Molecular genetic studies of cryptochrome 2 function in Arabidopsis thaliana.

摘要

Cryptochromes are flavo-protein blue light photoreceptors that regulate growth and development in plants, and the circadian clock in plants and animals. Arabidopsis CRY1 was shown to undergo light-dependent reduction of the flavin cofactor by intra-protein electron transfer through three adjacent Trp residues (Trp triad). This photoreduction reaction was proposed to be required for photoreceptor signaling. In this work we investigated the requirement for a redox active flavin cofactor in Arabidopsis CRY2. We showed that proper binding of the flavin cofactor is required for signaling and that photoreduction through the Trp triad is not required for CRY2 activity. Analysis was performed in vitro and in vivo, using CRY2 proteins with site-specific amino acid substitutions in the flavin binding pocket and the Trp triad. Some of the mutations resulted in gain of function which we concluded was the result of a small conformational change near the active site.;The structural basis of the active CRY dimer was investigated computationally using molecular models and the PONDR VL-XT algorithm. A surface region of the N-terminal (PHR) light-sensing and dimerization domain was identified by low VL-XT score and phylogenetic sequence conservation as a candidate for mediating a protein-protein interaction. Two CRY1 PHR monomers were manually positioned relative to one another using the Pymol program so that a topological complementarity was achieved at the candidate surface.;A region of 80 amino acids at the transition from the N- into the C-terminal domain of Arabidopsis CRY2 was shown to be required for photoreceptor activity. A structure-function analysis was performed using fusion proteins of C-terminal truncations linked to a heterologous dimerization domain. CRY2 is activated and phosphorylated in response to blue light. The 80 aa minimal domain of the GUS-NC80 fusion protein was constitutively active and unphosphorylated, suggesting that light activates CRY2 by inducing a conformational change that exposes NC-80.;The life-cycle of CRY2 was shown to take place in the nucleus using conditional nuclear localization by fusion of CRY2 to the gluccocorticoid receptor. Phosphorylation, signaling and degradation by the 26s proteasome occur only when CRY2 is in the nucleus.