The yeast two-hybrid system for beginners.

All you need to know:

The two-hybrid system is a molecular genetic tool which facilitates the study of protein-protein interactions. If your two proteins interact, then a reporter gene (e.g. gal1-lacZ - the beta-galactosidase gene) is transcriptionally activated. And you get a colour reaction on specific media. You can use this to (1) study the interaction between two proteins which you expect to interact and/or (2) find proteins (prey) which interact with a protein you have already (bait).

If you want to know more:

The intact Gal4 protein is a transcriptional activator which has two separate functions (a DNA-binding domain and an activating domain).

This application of this knowledge is achieved by splitting the yeast Gal4 gene in two parts - the activating domain (in yellow above and below) and the binding domain (in white above and below) . The split protein does not work unless the two parts are in physical contact. It is possible to make the two parts come in contact by making two chimeric proteins where each of the two parts are translationally fused to two other proteins which interact - i.e. an artificial gene which is transcribed to make a single protein). The chimera interact with each other resulting in a transcriptional activation of the beta-galactosidase. Simple isn’t it ? (-: see below to see how this is achieved.

How to make the chimeric proteins:

The chimeric proteins are made as translation gene fusions in yeast. Two plasmids are made, one in which a chimeric protein is fused to the binding domain, the other where the protein is fused to the activating domain.These plasmids are then transformed into a suitable yeast strain and the individual chimeric proteins are produced in this cell.

Applications

You can use the system to:

test for interactions between two proteins for which you have the genes.
as a means of cloning a gene encoding a protein which interacts with a protein for which you have the gene.

The gene you have is called the bait. The gene you catch with the bait is the prey. This is achieved by making a cDNA library in a special vector so that the yeast translates the gene you have cloned as a cDNA as a fusion with the part of the Gal4 protein.

For further reading see e.g. Old and Primrose 5^th edition p. 261.

Both these applications were used in the Pto story (lecture 24 in disease resistance, 2001) - references to the relevant papres there

Papers using this technique in our own work:

Finnie, C., Andersen, C.H., Borch, J., Gjetting, S., Christensen, A.B., De Boer, A.H., Thordal-Christensen, H. and Collinge, D.B. 14-3-3 Proteins are involved in an epidermis-specific response to the barley powdery mildew fungus. Plant Molecular Biology in press. abstract
Feys, B.J., Moisen, L.J., Newman, M.-A., and Parker J.E. 2001. Direct Interaction Between the Arabidopsis Disease Resistance Signaling Proteins, EDS1 and PAD4. EMBO J. 20:5400-5411. abstract
Jahn, T., Fuglsang, A.T., Olsson, A., Brüntrup, M., Collinge, D.B., Volkmann, D., Sommarin, M., Palmgren M.G. and Larsson, C. (1997) 14-3-3 protein interacts directly with the C- terminal region of the plant plasma membrane H⁺ATPase. The Plant Cell 9: 1805-1814. abstract

Limitations

This technique is prone to limitations and an interaction indicated by Y2H is not proof that this interaction really takes place. Further evidence for an interaction can be obtained by:

Affinity chromatography followed by protein identification to discover which proteins can bind to a known protein.
Gel overlay assays which is like a western blot.
Co-immunoprecipitation
Various more sophisticated techniques like Spin resonance.

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David B. Collinge, created 16/1-01 revised 04-12-01