High-throughput genetic interaction mapping in the fission yeast Schizosaccharomyces pombe

Epistasis analysis, which reports on the extent to which the function of one gene depends on the presence of a second, is a powerful tool for studying the functional organization of the cell. Systematic genome-wide studies of epistasis, however, have been limited, with the majority of data being collected in the budding yeast, Saccharomyces cerevisiae. Here we present two 'pombe epistasis mapper' strategies, PEM-1 and PEM-2, which allow for high-throughput double mutant generation in the fission yeast, S. pombe. These approaches take advantage of a previously undescribed, recessive, cycloheximide-resistance mutation. Both systems can be used for genome-wide screens or for the generation of high-density, quantitative epistatic miniarray profiles (E-MAPs). Since S. cerevisiae and S. pombe are evolutionary distant, this methodology will provide insight into conserved biological pathways that are present in S. pombe, but not S. cerevisiae, and will enable a comprehensive analysis of the conservation of genetic interaction networks.     

A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping

Despite the wide acceptance of yeast two-hybrid (Y2H) system for protein-protein interaction analysis and discovery, conventional Y2H assays are not well suited for high-throughput screening of the protein interaction network (“interactome”) on a genomic scale due to several limitations, including labor-intensive agar plating and colony selection methods associated with the use of nutrient selection markers, complicated reporter analysis methods associated with the use of LacZ enzyme reporters, and incompatibility of the liquid handling robots. We recently reported a robust liquid culture Y2H system based on quantitative analysis of yeast-enhanced green fluorescent protein (yEGFP) reporters that greatly increased the analysis throughput and compatibility with liquid handling robots. To further advance its utility in high-throughput complementary DNA (cDNA) library screening, we report the development of a novel surface display Y2H (sdY2H) library screening system with uniquely integrated surface display hemagglutination (sdHA) antigen and yEGFP reporters. By introduction of a surface reporter sdHA into the yEGFP-based Y2H system, positive Y2H targets are quickly isolated from library cells by a simple magnetic separation without a large plating effort. Moreover, the simultaneous scoring of multiple reporters, including sdHA, yEGFP, and conventional nutrient markers, greatly increased the specificity of the Y2H assay. The feasibility of the sdY2H assay on large cDNA library screening was demonstrated by the successful recovery of positive P53/T interaction pairs at a target-to-background ratio of 1:1,000,000. Together with the massive parallel DNA sequencing technology, it may provide a powerful proteomic tool for high-throughput interactome mapping on a genomic scale.     

A yeast two-hybrid smart-pool-array system for protein-interaction mapping

We present here a new two-hybrid smart pool array (SPA) system in which, instead of individual activation domain strains, well-designed activation domain pools are screened in an array format that allows built-in replication and prey-bait deconvolution. Using this method, a Saccharomyces cerevisiae genome SPA increases yeast two-hybrid screening efficiency by an order of magnitude.     

Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses

Background  

Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells. But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited. Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast two-hybrid mating system, will yield valuable information for functional biology on a proteomic scale.     

High-throughput analysis of the protein sequence-stability landscape using a quantitative yeast surface two-hybrid system and fragment reconstitution

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a yeast surface two-hybrid method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are recovered predominantly. Thus, it allows for the isolation of sequences that exhibit a desired level of stability. We identified more than 100 unique sequences for a β-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the β-bulge. Our method provides a powerful means for the rapid assessment of the stability of many variants, for the systematic assessment of the contribution of different factors to protein stability, and for enhancement of the protein stability.     

Potential target gene and protein interaction ofPimpinella brachycarpa HD-Zip protein, Phz4 by yeast two-hybrid system

The yeast two-hybrid system was used to investigate dimerization between proteins ofPhz2 andPhz4 clones of the homeodomain-leucine zipper family which were obtained by screening aPimpinella brachycarpa shoot-tip cDNA library. Assays showed that Phz4 formed a homo rather than a heterodimer with Phz2. In addition, we isolated cDNA clones,Phyb1, Phyb2, andPhyb3, that encode proteins interacting with Phz4. Although Phyb1 is not a HD-Zip protein, the activity of interaction between Phyb1 and Phz4 was, surprisingly, stronger than that of the homodimerization of Phz4. The analysis of interacting parts indicated that from 1 bp to 466 bp of Phyb1, there was no interaction with Phz4, but from 467 bp to 593 bp, interactions were found with the N-terminal and C-terminal regions, except for HD-Zip of Phz4. This region ofPhyb1 contained a nuclear localization signal. DNA-binding analysis showed that the Phz4 HD-Zip domain recognized the [T(C/G)ATTG] core sequence and the region containing the [TCATTG] motif, which is, in itself, a promoter in vitro.     

