Evidence of physical interactions of puroindoline proteins using the yeast two-hybrid system

The puroindoline proteins PINA and PINB play key roles in determining wheat grain texture and also have potential antimicrobial roles. Many recent studies show that their roles in grain texture involve some interaction or interdependence, and their antimicrobial activity may also involve formation of protein complexes. The issue of whether any homo- and/or heteromeric associations occur amongst the PIN proteins is thus critical for understanding their biological functions and exploiting them for grain texture modifications or antimicrobial applications, but is as yet unresolved. This work has utilised the well-established yeast two-hybrid system to directly address this issue. The results confirm occurrence of in vivo interactions between the two PIN proteins for the first time, and show that PINB interacts with itself and also interacts, although somewhat weakly, with PINA, while PINA is a weaker interactor. The results explain the many reported observations suggesting a co-operative interaction between the two proteins and provide a rapid and efficient tool for testing the effects of various alleles/mutations on the interactions and lipid binding properties of these proteins, which are of functional significance to grain texture and antimicrobial defence functions.     

Benchmarking yeast two‐hybrid systems using the interactions of bacterial motility proteins

Yeast two‐hybrid screens often produce vastly non‐overlapping interaction data when the screens are conducted in different laboratories, or use different vectors, strains, or reporter genes. Here we investigate the underlying reasons for such inconsistencies and compare the effect of seven different vectors and their yeast two‐hybrid interactions. Genome‐wide array screens with 49 motility‐related baits from Treponema pallidum yielded 77 and 165 interactions with bait vectors pLP‐GBKT7 and pAS1‐LP, respectively, including 21 overlapping interactions. In addition, 90 motility‐related proteins from Escherichia coli were tested in all pairwise combinations and yielded 140 interactions when tested with pGBKT7g/pGADT7g vectors but only 47 when tested with pDEST32/pDEST22. We discuss the factors that determine these effects, including copy number, the nature of the fusion protein, and species‐specific differences that explain non‐conserved interactions among species. The pDEST22/pDEST32 vectors produce a higher fraction of interactions that are conserved and that are biologically relevant when compared with the pGBKT7/pGADT7‐related vectors, but the latter appear to be more sensitive and thus detect more interactions overall.     

Protein interactome analysis of 12 mitogen‐activated protein kinase kinase kinase in rice using a yeast two‐hybrid system

The mitogen‐activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K‐interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two‐hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K‐interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full‐length cDNA in the rice KOME ( http://cdna01.dna.affrc.go.jp/cDNA/ ) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead‐associated domain‐containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K‐interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors.     

Lysine methylation modulates the protein–protein interactions of yeast cytochrome C Cyc1p

In recent years, protein methylation has been established as a major intracellular PTM. It has also been proposed to modulate protein‐protein interactions (PPIs) in the interactome. To investigate the effect of PTMs on PPIs, we recently developed the conditional two‐hybrid (C2H) system. With this, we demonstrated that arginine methylation can modulate PPIs in the yeast interactome. Here, we used the C2H system to investigate the effect of lysine methylation. Specifically, we asked whether Ctm1p‐mediated trimethylation of yeast cytochrome c Cyc1p, on lysine 78, modulates its interactions with Erv1p, Ccp1p, Cyc2p and Cyc3p. We show that the interactions between Cyc1p and Erv1p, and between Cyc1p and Cyc3p, are significantly increased upon trimethylation of lysine 78. This increase of interaction helps explain the reported facilitation of Cyc1p import into the mitochondrial intermembrane space upon methylation. This first application of the C2H system to the study of methyllysine‐modulated interactions further confirms its robustness and flexibility.     

A conditional two‐hybrid (C2H) system for the detection of protein–protein interactions that are mediated by post‐translational modification

The original bacterial two‐hybrid system is widely used but does not permit the study of interactions regulated by PTMs. Here, we have built a conditional two‐hybrid (C2H) system, in which bait and prey proteins can be co‐expressed in the presence of a modifying enzyme such as a methyltransferase, acetyltransferase, or kinase. Any increase or decrease in interaction due to the modification of the proteins can be measured by an increased or decreased level of reporter gene expression. The C2H system is comprised of eight new vectors based on the Novagen Duet co‐expression plasmids. These vectors include two multiple cloning sites per vector as well as a hexahistidine tag or S‐tag to aid in purification, if desired. We demonstrate the use of the C2H system to study the dimerization of the yeast protein Npl3, which is increased when methylated by the methyltransferase Hmt1.     

Application of Mu in vitro transposition for high-precision mapping of protein–protein interfaces on a yeast two-hybrid platform

High-precision mapping of regions involved in protein–protein interfaces of interacting protein partners is an essential component on a path to understand various cellular functions. Transposon-based systems, particularly those involving in vitro reactions, offer exhaustive insertion mutant libraries and high-throughput platforms for many types of genetic analyses. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision that is based on transposition-assisted construction, sampling, and analysis of a comprehensive insertion mutant library. The methodology integrates random pentapeptide mutagenesis of proteins, yeast two-hybrid screening, and high-resolution genetic footprinting. This straightforward strategy is general, and it provides a rapid and easy means to identify critical contact regions in proteins without the requirement of prior structural knowledge.     

