In 1983, while investigators had identified a few human proteins as important regulators of specific biological outcomes, how these proteins acted in the cell was essentially unknown in almost all cases. Twenty-five years later, our knowledge of the mechanistic basis of protein action has been transformed by our increasingly detailed understanding of protein-protein interactions, which have allowed us to define cellular machines. The advent of the yeast two-hybrid (Y2H) system in 1989 marked a milestone in the field of proteomics. Exploiting the modular nature of transcription factors, the Y2H system allows facile measurement of the activation of reporter genes based on interactions between two chimeric or “hybrid” proteins of interest. After a decade of service as a leading platform for individual investigators to use in exploring the interaction properties of interesting target proteins, the Y2H system has increasingly been applied in high-throughput applications intended to map genome-scale protein-protein interactions for model organisms and humans. Although some significant technical limitations apply, Y2H has made a great contribution to our general understanding of the topology of cellular signaling networks.

Introduction

Within the last 25 years, the yeast two-hybrid (Y2H) system has helped transform our understanding of the molecular landscape of a cell. By 1983, mammalian application of increasingly powerful molecular biological techniques began to identify novel genes that encoded proteins that were important for study based on their functional criteria. For example, mutation of the oncogene Ras (1) or amplification of the oncogene Myc (2) was known to cause cancer in humans. The proteins encoded by these and other important genes were then further characterized primarily by simple overexpression of intact, truncated, or mutated derivatives of the protein of interest, followed by measurements of changes in gross cellular processes such as proliferation or morphological change. This “isolationist,” single protein–centered approach was marked by the very limited nature of mechanistic insights that could be gained from the relationship between a primary protein of interest and its protein partner(s). For some proteins such as Ras that had known orthologs in lower eukaryotes (3), some insights into relevant signaling pathways could be gleaned by inference from genetic studies in those organisms (4). For proteins without orthologs such as Myc, options were more restricted. In 1983, the primary means of identifying interacting proteins was through biochemical co-purification and protein sequencing. These techniques were difficult, laborious, and expensive.

As databases of potentially interesting proteins burgeoned through the 1980s, this pattern continued. While most signaling proteins were thought or known to have at least one effector, existing signaling pathways were sparsely populated and drawn to indicate linear connections between interacting proteins. At its initial description in 1989 (5), it was not apparent that the Y2H assay was a technological advance that would transform our understanding of cell biology (6,7,8,9,10,11). Initially described as a simple yet powerful tool to detect protein-protein interactions (PPIs), Y2H technology evolved in order to address PPIs of increasing complexity and to ultimately be applied on a genome-wide scale. As most revolutionary technologies are judged by the physical or intellectual products they create, we believe that the Y2H system can be judged by the changing paradigm of protein interactions it helped usher in: from binary protein interactions of 25 years ago, to canonical pathways, and, most recently, interconnected protein interaction networks.

Y2H Basics

The original Y2H system was developed as an assay to study binary protein interactions between proteins already known or those strongly suspected to associate (5). The system capitalizes on the modular nature of transcription factors with split domains that can reconstitute protein function upon physical interaction (12). The protein of interest (X), or bait, is fused to the DNA-binding domain (DBD) of a transcription factor such as Gal4 or LexA. ((Figure 1)). The potential interactor protein (Y), or prey, is fused to a transcriptional activating domain (AD). DBD-X and AD-Y are co-expressed in an appropriate yeast strain that has been engineered to contain reporter genes with transcription depending on the association of DBD-X and AD-Y at the promoter. While the first described Y2H system used only a lacZ colorimetric reporter that was not amenable to high-throughput usage using 1980s technology, the subsequent addition (8,10) of secondary auxotrophic reporters opened the door for the use of Y2H for library screening. Some of the very earliest uses of Y2H for such screening involved oncoproteins such as Ras and Myc (11,13). The instructive case of screening with Ras set forth what was to become a typical paradigm: although for a number of years there had been accumulating evidence suggesting a functional relationship between Ras and a second oncoprotein, Raf, Y2H screening clarified the observation that a direct physical association underlies their functional relation (11). Suddenly, proteins could quickly find partners.

Figure 1.

The establishment of Y2H library screening capabilities allowed many investigators to try to identify other proteins that interacted with their protein of interest. In the approach that was standard in the early 1990s, a yeast strain expressing bait and reporter constructs that had passed certain optimization steps (see next section on Y2H limitations) would be supertransformed with a cDNA library expressing AD-fused proteins. Then 1–3 × 10⁶ transformants would be scored for activation of lacZ and the auxotrophic reporters. The basic technical manipulations required little more than low-cost microbiological growth media and minimal laboratory equipment, and were readily accomplishable by any laboratory with basic competence in molecular biology. The requisite plasmids and yeast strains were essentially free, distributed from the laboratories of the inventors of the technique. From start to finish, a screen could be performed in less than 6 weeks. These advantages underlie the essentially democratic nature of Y2H screening: with such a low investment cost, many could afford to attempt the approach.