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The yeast two-hybrid (Y2H) assay is a powerful technique to identify protein-protein interactions. However, the auxotrophic markers that are the most common Y2H reporters take several days to yield data and require subjective assessment of semiquantitative data to identify interactions. Several reporters have been developed to overcome these disadvantages, but there is still a need for a Y2H reporter that is objective, fast and able to be performed with common laboratory equipment. In this report, we replaced the ADE2 reporter in BK100 with NanoLuc luciferase to yield BK100Nano. We developed an optimized assay to measure NanoLuc activity in 96-well plates and analyzed a set of 74 pairs identified in Y2H library screens, which revealed 44 positive interactions using an unbiased cutoff based on the mean luminescence of negative control samples. The same set was also tested for growth on Y2H selection medium via expression of the HIS3 reporter. We found 91% agreement between the two assays, with discrepancies attributed to weak interactions that displayed variable growth on Y2H medium. Overall, the new BK100Nano strain establishes a quantitative and convenient method to identify Y2H interactions and has potential to be applied to a high throughput manner. [ABSTRACT FROM AUTHOR]

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structurefunction based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways. [ABSTRACT FROM AUTHOR]

Myzus persicae (Sulzer) (Homoptera: Aphididae) is one of the most important agricultural pests worldwide. In addition to sucking phloem sap, M. persicae also transmits Cucumber mosaic virus (CMV) as a vector in a nonpersistent manner. At present, the infection mechanism remains unclear, especially the process of aphid virus acquisition. In this study, we isolated four M. persicae cuticle protein genes (MPCP1, MPCP2, MPCP4, and MPCP5) from M. persicae. The relative amount of the gene encoding Cucumber mosaic virus capsid protein (CMV CP) and the transcript levels of these four cuticle protein genes were investigated in aphid virus acquisition by feeding the tobacco preinfested by CMV. The relative expression of MPCP1, MPCP2, and MPCP4 were significantly higher than MPCP5 at 24h after aphids feeding on virus-infested tobacco. Yeast two-hybrid assays demonstrated that the protein encoded by MPCP4 gene was closely associated with the CMV CP through the direct interaction. Moreover, the ability of M. persicae to acquire CMV was suppressed by RNA interference of MPCP4. All these lines of evidence indicate that MPCP4, as a viral putative receptor in the stylet of aphid, plays an important role in aphid acquisition of CMV. [ABSTRACT FROM AUTHOR]

Bioinformatic analysis indicates that sucrose phosphate synthase (SPS) contains a putative C-terminal sucrose phosphate phosphatase (SPP)-like domain that may facilitates the binding of SPP. If an SPS-SPP enzyme complex exists, it may provide sucrose biosynthesis with an additional level of regulation, forming a direct metabolic channel for sucrose-6-phosphate between these two enzymes. Herein, the formation of an enzyme complex between SPS and SPP was examined, and the results from yeast two-hybrid experiments suggest that there is indeed an association between these proteins. In addition, in planta bioluminescence resonance energy transfer (BRET) was observed in Arabidopsis seedlings, providing physical evidence for a protein interaction in live cells and in real time. Finally, bimo-lecular fluorescence complementation (BiFC) was employed in an attempt to detect SPS-SPP interactions visually. The findings clearly demonstrated that SPS interacts with SPP and that this interaction impacts soluble carbohydrate pools and affects carbon partitioning to starch. Moreover, a fusion construct between the two genes promotes plant growth in both transgenic Arabidopsis and hybrid poplar. [ABSTRACT FROM AUTHOR]

Background and Aims DEFICIENS (DEF)- and GLOBOSA (GLO)-like proteins constitute two sister clades of floral homeotic transcription factors that were already present in the most recent common ancestor (MRCA) of extant angiosperms. Together they specify the identity of petals and stamens in flowering plants. In core eudicots, DEF- and GLO-like proteins are functional in the cell only as heterodimers with each other. There is evidence that this obligate heterodimerization contributed to the canalization of the flower structure of core eudicots during evolution. It remains unknown as to whether this strict heterodimerization is an ancient feature that can be traced back to the MRCA of extant flowering plants or if it evolved later during the evolution of the crown group angiosperms. Methods The interactions of DEF- and GLO-like proteins of the early-diverging angiosperms Amborella trichopoda and Nuphar advena and of the magnoliid Liriodendron tulipifera were analysed by employing yeast two-hybrid analysis and electrophoretic mobility shift assay (EMSA). Character-state reconstruction, including data from other species as well, was used to infer the ancestral interaction patterns of DEF- and GLO-like proteins. Key Results The yeast two-hybrid and EMSA data suggest that DEF- and GLO-like proteins from early-diverging angiosperms both homo- and heterodimerize. Character-state reconstruction suggests that the ability to form heterodimeric complexes already existed in the MRCA of extant angiosperms and that this property remained highly conserved throughout angiosperm evolution. Homodimerization of DEF- and GLO-like proteins also existed in the MRCA of all extant angiosperms. DEF-like protein homodimerization was probably lost very early in angiosperm evolution and was not present in the MRCA of eudicots and monocots. GLO-like protein homodimerization might have been lost later during evolution, but very probably was not present in the MRCA of eudicots. Conclusions The flexibility of DEF- and GLO-like protein interactions in early-diverging angiosperms may be one reason for the highly diverse flower morphologies observed in these species. The results strengthen the hypothesis that a reduction in the number of interaction partners of DEF- and GLO-like proteins, with DEF–GLO heterodimers remaining the only DNA-binding dimers in core eudicots, contributed to developmental robustness, canalization of flower development and the diversification of angiosperms. [ABSTRACT FROM AUTHOR]

