RRC ID 1887
著者 Subramanian C, Woo J, Cai X, Xu X, Servick S, Johnson CH, Nebenführ A, von Arnim AG.
タイトル A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET).
ジャーナル Plant J
Abstract Bioluminescence resonance energy transfer (BRET) is a natural biophysical phenomenon that underlies an emerging technique to monitor protein-protein interactions in living cells in real time. Here, we present a series of technical advances to enhance the utility of the BRET assay in plants. A series of recombination cloning vectors was generated to accelerate the expression of proteins tagged with Renilla luciferase or yellow fluorescent protein under transient assay conditions and in stable transgenic plants. Working in stably transformed Arabidopsis or tobacco, we then detected BRET between three pairs of candidate interaction partners: dimerization of the E3 ubiquitin ligase COP1, interaction between COP1 and the B-box protein STH, and interaction between the light regulatory bZip transcription factors HY5 and HYH. A codon-optimized version of the Renilla luciferase gene resulted in improved expression in Arabidopsis. Renilla luciferase was active in a variety of subcellular organelles, including plastids, mitochondria, peroxisomes and Golgi stacks. In a survey of the Arabidopsis light signaling machinery as a model system, we estimated the likelihood that a known protein-protein interaction can be documented using BRET. Finally, we show that Renilla luciferase may serve as a reporter of protein stability in a cycloheximide chase assay.
巻・号 48(1)
ページ 138-52
公開日 2006-10-1
DOI 10.1111/j.1365-313X.2006.02851.x
PII TPJ2851
PMID 16925598
MeSH Arabidopsis / genetics Arabidopsis / metabolism Arabidopsis / ultrastructure Arabidopsis Proteins / analysis Basic-Leucine Zipper Transcription Factors / analysis Carrier Proteins / analysis DNA-Binding Proteins Genes, Reporter Light Luciferases / analysis Luminescent Measurements / methods* Nuclear Proteins / analysis Plant Proteins / analysis* Protein Interaction Mapping / methods* Recombinant Fusion Proteins / analysis Signal Transduction Tobacco / genetics Tobacco / metabolism Tobacco / ultrastructure Ubiquitin-Protein Ligases / analysis
IF 6.141
引用数 60
WOS 分野 PLANT SCIENCES
リソース情報
シロイヌナズナ / 植物培養細胞・遺伝子 pda01212 pda05441 pda01945