4.2.  Yeast two‐hybrid (Y2H) system and other two‐hybrid systems

A transcriptional activator in yeast GAL4 contains two distinct domains, a DNA‐binding domain (DBD) and an activation domain (AD), which independently are nonfunctional as a transcription factor, but reconstitute a functional GAL4 transcription factor when they are in close proximity.219 Inspired by this finding, Fields and Song first described the Y2H method in 1989.220 They fused two yeast proteins bait and prey to the DBD and AD of GAL4, respectively. As bait and prey were known to interact, co‐expression of these two hybrid proteins in yeast reconstituted proximity of the GAL4 domains, leading to transcription of a GAL1‐lacZ reporter gene that could be analyzed by β‐galactosidase activity measurement (Figure 3 B). Y2H is a powerful technique for identifying and analyzing PPIs in vivo. However, as Y2H requires the translocation of the interacting proteins into the nucleus, it′s not suitable for membrane proteins or proteins localized in other subcellular compartment. Other unsuited proteins include transcriptional activators, which produce spontaneous activation of the transcription of the reporter gene independent of bait–prey interaction. Moreover, Y2H cannot detect PPIs involving post‐translational modifications that do not take place in yeast.221

To circumvent the limitations of Y2H and extend its application, a number of variant two‐hybrid systems have been developed:

(1) The repressed transactivator (RTA) system was established for detection of interactions with transcriptional activator proteins.222 An auto‐activating bait protein is fused to DBD of GAL4, whereas prey is fused to the repression domain (RD) of a transcription repressor TUP1. Therefore, bait–prey interaction causes repression of a reporter gene. This method has been applied to screen for various transcriptional activators, such as VP16,222 c‐Myc,222, 223, 224 Mitf,225 and androgen receptor.226, 227, 228

(2) RNA polymerase III (Pol III) system can be used to assess the interactions between RNA polymerase II regulatory proteins and their partners, which cannot be analyzed by the classic, RNA polymerase II‐dependent Y2H. In the RNA Pol III system, protein prey is fused to τ138p, which can activate RNA polymerase III‐mediated transcription.229

(3) Sos recruitment system (SRS) is based on the Ras signaling pathway instead of transcriptional regulation in classic Y2H. In SRS, bait is fused to mammalian Sos, and prey is fused to a membrane localization signal. Interaction between bait and prey results in Sos membrane localization and Ras activation, allows a Cdc25‐2 temperature sensitive yeast strain grow at the restrictive temperature.230 Since SRS does not depend on a transcriptional readout, it can study interactions involving transcriptional activators or repressors. Furthermore, it is suitable for identifying proteins that do not target well to the nucleus or undergo post‐translational modifications in the cytoplasm.

(4) An improved version of SRS is the Ras recruitment system (RRS), in which bait is fused to constitutively active mammalian Ras instead of Sos.231

(5) The G protein fusion system allows the interaction between an integral membrane protein bait and a soluble prey to be studied. By this method, the prey fused to the G protein γ‐subunit is localized at the membrane upon interaction with bait, leading to the disruption of G protein signaling and downstream transcriptional events.232

(6) SCINEX‐P system is developed to analysis interactions between extracellular or transmembrane proteins in the endoplasmic reticulum (ER), in which bait and prey are fused to truncated Ire1p. Bait–prey interaction causes dimerization of Ire1p, which activates unfolded protein response signaling, leading to Hac1p‐dependent activation of reporter genes.233

(7) Reverse two‐hybrid system is an upside‐down version of Y2H, in which disruption of the bait–prey interaction decreases the expression of a counter‐selectable reporter gene, such as URA3,234 CYH2235 and GAL1,236 and allows cell growth under selective conditions. This method facilitates the detection of dissociation of PPIs, and the selection of small‐molecule inhibitors against PPIs that could be developed as therapeutic agents.237

(8) Bacterial two‐hybrid system using E. coli as a host organism offers some advantages over Y2H, including ease of use, lack of cellular compartmentalization, rapid library screening due to high transformation efficiency, and fast growth rate observed with E. coli, and increased sensitivity due to the absence of endogenous proteins that compete for the interactions.168 A number of bacterial two‐hybrid systems have been studied, which are based on recruitment of RNA polymerase to activate a lacZ reporter gene,238 complementation of signaling enzymes such as adenylate cyclase,239 dimerization of LexA derivatives to reconstitute a functional repressor,240 and reconstitution of polyhydroxyalkanoates synthesis regulatory protein PhaR.241

(9) Mammalian two‐hybrid system allows for the studies of interactions of proteins in native physiological context with proper folding and post‐translational modifications. One unique system termed MAPPIT (mammalian protein–protein interaction trap) is founded on type I cytokine signal transduction, in which bait–prey interaction restores JAK–STAT signaling and finally induces transcription of a reporter gene coupled to a STAT‐dependent promoter.242 This approach has been well validated for large‐scale interactomics studies and high‐throughput drug screening.243

(10) Fluorescent two‐hybrid (F2H) assay takes advantage of cell lines harboring a stable integration of a lac operator array. In such an assay, a fluorescently labeled bait is fused to a lac repressor and then immobilized. Interaction between the bait and a differently labeled prey leads to co‐localization of fluorescent signals. This technique enables visualization and analysis of PPIs from different subcellular compartments including nucleus, cytoplasm, and mitochondria in living cells in real time.244, 245, 246