PROTEIN INTERACTIONS: YEAST TWO HYBRID

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The yeast two-hybrid system (Fields and Song, 1989) is one of the most widely used and well-proven methods to detect biologically important protein-protein interactions (for review see (Brent and Finley, 1997; Fields and Sternglanz, 1994). In these systems the two proteins to be tested for interaction are expressed in the nucleus of a yeast cell, where their interaction leads to activation of reporter genes. Both proteins are expressed with amino-terminal fusion moieties taken from a transcription factor. One protein is fused to the DNA-binding domain (DBD) and the other is fused to a transcription activation domain (AD). If the two proteins interact, the AD activates transcription of reporter genes that contains binding sites for the DBD. Yeast two-hybrid systems offer several advantages over other approaches for detecting protein-protein interactions. First, the assay is conducted inside cells, which may mimic the appropriate physiological conditions for some interactions better than in vitro assays. Second, the two-hybrid assay is robust. It has been used successfully with a variety of different protein types including cytoplasmic and nuclear proteins, and surprisingly, even some proteins that are normally found in membranes or that are extracellular (e.g. ref. (Li et al., 1996; Paine and Snead, 1997). Third, the yeast two-hybrid assay is simple and does not require expensive reagents or instrumentation. Fourth, the assay can be conducted with proteins from organisms lacking robust expression systems, which are often necessary for alternative protein interaction assays; e.g., assays that require the availability of an affinity tag for at least one of the proteins, are often achieved by expressing an epitope- or affinity-tagged fusion protein. Finally, the yeast two-hybrid system is a mature technology with well-understood advantages and pitfalls. Some laboratories, including ours, have been working with the system for over 10 years. We and others have gained the necessary experience to understand the experimental nuances and, most importantly, to properly interpret the data, distinguishing, for example, true positives from likely false positives. In the last 7 years, over 2,800 papers have been published that describe the functional characterization of new proteins or protein interactions that were initially detected using a two-hybrid approach (unpublished analysis). Thus, the value of using yeast two-hybrid data to generate hypotheses and to guide functional studies is well established. This success inspired us and a handful of other researchers to develop approaches to scale-up the system to identify the interactions among large sets of proteins, including entire proteomes.