PARENT SESSION

Symposium S5B Light, redox and metabolic regulation: Dark reactions
Wednesday September 1st, 2004 10:20 AM-12:20 PM Room 510B
Chair: Peter Schürmann
Co-Chair: Toru Hisabori

Target proteins by chloroplast thioredoxin. —Efficient survey and characterization. Ken Motohashi^{*,1, 2}, Fumie Koyama^{1, 2}, Yoichi Nakanishi^{1, 2}, Hanayo Ueoka-Nakanishi^{1, 2}, Masasuke Yoshida^{1, 2}, Toru Hisabori^{1, 2}, ¹ ATP system project, Yokohama, Kanagawa, Japan² Chemical Resources Laboratory, Yokohama, Kanagawa, Japan

ABSTRACT- In chloroplasts, thioredoxin (Trx) regulates activities of various enzymes including ATP synthase and the four Calvin cycle enzymes, via thiol-disulfide exchange reaction. This regulation system is known as thiol modulation. Two Trxs, named Trx-m and Trx-f, in chloroplasts function for this redox regulation. On the process of the reduction of the target protein, a reduced form Trx exchanges the disulfide bond and di-thiols with the target thus gives the reduced form targets. We developed a new method to capture possible targets using mutant Trx-immobilized resin, and identified a number of targets for the chloroplast Trx (Motohashi, K. et al., Pro. Natl. Acad. Sci. USA 98, 11224-9 (2001)). Following our work, many target protein candidates for various Trxes and the related proteins were noted by using the similar strategy. We then characterized the interaction between Trx and several newly identified candidates. PrxQ, which is a novel type of peroxiredoxin and was captured by Trx-affinity chromatography, certainly showed peroxidase activity in Trx-dependent manner. In the case of chloroplast cyclophilin, we found that the peptidyl-prolyl cis-trans isomerase activity of this protein is fully inactivated in the oxidized state. Trx could reduce a disulfide bond on the oxidized cyclophilin and recovered the peptidyl-prolyl cis-trans isomerase activity. We finally determined two crucial disulfide bonds of chloroplast cyclophilin by disulfide-linked peptide mapping and the relevance of these cysteines for isomerase activity was confirmed by mutagenesis studies. This way, the knowledge on the candidate proteins of Trx is drastically accumulated. The physiological significance of redox regulation of the chloroplast proteins will be discussed based on the list of Trx candidates.

KEY WORDS: thioredoxin, cyclophilin, redox, peroxiredoxin