Yeast Two-Hybrid (Y2H) Screening System

      Y2H was first described by Fields and Song [1989] and is based on the modularity of binding domains in eukaryotic transcription factors. Eukaryotic transcription factors have at least two distinct domains: (1) the DNA binding domain (BD) which directs binding to a promoter DNA sequence (upstream activating sequence (UAS)); and (2) the transcription activating domain (AD) which activates the transcription of target reporter genes. Splitting the BD and AD domains inactivates transcription, but even indirectly connecting AD and BD can restore transcription resulting in activation of specific reporter genes. Plasmids are engineered to produce a protein product (chimeric or “hybrid”) in which the BD fragment is in-frame fused onto a protein of interest (the bait), while the AD fragment is in-frame fused onto another protein (the prey) (Figure 2.1). The plasmids are then transfected into cells chosen for the screening method, usually from yeast. If the bait and prey proteins interact, the AD and BD domains are indirectly connected, resulting in the activation of reporters within nuclei of cells. Typically, multiple independent yeast colonies are assayed for each combination of plasmids to account for the heterogeneity in protein expression levels and their ability to activate reporter transcription.

      This basic Y2H technique has been improved over the years to enable large library screening [Chien et al. 1991, Dufree et al. 1993, Gyuris et al. 1993, Finley and Brent 1994]. Interaction mating is one such protocol that can screen more than one bait against a library of preys, and can save considerable time and materials. In this protocol, the AD- and BD-fused proteins begin in two different haploid yeast strains with opposite mating types. These proteins are brought together by mating, a process in which two haploid cells fuse to form a single diploid cell. The diploids are then tested using conventional reporter activation for possible interactors. Therefore, different bait-expressing strains can be mated with a library of prey-expressing strains, and the resulting diploids can be screened for interactors. It is important to know how many viable diploids have arisen and to determine the false-positive frequency of the detected interactions. True interactors tend to come up in a timeframe specific for each given bait, with false positives clustering at a different timepoint. Multiple yeast colonies are assayed to confirm the interactors.

      Figure 2.1 Schematic representation of the yeast two-hybrid protocol to detect interaction between bait and prey proteins. If the bait and prey proteins interact, the DNA binding domain (BD) fused to the bait and the transcription activing domain (AD) fused to the prey are indirectly connected resulting in the activation of the reporter gene. UAS: upstream activating sequence (promoter) of the reporter gene.

      Y2H screens have been extensively used to detect protein interactions among yeast proteins, with two of the earliest studies reporting 692 [Uetz et al. 2000] and 841 [Ito et al. 2000] interactions for S. cerevisiae. In the bacteria Helicobacter pylori, one of the first applications of Y2H identified over 1,200 interactions, covering about 47% of the bacterial proteome [Rain et al. 2001]. Applications on fly proceeded on an even greater scale when Giot et al. [2003] identified 10,021 protein interactions involving 4,500 proteins in D. melanogaster. More recently, Vo et al. [2016] used Y2H to map binary interactions in the yeast S. pombe (fission yeast). This network, called FissionNet, consisted of 2,278 interactions covering 4,989 protein-coding genes in S. pombe. The Y2H system has also been applied for humans, with two initial studies [Rual et al. 2005, Stelzl et al. 2005] yielding over 5,000 interactions among human proteins. More recently, Rolland et al. [2014] employed Y2H to characterize nearly 14,000 human interactions.

      However, inherent to this type of library screening, the number of detected false-positive interactions is usually high. Among the possible reasons for the generation of false positives is that the experimental compartmentalization (within the nucleus) for bait and prey proteins does not correspond to the natural cellular compartmentalization. Moreover, proteins that are not correctly folded under experimental conditions or are “sticky” may show non-specific interactions. The third source of false positives is the interaction of the preys themselves with reporter proteins, which can turn on the reporter genes. Von Mering et al. [2002] estimated the accuracy of classic Y2H to be less than 10%, with subsequent evaluations suggesting the number of false positives to be between 50% and 70% in large-scale Y2H interaction datasets for yeast [Bader and Hogue 2002, Bader et al. 2004].

       Co-Immunoprecipitation/Affinity Purification (AP) Followed by Mass Spectrometry (Co-IP/AP followed by MS)

      Complementing the in vivo Y2H screens are the in vitro Co-IP/AP followed by MS screens that identify whole complexes of interacting proteins, from which the binary interactions between proteins can be inferred [Golemis and Adams 2002, Rigaut et al. 1999, Köcher and Superti-Furga 2007, Dunham et al. 2012]. The Co-IP/AP followed by MS screens consist of two steps: co-immunoprecipitation/affinity purification and mass spectrometry (Figure 2.2). In the first step, cells are lysed in a radioimmunoprecipitation assay (RIPA) buffer. The RIPA buffer enables efficient cell lysis and protein solubilization while avoiding protein degradation and interference with biological activity of the proteins. A known member of the set of proteins (the protein of interest or bait) is epitope-tagged and is either immunoprecipitated using a specific antibody against the tag or purified using affinity columns recognizing the tag, giving the interacting partners (preys) of the bait. Normally, this purification step is more effective when two consecutive purification steps are used with proteins that are doubly tagged (hence called tandem affinity purification or TAP). This results in an enrichment of native multi-protein complexes containing the bait. The individual components within each such purified complex are then screened by gel electrophoresis and identified using mass spectrometry.

      Figure 2.2 Schematic representation of the co-immunoprecipitation/affinity purification followed by mass spectrometry (Co-IP/AP followed by MS) protocol. The protein of interest (bait) is targeted with a specific antibody and pulled down with its interactors in a cell lysate buffer. The individual components of the pulled-down complex are identified using mass spectrometry. These days, liquid chromatography with mass spectrometry (LCMS) instead of running the gel is increasingly being used more frequently for as a combined physical-separation and MS-analysis technique [Pitt 2009].

      In one of the first applications of TAP/MS, Ho et al. [2002] expressed 10% of the coding open reading frames from yeast, and the identified interactions connected 25% of the yeast proteome as multi-protein complexes. Subsequently, Gavin et al. [2002], Gavin et al. [2006], and Krogan et al. [2006] purified 1,993 and 2,357 TAP-tagged proteins covering 60% and 72% of the yeast proteome, and identified 7,592 and 7,123 protein interactions from yeast, respectively. One of the first proof-of-concept studies for humans applied AP/MS to characterize interactors using 338 bait proteins that were selected based on their putative involvement in diseases, and the study identified 6,463 interactions between 2,235 proteins [Ewing et al. 2007].

       Comparison of Y2H and AP/MS Experimental Techniques

      A majority of the interaction data collected so far has come from Y2H screening. For example, approximately half of the data available in databases including IntAct [Hermjakob et al. 2004, Kerrien et al. 2012] and MINT [Zanzoni et al. 2002, Chatr-Aryamontri et al. 2007] are from Y2H screens [Brückner et al. 2009] (more sources of PPI data are listed in Table