Dsb M is a novel disulfide oxidoreductase that affects aminoglycoside resistance in Pseudomonas aeruginosa by an Oxy R-regulated process. However, the detailed mechanism of interaction between Dsb M and Oxy R had not yet been elucidated. In this study, we expressed Dsb M in Escherichia coli and showed that Dsb M can oxidize and reduce disulfide. We also used a yeast two-hybrid assay to identify interactions between Dsb M and Oxy R. A subsequent GSH oxidation experiment revealed that Dsb M could alter both the oxidized and reduced state of Oxy R. We hypothesized that Oxy R can be reduced by Dsb M, and thus Dsb M may be required for aminoglycoside resistance in P. aeruginosa. Our findings contribute to the understanding of the mechanisms underlying aminoglycoside resistance in P. aeruginosa. [ABSTRACT FROM AUTHOR]

We developed a high throughput yeast two-hybrid (Y2H) assay for screening pools of combinatorial cyclic peptide preys against pools of bait proteins. The assay used the PI (pooling with imaginary tags) deconvolution pooling strategy to generate pools of baits and a random pooling strategy to generate pools of cyclic peptide preys. Haploid yeast, expressing pools of baits or preys, were arrayed and mated to each other to generate diploid arrays, where the yeast express both baits and preys. Diploid arrays were scored for activation of the Y2H reporter genes. We used this Y2H pooling strategy, referred to as ‘PI-pool-on-random pool’, to screen a cyclic peptide library for interactions against Bcr-Abl domains. Seven Bcr-Abl domain baits and LexA control were pooled using the PI deconvolution pooling strategy. The cyclic peptide library was randomly arrayed into pools of ∼1000 members. Cyclic peptides were isolated for six of seven Bcr-Abl domain baits. The PI-pool-on-random pooling Y2H assay using high stringency Y2H reporter genes produced no false positives, while missing 20% of real interactions. The high specificity of the PI-pool-on-random pooling Y2H assay eliminates the need to validate interactions. Pooling of baits and preys allows large prey libraries to be screened against multiple baits and takes advantage of PI-deconvolution to determine protein interactions with high sensitivity and specificity. The scalability of this assay allows the peptide preys to be isolated in a high throughput manner against a large number of baits and provides an avenue for generating affinity agents against entire proteomes in the future. [ABSTRACT FROM PUBLISHER]

Understanding the flowering process in apple (Malus × domestica Borkh.) is essential for developing methods to shorten the breeding period and regulate fruit yield. It is known that FLOWERING LOCUS T (FT) acts as a transmissible floral inducer in the Arabidopsis flowering network system. To clarify the molecular network of two apple FT orthologues, MdFT1 and MdFT2, we performed a yeast two-hybrid screen to identify proteins that interact with MdFT1. We identified several transcription factors, including two members of the TCP (TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL FACTORs) family, designated MdTCP2 and MdTCP4, and an Arabidopsis thaliana VOZ1 (Vascular plant One Zinc finger protein1)-like protein, designated MdVOZ1. MdTCP2 and MdVOZ1 also interacted with MdFT2 in yeast. The expression domain of MdTCP2 and MdVOZ1 partially overlapped with that of MdFT1 and MdFT2, most strikingly in apple fruit tissue, further suggesting a potential interaction in vivo. Constitutive expression of MdTCP2, MdTCP4 and MdVOZ1 in Arabidopsis affected plant size, leaf morphology and the formation of leaf primordia on the adaxial side of cotyledons. On the other hand, chimeric MdTCP2, MdTCP4 and MdVOZ1 repressors that included the ethylene-responsive transcription factors (ERF)-associated amphiphilic repression (EAR) domain motif influenced reproduction and inflorescence architecture in transgenic Arabidopsis. These results suggest that MdFT1 and/or MdFT2 might be involved in the regulation of cellular proliferation and the formation of new tissues and that they might affect leaf and fruit development by interacting with TCP- and VOZ-family proteins.DDBJ accession nos. AB531019 (MdTCP2a mRNA), AB531020 (MdTCP2b mRNA), AB531021 (MdTCP4a mRNA), AB531022 (MdTCP4b mRNA) and AB531023 (MdVOZ1a mRNA). [ABSTRACT FROM AUTHOR]

