Yves Balmer

Proteomics gives insight into the regulatory function of chloroplast thioredoxins

Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.

Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics

Application of a thiol‐specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin‐linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed.

Proteomics uncovers proteins interacting electrostatically with thioredoxin in chloroplasts

The ability of thioredoxin f to form an electrostatic (non-covalent) complex, earlier found with fructose-1,6-bisphosphatase, was extended to include 27 previously unrecognized proteins functional in 11 processes of chloroplasts. The proteins were identified by combining thioredoxin f affinity chromatography with proteomic analysis using tandem mass spectrometry. The results provide evidence that an association with thioredoxin enables the interacting protein to achieve an optimal conformation, so as to facilitate: (i) the transfer of reducing equivalents from the ferredoxin/ferredoxin—thioredoxin reductase complex to a target protein; (ii) in some cases, to enable the channeling of metabolite substrates; (iii) to function as a subunit in the formation of multienzyme complexes.

Yet another plant thioredoxin

Thioredoxins are widely distributed proteins that function in a broad spectrum of cellular reactions. Plant cells have well characterized chloroplast and cytosolic thioredoxin systems, but, unlike animals and yeast, a mitochondrial counterpart has not been clearly defined. Recently, a complete thioredoxin system has been described in plant mitochondria, opening a new door for the study of thioredoxins as well as mitochondria.

Heterodimer formation between thioredoxin f and fructose 1,6‐bisphosphatase from spinach chloroplasts

Chloroplast fructose 1,6‐bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox‐active disulfide bridge. The goal of this study was to identify the Cys involved in the transient mixed disulfide. To stabilize this reaction intermediate, mutant proteins with modified active sites were used. We identified Cys‐155 of the FBPase as the one engaged in the formation of the mixed disulfide intermediate with Cys‐46 of Trx f.

Plant thioredoxins: role of the French School

Abstract A seminal discovery made by Pierre Gadal and his collaborators in Nancy, almost 30 years ago, has served as a nucleus for further development of research on plant thioredoxins. By attracting other investigators, research in the area has flourished and enabled France to assume a leadership position in the field. The chronology of development of this research is traced in the present review, from its origin in Nancy to its current distribution throughout France. A summary of the achievements of participating French scientists documents their influence on the field and the international thioredoxin community. The article closes with a personal perspective of Gadal’s scientific and professional contributions that was developed by the senior author during visits to Orsay over the past three decades.

Construction of Mutants for the Study of Reduction of Spinach Fructose-1,6-Bisphosphatase by Thioredoxin F

Chloroplast fructose-1, 6-bisphosphatase (FBPase) is a key enzyme of the Calvin cycle involved in regeneration of ribulose bisphosphate and it has the particularity, like several other chloroplastic enzymes, to be light regulated. Activation of these enzymes is due to an electron transfer from the dithiol of thioredoxin f (TRO to the disulfide of a regulatory site. The spinach TRf active site, formed between Cys 46 and 49, gets electrons from the photosynthetic electron flow through the ferredoxin-thioredoxin system and activates target enzymes by reduction (1).