a pair of interacting genes.

The yeast two-hybrid system has emerged as a powerful tool to study protein–protein interactions. It is a genetic method based on the modular structure of transcription factors wherein close proximity of the DNA-binding domain to the activation domain induces increased transcription of a set of genes. The yeast hybrid system uses ORFs fused to the DNA-binding or -activation domain of GAL4 such that increased transcription of a reporter gene results when the proteins encoded by two ORFs interact in the nucleus of the yeast cell. One of the main consequences of this is that once a positive interaction is detected, the ORF is identified simply by sequencing the relevant clones. For these reasons it is a generic method that is simple and amenable to high-throughput screening of protein–protein interactions.

On a large scale, this strategy has been used in two formats. In the array method, yeast clones containing ORFs as fusions to DNA or activation domains are arrayed onto a grid and the ORFs to be tested (as reciprocal fusions) are screened against the entire grid to identify interacting clones. In the library screening method, one set of ORFs are first pooled to generate a library and then the reciprocal ORF–fusions are mated with the library one by one or several at a time. 

Such analyses on a genome-wide scale have already been reported in Saccharomyces cerevisiae and to a more limited extent in Caenorhabditis elegans. In yeast, the array method was performed on 192 ORFs and the library screening method for 87% of the yeast genome. Together, this experiment resulted in 957 putative interac-tions. Another group analysed the results of 10% of an exhaustive library screen in yeast, resulting in 183 putative interactions. The vast majority of the interactions found in these two large-scale studies were new. Several of these interactions seem plausible based on previous genetic or biochemical studies, whereas the relevance of most others cannot easily be determined. Therefore, such studies provide only potential interactions that have to be confirmed or eliminated by further biological experimentation. The main advantage of these methods is that they can be performed with a high throughput and in an automated manner. A recently described modification of the yeast two-hybrid method, termed ‘reverse’ two hybrid, can be used for identification of compounds and peptides that disrupt protein–protein interactions. This can lead to development of drugs that have activities in vivo as opposed to drug screens that are conventionally done in vitro.

The following references may tell you a lot about this technology and its application in proteomics:

Two-hybrid analysis of genetic regulatory networks   (Finley Lab)
This is one of the most comprehensive and detailed guide to yeast two-hybrid system technique with introduction to its background.

Protein-interaction mapping for functional proteomics

Yeast Two-Hybrid: State of the Art

Genome-wide protein interaction maps using two-hybrid systems  (PDF, Recommended)

From the analysis of protein complexes to proteome-wide linkage maps (PDF)