Subunit interactions in the carboxy-terminal domain of phytochrome

Abstract

We have produced defined fragments of the oat PhyA AP3 protein using an in vitro translation system and analyzed the quaternary structure of these fragments by size exclusion chromatography. Sequences between amino acids S599 and L683 are shown to dimerize by this in vitro assay and by a lambda repressor-based in vivo assay. A subset of this dimerization region, V623-S673, which has previously been identified as being involved in interdomain interactions on the basis of the behavior of overlapping constructs in a lambda repressor assay for protein-protein interaction, is shown by both assays to be necessary but insufficient for dimerization. Sequences between L685 and R815, which are unable to dimerize by themselves, are shown to interact with sequences between S599 and L683. Sequences E1069-Q1129, also previously suggested to be involved in dimerization, are shown here not to be required for phytochrome dimerization. These results based on an in vitro assay have confirmed some of the results previously obtained using an in vivo assay and extend these earlier results by revealing new protein-protein interactions. This dissection of sequences involved in phytochrome dimerization taken together with previous work has enabled us to propose a model for the behavior of the dimerization region where the core structure involved in dimerization is located on both sides of a region around residue 750 found at the surface.

Sequence similarities of phytochrome to protein kinases: implication for the structure, function and evolution of the phytochrome gene family

“…Possibly, this short region has an important function for the activity of the different proteins. Because of the predicted amphipatic s-helix nature of this region in conventional phytochromes, it was first speculated that it could be involved in phytochrome dimerization [30,31]; later, this hypothesis could not be confirmed with overexpression of deletion mutants of oat PhyA in transgenic tobacco [6] or by experiments with in vitro-translated fragments of oat PhyA performed by Edgerton and Jones [32].…”

The phytochrome apoprotein family inArabidopsis is encoded by five genes: the sequences and expression ofPHYD andPHYE

“…These portions of phytochrome have been shown to be important for chromophore attachment to the apoprotein and for spectral integrity of holo-phytochrome [4,22]. A candidate region for involvement in dimerization of the PHYA form of phytochrome [11,12] contains regions of comparatively high sequence conservation (positions 650-735 in Fig. 4), while the most amino-and carboxy-terminal sequences, which are important for biological activity of PHYA phytochrome in producing overexpression phenotypes [4,5,42], are poorly conserved among the five proteins.…”

Chromophore-bearing NH2-terminal domains of phytochromes A and B determine their photosensory specificity and differential light lability.

“…Each subunit has two major structural domains (3): (i) a globular NH2-terminal domain that carries the covalently bound chromophore (3) and (ii) an extended COOH-terminal domain that carries the dimerization sites (4,5). After synthesis in the red-absorbing phytochrome (Pr) form, photoconversion to the active far-red-absorbing phytochrome (Pfr) form is required for most responses.…”