The 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. 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(L105A), 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.
Subramanian
Chitra
C
Department of Botany, University of Tennessee, Knoxville, TN 37996-1100, USA.
Kim
Byung-Hoon
BH
Lyssenko
Nicholas N
NN
Xu
Xiaodong
X
Johnson
Carl Hirschie
CH
von Arnim
Albrecht G
AG
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism*; D019281:Dimerization; D004735:Energy Transfer; D005816:Genetic Complementation Test; D008027:Light; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases
D029681:Arabidopsis Proteins; C471983:AT2G32950 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases