Exploring protein interactions: yeast two hybrid systems

>Proteins are one of the key molecules inside cells; involved in signalling, intracellular transport, metabolism and gene control. They rarely work alone, most of the proteins in the cell are part of large complex networks consisting of many interacting proteins. Various techniques exist in order to find these interactions, and one of the most common is the use of yeast two hybrid systems.

Yeast is apparently quite a nice organism to work with (I've never worked with it myself, I must say, apart from a few practicals in second year, where I almost set my lab-partners hand on fire). The genome is fully annotated, the organism is well characterised, and yeast grows and responds quite fast, so experiments shouldn't take too long.

The yeast-two hybrid system is based around molecules called transcription factors, which are normally used by the cell to active gene expression. In yeast, there are several transcription factors which consist of two separate molecules, which need to be in close proximity in order for a gene to be expressed. If you attach an experimental protein to one half of the transcription factor, and another experimental protein (that you think interacts with the first) to the other half you can test for interactions. If the two proteins do interact then the transcription factors will be brought close together and the gene downstream of them (which acts as a reporter gene) will be expressed:The diagram above (taken from the reference below) shows this process. The blue jigsaw-shaped 'X' and 'Y' proteins are the experimental proteins, being tested for interactions and the yellow shapes are the two parts of the transcription factor. The big white cloud is the polymerase, which begins the process of turning the DNA into protein. The reporter gene can be set to code for a vital compound such as histidine; stick the whole system into a histidine deficient mutant and you have a marker system to see if the proteins interact. If they do, the histidine is produced and the cells can grow, if they don't, the cells die.

One of the most useful things about this technique is that it can be automated, and used to scan whole libraries of proteins to see if they interact. By using a matrix, each protein X (the 'bait' protein) can be given a defined position and then systematically exposed to a number of different protein Y (the 'prey' protein). If you're taking Y from a clonal library, and have a sufficiently intelligent robot, the whole procedure can be carried out with minimal human input.

The yeast two-hybrid system has been invaluable for determining many important protein-protein interactions however there are some problems with it. Firstly, this is a yeast two component system, and most protein complexes consist of many interacting proteins, certainly more than two! Secondly, this whole system relies on two soluble proteins interacting in the nucleus (where the DNA is) and so doesn't work for membrane bound protein interactions.

In view of this, several modifications have been made to the original methodology to make it more useful for trapping a wider range of protein interactions. It's been expanded into the three component system, which identifies proteins that interact with (or inhibit) both the the bait and the prey. Using another natural yeast system (the G protein system) has allowed transmembrane proteins to be identified as well:
In this system protein Y contains a binding site for a subunit of the G protein, while X is a membrane-spanning protein. G proteins are membrane bound proteins that activate transcription factors inside the nucleus. If the two proteins interact then the G protein subunit bound to the Y is sequestered away from the rest of the complex, and the G protein signal cannot be transmitted. There is therefore no signal to the transcription factors, meaning reporter genes (in this case genes for pigmentation rather than for death) will be turned off.

Even with these modifications there still are problems with the yeast two hybrid system. There is very little quantitative analysis involved to see how strongly the protein is binding, and the discovery of new interactions will always be limited by the choice of proteins to screen. However one of the largest advantages of yeast two hybrid systems is that it carries out protein binding analysis inside the cell, in proper cellular conditions, unlike affinity binding which, while it can identify much larger protein complexes, involves taking proteins out of the cell and handling them in vitro.

Yeast two hybrid systems are therefore still one of the main practical methods used for determining protein interactions. The field of systems biology is a fascinating one, and while attempting to catalogue the whole spectrum of cellular interactions may seem like a daunting task ('interactomics' for the funding people) working towards it will only reveal more and more useful information about the complex and fascinating networks of proteins within the cell.

[btw: The reference below is a great source of information about the many different modifications made to the basic yeast two hybrid system, thoroughly recommended for anyone interested in protein interactions or systems biology]

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Brückner A, Polge C, Lentze N, Auerbach D, & Schlattner U (2009). Yeast two-hybrid, a powerful tool for systems biology. International journal of molecular sciences, 10 (6), 2763-88 PMID: 19582228