用于蛋白质间相互作用研究的新型双杂交系统

近年来,一些不依赖于转录因子活性的新型双杂交系统相继建立,如分离的泛素系统、蛋白质片段互补分析、阻遏物重构分析和SOS恢复系统等。同利用转录因子活性的酵母双杂交系统相似,这些方法也利用了一些活性蛋白的结构与功能特点来研究蛋白质间相互作用,这些活性蛋白不是转录因子,但也可在结构上进行分离可通过重构使其生物活性得以恢复。由于这些新型双杂交系统的各自特点,使得它们成为酵母双杂交系统的有益补充和研究蛋白质间相互作用的有力工具。     

Genetic approaches to the identification of interactions between membrane proteins in yeast

The recent sequencing of entire eukaryotic genomes has renewed the interest in identifying and characterizing all gene products that are expressed in a given organism. The characterization of unknown gene products is facilitated by the knowledge of its binding partners. Thus, a novel protein may be classified by identifying previously characterized proteins that interact with it. If such an approach is carried out on a large scale, it may allow the rapid characterization of the thousands of predicted open reading frames identified by recent sequencing projects. Currently, the yeast two-hybrid system is the most widely used genetic assay for the detection of protein-protein interactions. The yeast two-hybrid system has become popular because it requires little individual optimization and because, as compared to conventional biochemical methods, the identification and characterization of protein-protein interactions can be completed in a relatively short time span. In this review, we briefly discuss the yeast two-hybrid system and its application to large scale screening studies that aim at deciphering all protein-protein interactions taking place in a given cell type or organism. We then focus on a class of proteins that is unsuitable for conventional yeast two-hybrid systems, namely integral membrane proteins and membrane-associated proteins, and describe several novel genetic systems that combine the advantages of the yeast two-hybrid system with the potential to identify interaction partners of membrane-associated proteins in their natural setting.     

Reporter gene imaging of protein-protein interactions in living subjects

In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene expression within living subjects. An important application of this approach is the molecular imaging of interacting protein partners, which could pave the way to functional proteomics in living animals and might provide a tool for the whole-body evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies. In the future, these innovative approaches are likely to enhance our appreciation of entire biological pathway systems and their pharmacological regulation.     

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.     

Re-engineering a split-GFP reassembly screen to examine RING-domain interactions between BARD1 and BRCA1 mutants observed in cancer patients

Identification of protein-protein interactions is critical for understanding protein function and regulation. Split protein reassembly is an in vivo probe of protein interactions that circumvents some of the problems with yeast 2-hybrid (indirect interactions, false positives) and co-immunoprecipitation (loss of weak and transient interactions, decompartmentalization). Split GFP reassembly, also called Bimolecular Fluorescence Complementation (BiFC), is especially attractive because the GFP chromophore forms spontaneously on protein folding in virtually every cell type tested. However, cellular fluorescence evolves slowly in bacteria and fails to evolve at all for some interactions. We aimed to use split-GFP reassembly to examine the determinants of association for a heterodimeric four-helix bundle, and we chose the N-terminal RING domains of BARD1 and the tumor suppressor BRCA1 as our test system. The wild-type interaction failed to give fluorescence with the split sg100 GFP variant. We found that split folding-reporter GFP (a hybrid of EGFP and GFPuv) evolves fluorescence much faster (overnight) with associating peptides and also evolves fluorescence for the BRCA1/BARD1 wild-type pair. Six cancer-associated BRCA1 interface mutants were examined with the system, and only two resulted in a significant reduction in complex reassembly. These results are generally in accord with Y2H studies, but the differences highlight the utility of complementary approaches. The split frGFP system may also be generally useful for other proteins and cell types, as the split-Venus system has proven to be in mammalian cells.     

Quenching accumulation of toxic galactose-1-phosphate as a system to select disruption of protein-protein interactions in vivo

The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.     

Analysing protein-protein interactions with the yeast two-hybrid system

Plant research is moving into the post-genomic era. Proteomic-based strategies are now being developed to study functional aspects of the genes predicted from the various genome-sequencing initiatives. All biological processes depend on interactions formed between proteins and the mapping of such interactions on a global scale is providing interesting functional insights. One of the techniques that has proved itself invaluable in the mapping of protein-protein interactions is the yeast two-hybrid system. This system is a sensitive molecular genetic approach for studying protein-protein interactions in vivo. In this review we will introduce the yeast two-hybrid system, discuss modifications of the system that may be of interest to the plant science community and suggest potential applications of the technology.     

酵母三杂交系统的原理和应用

酵母双杂交系统自出现以来,广泛用于研究蛋白质之间的相互作用,它是一种具有高灵敏度的研究蛋白质之间关系的技术.在酵母双杂交系统基础上发展的酵母三杂交系统将应用范围扩展到蛋白质-蛋白质、蛋白质-RNA、蛋白质-小分子化合物等更广阔的研究领域.本文着重介绍酵母三杂交系统的原理、应用及局限性.     

Identification of a S-ribonuclease-binding protein in Petunia hybrida

To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBP1 may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.     

Foreword: The structural biology of membrane proteins

Membrane protein-protein interactions are important for regulation, targeting, and activity of proteins in membranes but are difficult to detect and analyse. This review covers current approaches to studying membrane protein interactions. In addition to standard biochemical and genetic techniques, the classic yeast nuclear two-hybrid system has been highly successful in identification and characterization of soluble protein-protein interactions. However, classic yeast two-hybrid assays do not work for membrane proteins because such yeast-based interactions must occur in the nucleus. Here, we highlight recent advances in yeast systems for the detection and characterization of eukaryote membrane protein-protein interactions. We discuss these implications for drug screening and discovery.     

In vivo quantification of protein-protein interactions in Saccharomyces cerevisiae using bimolecular fluorescence complementation assay

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.