Yeast two-hybrid, a powerful tool for systems biology

Abstract

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.

Keywords: interactomics; mass spectrometry; protein-protein interaction; systems bioenergetics; yeast two-hybrid.

Figure 1.

The classical yeast two-hybrid system. (A) The protein of interest X, is fused to the DNA binding domain (DBD), a construct called bait. The potential interacting protein Y is fused to the activation domain (AD) and is called prey. (B) The bait, i.e. the DBD-X fusion protein, binds the upstream activator sequence (UAS) of the promoter. The interaction of bait with prey, i.e. the AD-Y fusion protein, recruits the AD and thus reconstitutes a functional transcription factor, leading to further recruitment of RNA polymerase II and subsequent transcription of a reporter gene.

Figure 2.

Yeast two-hybrid systems, their subcellular location within a yeast cell, and their operating mode (represented at the moment of bait-prey interaction). Protein X (dark blue puzzle piece, part of bait construct) and protein Y (light blue puzzle piece, part of prey construct) directly interact (fitting puzzle pieces), thus inducing reconstitution of split-proteins (puzzle pieces of different colors in A, D, E), membrane recruitment (B, C) or protein dimerization (F). Protein fusions in bait or prey constructs are shown as solid black lines between puzzle pieces. Bait-prey interaction activates further downstream events (arrows) that directly (A) or indirectly (B, C, D, F) lead to transcriptional activation, or are independent of transcriptional activation (D, E), finally yielding screenable readouts like growth on specific media or color reactions. (A) Nuclear Y2H systems all require protein recruitment and bait-prey interaction at nuclear DNA. The classic Y2H and RTA Y2H both engage RNA polymerase II (RNA Pol II) transcription either by its activation or its inhibition. By contrast, the Pol III Y2H, involves RNA polymerase III (RNA Pol III) transcription. (B) Ras signalling based Y2H at the plasma membrane. The SRS Y2H, RRS Y2H, and rRRS Y2H are all based on protein recruitment to the plasma membrane via bait-prey interaction and subsequent activation of MAPK downstream signalling. While in the SRS and RRS Y2H the prey constructs harboring protein Y are anchored at the membrane via myristoylation to analyze interactions with cytosolic bait constructs harboring protein X, the rRRS is used to analyze interactions between soluble preys containing protein Y and partner X being a membrane protein. (C) G-protein signalling-based Y2H at the plasma membrane. In the G-protein fusion Y2H, bait X is a membrane or membrane-associated protein whose interaction with the prey construct disrupts protein G downstream signalling. (D) Split-ubiquitin based Y2H systems involve reconstitution of ubiquitin from two domains upon bait-prey interaction. Their subcellular localization depends on the nature of interacting proteins X or Y, and on the reporter proteins used. The Split ubiquitin Y2H uses non-transcriptional reporting of protein interactions in the cytosol, but can also be used for membrane proteins (not shown). The MbY2H is used for interaction analysis with membrane baits and thus occurs at the membrane location of protein X, e.g. the plasma membrane. The CytoY2H is used for membrane anchored cytosolic baits and occurs close to the ER membrane (E) Split-protein sensor Y2H. The Split-Trp Y2H is used to assay cytosolic bait-prey interactions based on reconstitution of an enzyme in tryptophan synthesis, allowing for non-transcriptional reporting. (F) ER Y2H system. The SCINEX-P Y2H allows bait-prey interaction analysis in the reducing environment of the ER, based on protein dimerization in unfolded protein signalling. ER, endoplasmic reticulum; for further abbreviations and details see chapter 3.2.