Arabidopsis repressor of light signaling, COP1, is regulated by nuclear exclusion: mutational analysis by bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET) between Renilla luciferase and yellow fluorescent protein has been adapted to serve as a real-time reporter on protein-protein interactions in live plant cells by using the Arabidopsis Constitutive photomorphogenesis 1 (COP1) protein as a model system...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 101; no. 17; pp. 6798 - 6802
Main Authors Subramanian, C, Kim, B.H, Lyssenko, N.N, Xu, X, Johnson, C.H, Arnim, A.G. von
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 27.04.2004
National Acad Sciences
Subjects
Alleles
Amino Acid Sequence
Analysis
Arabidopsis - metabolism
Arabidopsis - physiology
Arabidopsis Proteins - chemistry
Arabidopsis Proteins - genetics
Arabidopsis Proteins - metabolism
Arabidopsis Proteins - physiology
Arabidopsis thaliana
Biological Sciences
Bioluminescence
Botany
Cell Nucleus - metabolism
Dimerization
Energy
Energy Transfer
Epidermal cells
Flowers & plants
Fluorescence
Genetic Complementation Test
Hypocotyls
Light
Luminescent Measurements
Molecular Sequence Data
Mutant proteins
Mutation
Onions
Plant cells
Sequence Homology, Amino Acid
Signal Transduction - physiology
Transgenes
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Summary:Bioluminescence resonance energy transfer (BRET) between Renilla luciferase and yellow fluorescent protein has been adapted to serve as a real-time reporter on protein-protein interactions in live plant cells by using the Arabidopsis Constitutive photomorphogenesis 1 (COP1) protein as a model system. COP1 is a repressor of light signal transduction that functions as part of a nuclear E3 ubiquitin ligase. COP1 possesses a leucine-rich nuclear-exclusion signal that resides in a domain implicated in COP1 dimerization. BRET was applied in conjunction with site-directed mutagenesis to explore the respective contributions of the nuclear-exclusion and dimerization motifs to the regulation of COP1 activity in vivo. One specific mutant protein, COP1 L105 A, showed increased nuclear accumulation but retained the ability to dimerize, as monitored by BRET, whereas other mutations inhibited both nuclear exclusion and COP1 dimerization. Mutant rescue and overexpression experiments indicated that nuclear exclusion of COP1 protein is a rate-limiting step in light signal transduction.
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Abbreviations: COP1, Constitutive photomorphogenesis 1; CLS, cytoplasmic localization signal; CC, coiled coil; BRET, bioluminescence resonance energy transfer; RLUC, Renilla luciferase; YFP, yellow fluorescent protein.
Present address: Huck Institute for Life Sciences and Department of Biochemistry and Molecular Biology, Pennsylvania State University, 513 Wartik Laboratory, University Park, PA 16802.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AY189980 (35S:RLUC) and AY189981 (35S:YFP)].
This paper was submitted directly (Track II) to the PNAS office.
Edited by J. Woodland Hastings, Harvard University, Cambridge, MA, and approved March 12, 2004
To whom correspondence should be addressed. E-mail: vonarnim@utk.edu.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0307964101