In vitro assembly of apophytochrome and apophytochrome deletion mutants expressed in yeast with phycocyanobilin.
Recombinant pea type I phytochrome apoprotein expressed in yeast is shown to assemble in vitro with phycocyanobilin to produce a photoreversible phytochrome-like adduct. As an initial investigation of the amino acid sequence requirements for chromophore incorporation, three phyA gene product deletion mutants were produced in yeast. Truncation of the N-terminal tail to residue 46 demonstrates that this region is not critical to bilin attachment, but a deletion mutant lacking 222 amino acids from the N terminus failed to yield holophytochrome in vitro, under the same conditions. A mutant comprising a deletion of the C terminus to residue 548 showed bilin incorporation and red/far-red photoreversibility, indicating that bilin-apophytochrome assembly still occurred even when the entire C-terminal domain was truncated.
Deforce
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Plant Biological Regulation, Frontier Research Program, RIKEN, Saitama, Japan.
Tomizawa
K
K
Ito
N
N
Farrens
D
D
Song
P S
PS
Furuya
M
M
D001059:Apoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D052980:Phycobilins; D010798:Phycocyanin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010834:Phytochrome / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010946:Plants, Medicinal; D011758:Pyrroles / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013053:Spectrophotometry; D045725:Tetrapyrroles
D001059:Apoproteins; D009838:Oligodeoxyribonucleotides; D052980:Phycobilins; D011758:Pyrroles; D011993:Recombinant Fusion Proteins; D045725:Tetrapyrroles; C071907:apophytochrome; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin