Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different

Abstract

Light-dependent regulation of a growing number of chloroplast enzymatic activities has been found to occur through the reversible reduction of intra-or intermolecular disulphides by thioredoxins. In cyanobacteria, despite their similarity to chloroplasts, no proteins have hitherto been shown to interact with thioredoxins, and the role of the cyanobacterial ferredoxin͞thioredoxin system has remained obscure. By using an immobilized cysteine 35-to-serine site-directed mutant of the Synechocystis sp. PCC 6803 thioredoxin TrxA as bait, we screened the Synechocystis cytosolic and peripheral membrane protein complements for proteins interacting with TrxA. The covalent bond between the isolated target proteins and mutated TrxA was confirmed by nonreducing͞reducing twodimensional SDS͞PAGE. Thus, we have identified 18 cytosolic proteins and 8 membrane-associated proteins as candidate thioredoxin substrates. Twenty of these proteins have not previously been associated with thioredoxin-mediated regulation. Phosphoglucomutase, one of the previously uncharacterized thioredoxinlinked enzymes, has not earlier been considered a target for metabolic control through disulphide reduction. In this article, we show that phosphoglucomutase is inhibited under oxidizing conditions and activated by DTT and reduced wild-type TrxA in vitro. The results imply that thioredoxin-mediated redox regulation is as extensive in cyanobacteria as in chloroplasts but that the subjects of regulation are largely different. Dithiol͞disulphide exchange catalyzed by thioredoxin (Trx) forms the molecular basis for light-dependent regulation of many enzymes in the chloroplasts of higher plants and algae (1-3). Ferredoxin receives reducing equivalents from the photosynthetic electron transport in the light and reduces Trx m and f by means of ferredoxin-Trx reductase. The Trxs, in turn, convert disulphides to dithiols in their respective target enzymes, thereby modulating their activities. The earliest discovered targets for Trx-mediated regulation belong to the Calvin cycle of CO 2 assimilation (1). Since the initial discoveries, several more chloroplast enzymes have been recognized as substrates for Trx (3). A breakthrough in the investigation of chloroplast redox regulation came with the introduction of a new method to isolate Trx target proteins (4-6). The method involves mutation of the buried redox-active cysteine of Trx, which favors the formation of stable mixed disulphides with its target proteins. This experimental approach confirmed the interaction between Trx and its known targets and more than doubled the number of potential Trx-regulated proteins (6).Cyanobacteria are oxygenic photosynthetic prokaryotes that probably share a common ancestor with the chloroplast. The complete sequence of the cyanobacterium Synechocystis sp. PCC 6803 genome (Cyanobase, www.kazusa.or.jp͞cyano͞cyano. html) reveals that this organism, hereafter referred to as Synechocystis, contains ferredoxin-Trx reductase and at least four different Trxs. Nevertheless, attempts to ...

New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii

“…In C. reinhardtii we found six potential targets involved in biosynthesis of arginine (argininosuccinate synthase), threonine (threonine synthase), lysine (diaminopimelate epimerase), branched amino acids valine and isoleucine (dihydroxyacid dehydratase and ketolacid reductoisomerase), and leucine (isopropylmalate dehydrogenase). Synechocystis argininosuccinate synthase (16) and Arabidopsis dihydroxyacid dehydratase (14) have already been proposed as TRX targets. The four other proteins are new targets.…”

“…A single study was led with cyanobacteria and revealed targets mostly different from those of higher plants (16). The unicellular eukaryotic green alga Chlamydomonas reinhardtii possesses a TRX system in chloroplasts and cytosol (17) and very likely in mitochondria (3).…”

Proteomic analyses of the photoauto- and diazotrophically grown cyanobacterium Nostoc sp. PCC 73102

“…Trxs are small, multifunctional, widely distributed redoxactive proteins operating through a reversible reduction of intra-or intermolecular disulfides Lindahl & Florencio, 2003). Recently, numerous putative Trx targets have been identified in plant chloroplasts (Balmer et al, 2003Marchand et al, 2004), in green algae (Lemaire et al, 2004) and in cyanobacteria (Lindahl & Florencio, 2003).…”

“…Recently, numerous putative Trx targets have been identified in plant chloroplasts (Balmer et al, 2003Marchand et al, 2004), in green algae (Lemaire et al, 2004) and in cyanobacteria (Lindahl & Florencio, 2003). About 200 proteins appear to be linked to Trx in plants (Buchanan & Balmer, 2005).…”

“…Putative thioredoxin (Trx) targets were predicted based on homology (BLAST) to sequences of known Trx-linked proteins. To determine the number of conserved cysteines in cyanobacterial Trx targets, homologues (see Supplementary Table S1, available with the online version of this paper) from cyanobacteria, plants or green alga were compared and analysed by CLUSTALW (Lemaire et al, 2004;Lindahl & Florencio, 2003) (Supplementary Fig. S1).…”