Comprehensive survey of proteins targeted by chloroplast thioredoxin

DOI Web Site PubMed 38 Citations Open Access
  • Ken Motohashi
    Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan; and Core Research for Evolutional Science and Technology (CREST), Genetic Programming Team 13, Nogawa 907, Miyamae-ku, Kawasaki 216-0001, Japan
  • Aiko Kondoh
    Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan; and Core Research for Evolutional Science and Technology (CREST), Genetic Programming Team 13, Nogawa 907, Miyamae-ku, Kawasaki 216-0001, Japan
  • Michael T. Stumpp
    Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan; and Core Research for Evolutional Science and Technology (CREST), Genetic Programming Team 13, Nogawa 907, Miyamae-ku, Kawasaki 216-0001, Japan
  • Toru Hisabori
    Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan; and Core Research for Evolutional Science and Technology (CREST), Genetic Programming Team 13, Nogawa 907, Miyamae-ku, Kawasaki 216-0001, Japan

Description

<jats:p> 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 <jats:italic>m</jats:italic> -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- <jats:italic>bis</jats:italic> phosphatase, 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 <jats:sub>2</jats:sub> -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 <jats:italic>Arabidopsis thaliana</jats:italic> 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. </jats:p>

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