Comprehensive survey of proteins targeted by chloroplast thioredoxin.
Possible target proteins of chloroplast thioredoxin (Trx) have been investigated in the stroma lysate of spinach chloroplasts. For that purpose, we immobilized a mutant of m-type Trx in which an internal cysteine at the active site was substituted with serine, on cyanogen bromide-activated resin. By using this resin, the target proteins in chloroplast were efficiently acquired when they formed the mixed-disulfide intermediates with the immobilized Trxs. We could acquire Rubisco activase (45 kDa) and 2-Cys-type peroxiredoxin (Prx), which were recently identified as targets of chloroplast Trxs. Glyceraldehyde-3-phosphate dehydrogenase and sedoheputulose 1,7-bisphosphatase, well-known thiol enzymes in the Calvin cycle, also were recognized among the collected proteins, suggesting the method is applicable for our purpose. Furthermore, four proteins were identified from a homology search of the NH(2)-terminal sequence of the acquired proteins: glutamine synthetase, a protein homologous to chloroplast cyclophilin, a homolog of Prx-Q, and the Rubisco small subunit. The Trx susceptibilities of the recombinant cyclophilin and Prx-Q of Arabidopsis thaliana were then examined. The method developed in the present study is thus applicable to investigate the various redox networks via Trxs and the related enzymes in the cell.
Motohashi
K
K
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan.
Kondoh
A
A
Stumpp
M T
MT
Hisabori
T
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000378:metabolism; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D021983:Cyclophilins / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D054464:Peroxiredoxins; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012116:Resins, Plant; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D012694:Serine; D018724:Spinacia oleracea / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism*
D004220:Disulfides; D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D012116:Resins, Plant; C054765:rca protein, plant; D012694:Serine; D013879:Thioredoxins; D010544:Peroxidases; D054464:Peroxiredoxins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010744:Phosphoric Monoester Hydrolases; C022627:sedoheptulose-bisphosphatase; D012273:Ribulose-Bisphosphate Carboxylase; D021983:Cyclophilins; D005974:Glutamate-Ammonia Ligase; D003545:Cysteine