Transcription factors play key roles in plant development through their interaction with cis-elements and/or other transcription factors. A HD-Zip IV family transcription factor, G̲ossypium ḇarbadense M̲eristem Ḻayer 1 (GbML1) has been identified and characterized here. GbML1 specifically bound to the L1 box and the promoters of GbML1 and GbRDL1. GbML1 physically interacted with a key regulator of cotton fibre development, GbMYB25. Truncated and point mutation assays indicated the START-SAD domain was required for the binding to the C terminal domain (CTD) of GbMYB25. GbML1 overexpression in Arabidopsis increased the number of trichomes on stems and leaves and increased the accumulation of anthocyanin in leaves. Taken together, the L1 box binding protein, GbML1 was identified as the first partner for GbMYB25 and the role of START domain was discovered to be a protein binding domain in plants. Our findings will help the improvement of cotton fibre production and the understanding of the key role of HD-Zip family and MYB family in plants. [ABSTRACT FROM AUTHOR]

The article focuses on the application of a yeast bioassay to study the interaction between NH2- and COOH (N–C) terminals of androgen receptors induced by androgens and anti-androgens. The bioassay, accordingly, uses a green fluorescent protein (GFP) as a reporter gene which however produces higher 50% effective concentration (EC50) values of various steroids. It adds the method was also effective in detecting compounds which inhibit the N–C interaction, such as resveratrol.

One of the greatest challenges of the post-genomic era is the construction of a more comprehensive human protein interaction map. While this process may take many years to complete, the development of stringent high throughput techniques and the emergence of complementary assays mean that the aim of building a detailed binary map of the human interactome is now a very realistic goal. In particular, methods which facilitate the analysis of large numbers of membrane-protein interactions mean that it will be possible to construct more extensive networks, which in turn provide new insights into the functional connectivity between intra- and extra-cellular processes. This is important as many therapeutic strategies are designed to elicit effects via 'tractable' cell-surface proteins. Therefore, the construction of maps depicting the complexity of trans-cellular communication networks will not only improve our understanding of physiological processes, it will also aid the design of rational therapeutic strategies, with fewer potential side effects. This review aims to provide a basic insight into the approaches currently being used to construct binary human protein interaction networks, with particular reference to newer techniques, which have the potential to extend network coverage and aid the conditional annotation of interactome-scale protein interaction maps. [ABSTRACT FROM AUTHOR]

MADS-domain proteins are important transcription factors involved in many biological processes of plants. Interactions between MADS-domain proteins are essential for their functions. In tomato (Solanum lycopersicum), the number of MIKCc-type MADS-domain proteins identified has totalled 36, but a large-scale interaction assay is lacking. In this study, 22 tomato MADS-domain proteins were selected from six functionally important subfamilies of the MADS-box gene family, to create the first large-scale tomato protein interaction network. Compared with Arabidopsis and petunia (Petunia hybrida), protein interaction patterns in tomato displayed both conservation and divergence. The majority of proteins that can be identified as putative orthologues exhibited conserved interaction patterns, and modifications were mostly found in genes underlining traits unique to tomato. JOINTLESS and RIN, characterized for their roles in abscission zone development and fruit ripening, respectively, showed enlarged interaction networks in comparison with their Arabidopsis and petunia counterparts. Novel interactions were also found for members of the expanded subfamilies, such as those represented by AP1/FUL and AP3/PI MADS-domain proteins. In search for higher order complexes, TM5 was found to be the preferred bridge among the five SEP-like proteins. Additionally, 16 proteins with the MADS-domain removed were used to assess the role of the MADS-domain in protein–protein interactions. The current work provides important knowledge for further functional and evolutionary study of the MADS-box genes in tomato. [ABSTRACT FROM PUBLISHER]