酵母双杂交在植物功能基因组研究中的应用

扼要介绍了酵母双杂交技术的原理,详细评述了该技术在植物功能基因组中的研究进展,并结合自己研究领域对该技术在植物领域发展方向作了展望。     

Systematic prediction of human membrane receptor interactions

Membrane receptor‐activated signal transduction pathways are integral to cellular functions and disease mechanisms in humans. Identification of the full set of proteins interacting with membrane receptors by high‐throughput experimental means is difficult because methods to directly identify protein interactions are largely not applicable to membrane proteins. Unlike prior approaches that attempted to predict the global human interactome, we used a computational strategy that only focused on discovering the interacting partners of human membrane receptors leading to improved results for these proteins. We predict specific interactions based on statistical integration of biological data containing highly informative direct and indirect evidences together with feedback from experts. The predicted membrane receptor interactome provides a system‐wide view, and generates new biological hypotheses regarding interactions between membrane receptors and other proteins. We have experimentally validated a number of these interactions. The results suggest that a framework of systematically integrating computational predictions, global analyses, biological experimentation and expert feedback is a feasible strategy to study the human membrane receptor interactome.     

A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways

Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed.     

Mass spectrometry-based footprinting of protein–protein interactions

We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein–protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein–protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein–protein interactions that are less amenable for crystallographic or NMR analysis.     

Macromolecular interactions of the bacterial division FtsZ protein: from quantitative biochemistry and crowding to reconstructing minimal divisomes in the test tube

The division of Escherichia coli is an essential process strictly regulated in time and space. It requires the association of FtsZ with other proteins to assemble a dynamic ring during septation, forming part of the functionally active division machinery, the divisome. FtsZ reversibly interacts with FtsA and ZipA at the cytoplasmic membrane to form a proto-ring, the first molecular assembly of the divisome, which is ultimately joined by the rest of the division-specific proteins. In this review we summarize the quantitative approaches used to study the activity, interactions, and assembly properties of FtsZ under well-defined solution conditions, with the aim of furthering our understanding of how the behavior of FtsZ is controlled by nucleotides and physiological ligands. The modulation of the association and assembly properties of FtsZ by excluded-volume effects, reproducing in part the natural crowded environment in which this protein has evolved to function, will be described. The subsequent studies on the reactivity of FtsZ in membrane-like systems using biochemical, biophysical, and imaging technologies are reported. Finally, we discuss the experimental challenges to be met to achieve construction of the minimum protein set needed to initiate bacterial division, without cells, in a cell-like compartment. This integrated approach, combining quantitative and synthetic strategies, will help to support (or dismiss) conclusions already derived from cellular and molecular analysis and to complete our understanding on how bacterial division works.     

Transmembrane peptides used to investigate the homo-oligomeric interface and binding hotspot of latent membrane protein 1

Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. Although normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein latent membrane protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here, we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full-length LMP-1 variant harboring the D150A substitution is deficient in NFκB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.     

The structural analysis of protein–protein interactions by NMR spectroscopy

A comprehensive understanding of protein–protein interactions is an important next step in our quest to understand how the information contained in a genome is put into action. Although a number of experimental techniques can report on the existence of a protein– protein interaction, very few can provide detailed structural information. NMR spectroscopy is one of these, and in recent years several complementary NMR approaches, including residual dipolar couplings and the use of paramagnetic effects, have been developed that can provide insight into the structure of protein–protein complexes. In this article, we review these approaches and comment on their strengths and weaknesses.     

Reconstitution of fibroblast growth factor receptor interactions in the yeast two hybrid system

Fibroblast growth factors (FGF) activate their receptors through the formation of trimolecular complexes, composed of a ligand, a receptor, and a heparan sulfate oligosaccharide, all of which are members of particularly large families capable of multiple interactions in a combinatorial fashion. Understanding this large network of interactions not only presents a great challenge, but is practically beyond the capacity of most classical techniques routinely used to study ligand receptor interactions. We have used the yeast two hybrid system to study protein-protein interaction in the FGF family. Both ligand and receptor ectodomains are properly folded and functional in the yeast. Basic FGF (bFGF) expressed in the yeast dimerizes spontaneously. This self-assembly occurs at low affinity, which can be greatly enhanced by the introduction of heparin, supporting a defined role for heparin in bFGF dimerization. Screening a rat embryo cDNA library with bFGF in the yeast two hybrid system identified a short variant of FGF receptor 1, found most frequently in embryonal and tumor cells and which possesses affinity toward bFGF that is significantly greater than that of the more abundant, full-length receptor. We find the yeast two hybrid system, a most suitable alternative method for the analysis of growth factor-receptor interactions as well as for screening for novel interacting proteins and modulators of FGF and its receptors.     

Application of SVM to predict membrane protein types

As a continuous effort to develop automated methods for predicting membrane protein types that was initiated by Chou and Elrod (PROTEINS: Structure, Function, and Genetics, 1999, 34, 137-153), the support vector machine (SVM) is introduced. Results obtained through re-substitution, jackknife, and independent data set tests, respectively, have indicated that the SVM approach is quite a promising one, suggesting that the covariant discriminant algorithm (Chou and Elrod, Protein Eng. 12 (1999) 107) and SVM, if effectively complemented with each other, will become a powerful tool for predicting membrane protein types and the other protein attributes as well.     

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.