Construction of a reading frame-independent yeast two-hybrid vector system for site-specific recombinational cloning and protein interaction screening

The yeast two-hybrid (Y2H) system is a powerful method to identify protein-protein inter-actions (PPI) in vivo, requiring minimal prior information of the putative interactors. The time and effort required for each experiment can be significantly reduced if the "bait" and the "prey" proteins are cloned into specific recombination-amenable two-hybrid vectors. We describe the construction of a reading frame-independent vector system for Y2H PPI studies. The described vector system knits together the advantages of site-specific recombination cloning with the Y2H system. The produced plasmids enable recombination-based cloning of genes or gene fragments in all possible reading frames into Y2H library vectors. Thus, Y2H screening libraries can be rapidly constructed and will present more amino termini in the correct reading frame. Additionally, advantageous for small-scale Y2H studies, there is no need to know the natural reading frame of the genes of interest, because the bait and prey genes can be transferred into the vectors by a single reaction and are present in all possible reading frames. Since the Y2H system per se is a positive selection system, only pairs of bait and prey genes harboring the correct reading frames will emerge. We tested the new vectors within the Y2H system and demonstrated full functionality without any undesired effects on the Y2H system itself. Besides the vector construction, we investigated the utility of the system for Y2H analysis and demonstrated clearly its practicability in genome-wide Y2H screenings and the advantage of using additional reading-frame Y2H cDNA libraries. We performed a series of genome-wide Y2H library screenings with the human vitamin D receptor protein (VDR) as bait. We investigated: (i) whether more protein interactors are found by using three instead of one reading-frame destination vectors; (ii) how much overlap between the different reading-frame libraries exists; and (iii) the rate of possible additional autoactivators. We conclude that our vectors deliver significantly more interactors and outperform a single reading-frame library. This new system could enable simple and fast large-scale PPI studies and the construction of high-quality screening libraries.     

High-throughput assays for promiscuous inhibitors

High-throughput screening (HTS) searches large libraries of chemical compounds for those that can modulate the activity of a particular biological target; it is the dominant technique used in early-stage drug discovery. A key problem in HTS is the prevalence of nonspecific or 'promiscuous' inhibitors. These molecules have peculiar properties, act on unrelated targets and can dominate the results from screening campaigns. Several explanations have been proposed to account for promiscuous inhibitors, including chemical reactivity, interference in assay read-out, high molecular flexibility and hydrophobicity. The diversity of these models reflects the apparently unrelated molecules whose behaviors they seek to explain. However, a single mechanism may explain the effects of many promiscuous inhibitors: some organic molecules form large colloid-like aggregates that sequester and thereby inhibit enzymes. Hits from HTS, leads for drug discovery and even several drugs appear to act through this mechanism at micromolar concentrations. Here, we report two rapid assays for detecting promiscuous aggregates that we tested against 1,030 'drug-like' molecules. The results from these assays were used to test two preliminary computational models of this phenomenon and as benchmarks to develop new models.     

High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions

The analysis of protein-protein interactions is a key focus of proteomics efforts. The yeast two-hybrid system (Y2H) has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale Y2H assays, we report a novel array approach by coupling a GFP reporter based Y2H system with high throughput flow cytometry that enables the processing of a 96-well plate in as little as 3 min. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hr postmating compared with the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation.     

Search for protein-protein interaction partners of peroxiredoxin 6 with the yeast two-hybrid system

Peroxiredoxins (Prx) are a family of nonselenium peroxidases that are involved in cell defense against oxidative stress and in redox regulation of intracellular signaling. Mammalian peroxiredoxin 6 (Prx6) belongs to the 1-Cys Prx subfamily. The protein--protein interactions of human Prx6 were studied using a yeast two-hybrid system. Hybrid plasmid pHybLex/Zeo/Prx6, which directed synthesis of a chimeric protein consisting of the DNA-binding domain (BD) of LexA and a Prx6 sequence, was used to screen a two-hybrid cDNA library Hybrid Hunter (Invitrogen). The screening identified two potential interaction partners of Prx6: the calcium-activated cysteine endopeptidase calpain and the p50RhoGAP protein of the family of Sec14-like proteins. The possibility for the interactions observed in the two-hybrid system to occur in oxidative stress in vivo is discussed.     

High-throughput assays for lipases and esterases

In the past few years a considerable number of high-throughput screening (HTS) systems have been developed, especially for lipases and esterases. In this review, a range of HTS methods for the directed evolution of these hydrolases are covered. This includes spectrophotometric and fluorimetric formats as well as other approaches to allow for fast, efficient and reliable identification of desired enzyme variants within large mutant libraries. In addition, methods for library creation and application of lipases and esterases are briefly covered.     

Use of yeast two-hybrid system for detection ofBacillus subtilis FtsZ protein partners

Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions. Three plasmids (pGBR1, pGBR2, pGBR3) with theClaI restriction site shifted in the three possible reading frames in fusion withGAL4 activating domain were constructed. The modified plasmids were used for identification of protein partners of FtsZ fromBacillus subtilis. Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified. The protein interactions were quantified by measurements of β-galactosidase activity in yeast cells using 4-methylumbelliferyl β-d-glactopyranoside as fluorogenic substrate.     

Systematic and large-scale two-hybrid screens

The increasing rate at which complete genome sequences become available necessitates rapid and robust methods for investigating the functions of their encoded proteins. Efforts have been made to study protein function by systematically screening large sets of proteins using the two-hybrid method. Analyses of the complete proteomes of baceriophage T7, the mammalian viruses hepatitis C and vaccinia, as well as of several protein complexes including RNA splicing proteins and RNA polymerase III from yeast, have been undertaken. Saccharomyces cerevisiae has been studied extensively by two-hybrid methods, with more than 2500 protein-protein interactions described. Systematic studies on metazoan proteomes are, however, still in their infancy.