Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins. For example, Arabidopsis thaliana has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins. The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions. Because most of these proteins display very similar activities on artificial or biological substrates in vitro, we developed an in vivo approach to answer this question. The disruption of both of the two Saccharomyces cerevisiae thioredoxin genes leads to pleiotropic effects including methionine auxotrophy, H 2 O 2 hypersensitivity, altered cell cycle characteristics, and a limited ability to use methionine sulfoxide as source of methionine. We expressed eight plant thioredoxins (six cytoplasmic and two chloroplastic) in yeast trx1, trx2 double mutant cells and analyzed the different phenotypes. Arabidopsis type h thioredoxin 2 efficiently restored sulfate assimilation whereas Arabidopsis type h thioredoxin 3 conferred H 2 O 2 tolerance. All thioredoxins tested could complement for reduction of methionine sulfoxide, whereas only type h thioredoxins were able to complement the cell cycle defect. These findings clearly indicate that specific interactions between plant thioredoxins and their targets occur in vivo.Thioredoxins are small oxidoreductases (molecular mass Ϸ12 kDa) that all contain two redox-active half-cystine residues in an exposed active center, having the amino acid sequence WCXPC (1). In its reduced form, thioredoxin can function as hydrogen donor for a variety of target proteins. The oxidized form of thioredoxin, having an intramolecular disulfide bridge between the two cysteine residues from the catalytic center, is generally reduced by a thioredoxin reductase. Thioredoxins are ubiquitous and have been described in prokaryotes as well as in eukaryotes, including, fungi, plants, invertebrates, and vertebrates (2-4). Higher plants are known to possess at least three types of thioredoxins: thioredoxin f and m, which are both encoded by the nuclear genome but are located within the chloroplast, and thioredoxin h, which is a cytosolic protein.The chloroplastic thioredoxins f and m are thought to provide a functional link between the light-absorbing pigments and several key metabolic enzymes such as fructose-1,6-bisphosphatase, phosphoribulose kinase, and malate dehydrogenase (5-7). Both oxidized thioredoxin f and thioredoxin m are reduced by a ferredoxin-dependent thioredoxin reductase (8-10). In contrast, the cytosolic thioredoxins h are reduced by an NADPH-dependent thioredoxin reductase (11,12). Recent progress in the systematic sequencing of plant genomes provides evidence that the diversity of thioredoxins is much greater than previously expected, each class of thioredoxin being encoded by multigene families. For instance, Arabidopsis thaliana has been shown to express at least two thioredoxins f (L.V. and...

By using a yeast functional complementation assay, we have identified AtTDX, a new Arabidopsis thaliana gene, encoding a two-domain 42-kDa protein. The amino-terminal domain of AtTDX is closely related to the co-chaperone Hsp70-interacting protein HIP, whereas its carboxyl-terminal part contains a thioredoxin domain. Both in vivo and in vitro assays showed that At-TDX is a protein-disulfide reductase. We next found that the HIP domain of AtTDX is capable of interacting with the ATPase domain of Ssb2, a yeast heat-shock protein 70 chaperone. Strikingly, the AtTDX-Ssb2 interaction can be released under oxidative stress, a redox-dependent regulation involving the thioredoxin activity of At-TDX. A mutation inactivating the cysteine 20 of the ATPase domain of Ssb2 was found to stabilize the At-TDX-Ssb2 interaction that becomes redox-insensitive. As cysteine 20 is conserved in virtually all the Hsp70 chaperones, our results suggest that this residue might be more generally the target of redox regulations of chaperone binding activity. Thioredoxin (TRX)1 is a small 12-kDa ubiquitous protein containing two redox-active half-cystine residues in an active center with conserved amino acid sequence Cys-X-X-Cys (where X indicates various amino acids) that functions as a protein-disulfide reductase. The two cysteine residues in the active site provide the sulfhydryl groups involved in the thioredoxin-dependent reducing activity. Under an oxidized form, the TRX-S 2 protein contains a disulfide bridge within the active site that is reduced to a TRX-(SH 2 ) dithiol by NADPH and the flavoprotein TRX reductase (for review, see Ref.