Yeast two-hybrid (Y2H) screening methods are an effective means for the detection of protein-protein interactions. Optimisation and automation has increased the throughput of the method to an extent that allows the systematic mapping of protein-protein interactions on a proteome-wide scale. Since two-hybrid screens fail to detect a great number of interactions, parallel high-throughput approaches are needed for proteome-wide interaction screens. In this review, we discuss and compare different approaches for adaptation of Y2H screening to high-throughput, the limits of the method and possible alternative approaches to complement the mapping of organism-wide protein-protein interactions. [ABSTRACT FROM AUTHOR]

We find accurate approximations for the expected number of three-cycles and unchorded four-cycles under a stochastic distribution for graphs that has been proposed for modelling yeast two-hybrid protein--protein interaction networks. We show that unchorded four-cycles are characteristic motifs under this model and that the count of unchorded four-cycles in the graph is a reliable statistic on which to base parameter estimation. Finally, we test our model against a range of experimental data, obtain parameter estimates from these data and investigate possible improvements in the model. Characterization of this model lays the foundation for its use as a prior distribution in a Bayesian analysis of yeast two-hybrid networks that can potentially aid in identifying false-positive and false-negative results. [ABSTRACT FROM AUTHOR]

Presents a study on the identification of Rab guanosine triphosphatase-activating protein-like protein to merlin-associated protein. Implication of crosstalk of membrane traffic and cell adhesion; Role of Alix, a multifunctional adaptor protein, in endosomal protein sorting of the endocytosed epidermal growth factor receptor; Interaction between endophilin B2 and Alix.

The mapping of protein-protein interactions is key to understanding biological processes. Many technologies have been reported to map interactions and these have been systematically applied in yeast. To date, the number of reported yeast protein interactions that have been truly validated by at least one other approach is low. The mapping of human protein interaction networks is even more complicated. Thus, it is unreasonable to try to map the human interactome; instead, interaction mapping in human cell lines should be focused along the lines of diseases or changes that can be associated with specific cells. In this paper, an approach for combining different 'omics' technologies to achieve efficient mapping and validation of protein interactions in human cell lines is presented. [ABSTRACT FROM AUTHOR]

The gene HCAP1 (HCC-associated Protein 1), one variant of GEMIN4, has been mapped in a minimum LOH region on chromosome 17p13.3 and encodes a 1047-amino acid protein. Function predictions based on the amino acid sequence of protein HCAP1 revealed it to contain one helix-loop-helix motif and one leucine zipper domain. Using yeast two-hybrid screening, five zinc-finger proteins were identified as HCAP1-interacting proteins. Among them, NDP52 (nuclear dot protein 52) appeared most frequently in positive clones and was the most strongly interacting protein. Then, the interaction between HCAP1 and NDP52 was confirmed by GST pull-down assay and a coimmunoprecipitation experiment. Moreover, an immunofluorescent staining assay indicated that NDP52 colocalizes with HCAP1 in the cytoplasm. By deletion analysis, the leucine zipper domain of HCAP1 and the zinc finger domain of NDP52 were identified as important regions responsible for the interaction. [ABSTRACT FROM PUBLISHER]

The activity of the Arabidopsis heat shock transcription factor (HSF) is repressed at normal conditions but activated by cellular stresses. Circumstantial evidence suggests that HSP70 may function as a negative feedback regulator of HSF activity. Here the interaction between HSF and HSP70 is reported using electrophoretic mobility shift and yeast two-hybrid assays. Subdomain mapping indicates an interaction of the activation domain and DNA-binding domain of HSF1 with HSP70. [ABSTRACT FROM AUTHOR]

The yeast UBC9 and hus5 gene products have been identified as putative E2 members of the ubiquitin-conjugating enzyme (UBC) family and have been shown to play an essential role in cell cycle progression. We have identified a Drosophila Ubc9/Hus5 homologue (termed dUBC9) in an attempt to identify proteins that interact with the amino-terminal tran-scriptional repression domain of the Groucho corepressor by use of the yeast two-hybrid system. The predicted dUBC9 protein consists of 159 amino acids and shows 85,68, and 54% amino acid sequence identities with human UBC9 homologue, Schizosaccharomyces pombe Huso, and Saccharomyces cerevisiae Ubc9 proteins, respectively. Expression of dUBC9 cDNA complements a temperature-sensitive ubc9–l mutation of S. cerevisiae to fully restore normal growth, indicating that the dUBC9 protein can act as a substitute for the yeast Ubc9 protein. The dUBC9 transcripts were about 1.2 kb and were detected at all stages of Drosophila development and in ovaries and Schneider cells. However, an increased level was observed in early embryos and ovaries. The dUBC9 gene is present as a single copy in the genome and localized in segment 21C–D on the left arm of the second chromosome. [ABSTRACT FROM PUBLISHER]