title: A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET).
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.
authors.affiliation: Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA.
authors.forename: Chitra
authors.initials: C
authors.lastname: Subramanian
authors.forename: Jongchan
authors.initials: J
authors.lastname: Woo
authors.forename: Xue
authors.initials: X
authors.lastname: Cai
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xu
authors.forename: Stein
authors.initials: S
authors.lastname: Servick
authors.forename: Carl H
authors.initials: CH
authors.lastname: Johnson
authors.forename: Andreas
authors.initials: A
authors.lastname: Nebenführ
authors.forename: Albrecht G
authors.initials: AG
authors.lastname: von Arnim
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000032:analysis; D050976:Basic-Leucine Zipper Transcription Factors / Q000032:analysis; D002352:Carrier Proteins / Q000032:analysis; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D008027:Light; D008156:Luciferases / Q000032:analysis; D008163:Luminescent Measurements / Q000379:methods*; D009687:Nuclear Proteins / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis*; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D044767:Ubiquitin-Protein Ligases / Q000032:analysis
chemical_list: D029681:Arabidopsis Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C109254:HY5 protein, Arabidopsis; C457459:HYH protein, Arabidopsis; D009687:Nuclear Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; C471983:AT2G32950 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases