Subunit interactions in the carboxy-terminal domain of phytochrome.
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.
Edgerton
M D
MD
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
Jones
A M
AM
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D002523:Edible Grain / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry*; D010834:Phytochrome / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000235:genetics; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D010834:Phytochrome