A Fluorescent Two-hybrid Assay for Direct Visualization of Protein Interactions in Living Cells

Abstract

Genetic high throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm, and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the fluorescent two-hybrid assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real time. Molecular & Cellular Proteomics 7: 2279 -2287, 2008.After sequencing the human genome the next challenge is now to analyze the complex protein networks underlying cellular functions. In the last decade a wide variety of methods to study protein-protein interactions ranging from biochemical to genetic or cell-based approaches have been developed. Biochemical methods like affinity purification or co-immunoprecipitation (Co-IP) 1 allow the detection of protein complexes in vitro. Genetic methods, such as the yeast two-hybrid system (1), enable efficient high throughput screening of interactions within the cellular environment (2). However, the analysis of mammalian protein interactions in yeast may suffer from the absence or insufficient conservation of cellular factors modulating protein-protein interactions, e.g. through posttranslational modifications (3).In the last years new fluorescence-based methods for incell visualization of protein-protein interactions have been introduced. Two established techniques, fluorescence resonance energy transfer (4, 5) and bimolecular fluorescence complementation (6), are based on the expression of fluorescently labeled proteins or fragments thereof. However, fluorescence resonance energy transfer requires costly instrumentation and advanced technical expertise, whereas bimolecular fluorescence complementation is based on the irreversible complementation and slow maturation of fluorophores, which does not allow real time detection of proteinprotein interactions (6). Another strategy is based on the relocation of proteins to either cell membranes (7) or cytoplasmic aggregates of viral proteins (8).All these methods have inherent shortcomings and are typically combined to obtain more reliable results. We have now developed a novel fluorescent two-hybrid (F2H) assay for the direct visualization of protein-protein interactions in living mammalian cells. The simple optical readout of this F2H assay allows observation of protein-protein interactions in real time and should also be suitable for high throughput screens. EXPERIMENTAL PROCEDURESExpression Construct...

Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins

“…The virtually inexhaustible source of the anti-GFP antibody makes it possible to perform interaction assays on a large scale. Although previous studies have made use of anti-GFP nanobody to perform various interaction assays with low or medium throughput (Pichler et al, 2012;Rothbauer et al, 2008;Zolghadr et al, 2008), those studies were all limited to binary interactions. In this study, we made use of anti-GFP nanobody to co-immunoprecipitate up to eight proteins fused to up to four distinct fluorescent proteins, thereby elucidating the architectures of the BBSome and exocyst complexes.…”

De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation

“…Different GFP-tagged proteins were recruited to the lacO arrays through a fusion of LacI repressor to a highaffinity GFP-binding domain (GBP) and a red fluorescent protein domain (GBP-LacI-RFP). Endogenous interaction partners of the GFP-labeled protein were then identified by subsequent immunostaining and evaluation of the colocalization of the fluorescence signals by confocal laser scanning microscopy (Rothbauer et al, 2006;Zolghadr et al, 2008). (B) The F6B2 U2OS cell line was co-transfected with GFP-PML and GBP-LacI expression vectors.…”

“…To address this issue, we investigated the de novo formation of APBs. Protein components of APBs were recruited to telomeres tagged with stable integrations of bacterial lac operator DNA sequence (lacO) repeats in the ALT-positive human osteosarcomatelomeric lacO arrays) directly adjacent to the telomeres of chromosomes 6q, 11p and 12q (Jegou et al, 2009), was transfected with a bacterial LacI repressor fused to a GFPbinding protein (GBP) (Rothbauer et al, 2006;Zolghadr et al, 2008). The LacI construct with (GBP-LacI-RFP) or without (GBP-LacI) an additional red fluorescent mRFP1 marker was used to recruit GFP-or YFP-tagged proteins and interacting factors to the telomere-associated lacO arrays (Fig.…”

Regulation of DNA methyltransferase 1 by interactions and modifications

“…Indeed, DNMT1 associates with the replication machinery 15 by directly binding to PCNA, a homotrimeric The function of proteins is according to the UniProt database 38,39 and the applied method is according to the BioGriD 40 database apart from the fluorescent two-hybrid (F2H) assay. 41 All protein names are according to the human nomenclature unless noted otherwise. Proteins involved in the post-translational modification of DNMT1 are highlighted in green.…”