Comprehensive survey of proteins targeted by chloroplast thioredoxin

Abstract

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.

Light Regulation of Nitrate uptake, Assimilation and Metabolism

“…GS2 was activated by the sulfhydryl-reducing agent DTT like enzymes activated by photosynthetic electron transport by the ferredoxin-thioredoxin system (Choi et al 1999). Regulation of GS2 by a redox change was further confirmed by identification of Arabidopsis GS as a binding target for thioredoxin (Motohashi et al 2001).…”

Proteomics Uncovers Proteins Interacting Electrostatically with Thioredoxin in Chloroplasts

“…The proteins identified in the present study function in a spectrum of chloroplast processes (Table 2). In addition to FBPase, four other enzymes of the Calvin cycle, all known targets, showed an electrostatic interaction with thioredoxin -glyceraldehyde 3-phosphate dehydrogenase, rubisco activase, rubisco small subunit and transketolase (Schürmann and Jacquot 2000;Motohashi et al 2001;Balmer et al 2003). These enzymes function in the three phases of the Calvin cycle: carboxylation, reduction and regeneration.…”

“…The chloroplast ferredoxin/thioredoxin system -composed of ferredoxin, ferredoxin-thioredoxin reductase (FTR) and several thioredoxins (f, m and the recently added x and y) -links photosynthetic electron flow to the regulation of a growing number of biochemical processes (Buchanan 1980;Mestres-Ortega and Meyer 1999;Schürmann and Jacquot 2000;Motohashi et al 2001;Buchanan et al 2002;Balmer et al 2003;Lemaire et al 2003;Marx et al 2003). The iron-sulfur/disulfide enzyme FTR transfers electrons from ferredoxin to a thioredoxin which, in turn, activates (or deactivates) enzymes and related functional proteins by reduction of a specific disulfide site (Dai Dedicated to Myroslawa Miginiac-Maslow and Peter Schürmann on the occasion of their retirement.…”

“…The recent identification of new potential thioredoxin targets in chloroplasts raises the possibility that other proteins could form such complexes (Verdoucq et al 1999;Motohashi et al 2001;Goyer et al 2002;Balmer et al 2003). To pursue this question, we have analyzed chloroplast stroma with affinity chromatography and proteomic techniques.…”

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

“…The c domain of animal PDIs has been proposed to serve as lowaffinity, high-capacity Ca 2+ binding domain (Lucero and Kaminer, 1999). PDI utilizes the thioredoxin domains for catalysis, which places them in the superfamily of thioredoxin-like proteins (Motohashi et al, 2001). However, PDIs are several-fold larger than thioredoxins (Ferrari and Soling, 1999;Lu and Christopher, 2008) and have other functions in addition to protein folding.…”