Blue Light-induced Dimerization of Monomeric Aureochrome-1 Enhances Its Affinity for the Target Sequence [Gene Regulation]

May 1st, 2014 by Hisatomi, O., Nakatani, Y., Takeuchi, K., Takahashi, F., Kataoka, H.

Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in stramenopile alga, Vaucheria frigida. AUREO1 contains a basic leucine zipper (bZIP) domain in the central region and a light-oxygen-voltage-sensing (LOV) domain at the C-terminus, and has been suggested to function as a light-regulated transcription factor. We have previously reported that preparations of recombinant AUREO1 contained the complete coding sequence (FL) and N-terminal truncated protein (ZL) containing bZIP and LOV domains, and suggested that wild-type ZL (ZLwt2) was in a dimer form with intermolecular disulfide-linkages at Cys162 and Cys182 [Hisatomi et al., (2013) Plant Cell Physiol., 54, 93-106]. In the present study, we report the photoreactions, oligomeric structures, and DNA binding of monomeric cysteine-to-serine mutated ZL (ZLC2S), DTT-treated ZL (DTT-ZL), and FL (DTT-FL). Recombinant AUREO1 showed similar spectral properties and dark regeneration kinetics to those of dimeric ZLwt2. Dynamic light scattering and size exclusion chromatography revealed that ZLC2S and DTT-ZL were monomeric in the dark state. Dissociation of intermolecular disulfide bonds of ZLwt2 was in equilibrium with a midpoint oxidation-redox potential of approximately −250 mV. BL induced the dimerization of monomeric ZL, which subsequently increased its affinity for the target sequence. Also, DTT-FL was monomeric in the dark state and underwent BL-induced dimerization, which led to formation of the FL2/DNA complex. Taken together, our results suggest that monomeric AUREO1 is present in vivo, with dimerization playing a key role in its role as a BL-regulated transcription factor.