Abstract
All organisms contain thioredoxin (TRX), a regulatory thiol:disul-fide protein that reduces disulfide bonds in target proteins. Unlike animals and yeast, plants contain numerous TRXs for which no function has been assigned in vivo. Recent in vitro proteomic approaches have opened the way to the identification of >100 TRX putative targets, but of which none of the numerous plant TRXs can be specifically associated. In contrast, in vivo methodologies, including classical yeast two-hybrid (Y2H) systems, failed to reveal the expected high number of TRX targets. Here, we developed a yeast strain named CY306 designed to identify TRX targets in vivo by a Y2H approach. CY306 contains a GAL4 reporter system but also carries deletions of endogenous genes encoding cytosolic TRXs (TRX1 and TRX2) that presumably compete with TRXs introduced as bait. We demonstrate here that, in the CY306 strain, yeast TRX1 and TRX2, as well as Arabidopsis TRX introduced as bait, interact with known TRX targets or putative partners such as yeast perox-iredoxins AHP1 and TSA1, whereas the same interactions cannot be detected in classical Y2H strains. Thanks to CY306, we also show that TRXs interact with the phosphoadenosine-5-phosphosulfate (PAPS) reductase MET16 through a conserved cysteine. Moreover, interactions visualized in CY306 are highly specific depending on the TRX and targets tested. CY306 constitutes a relevant genetic system to explore the TRX interactome in vivo and with high specificity, and opens new perspectives in the search for new TRX-interacting proteins by Y2H library screening in organisms with multiple TRXs. affinity chromatography ͉ gene disruption ͉ thioredoxin targets ͉ two-hybrid system ͉ redox T hioredoxins (TRXs) are small heat-stable oxidoreductases containing two redox-active half-cysteine residues in an active site with a conserved amino acid sequence CXXC (where X indicates various amino acids) (1). TRX was originally identified as hydrogen donor for the reduction of methionine sulfoxide (MetSO) (2) and of sulfate (3) in yeast and received its name when it was characterized as a small protein dithiol hydrogen donor to Escherichia coli ribonucleotide reductase (4). TRX is a hydrogen donor to perox-iredoxins (5) and is also required for a number of metabolic enzymes as part of their catalytic cycle. TRX is implicated in many cellular processes, including protein folding and regulation of transcription factors (6), protein repair after damage by oxidation, sulfur metabolism (7, 8), reduction of dehydroascorbate (9), germination , or reduction of the Calvin cycle and stromal enzymes in plants (10, 11). Occurrence of TRXs in all genomes is highly variable and somewhat complex, depending on the organisms concerned. Most organisms, such as E. coli, the yeast Saccharomyces cerevisiae, and mammals (12) contain a limited number of TRX or TRX-like genes (usually fewer than five). In contrast, plants possess numerous TRX isoforms (13, 14) or TRX-like proteins (11, 13-15). During the latest inventories in the Arabidopsis genome, Ͼ40 TRX isoforms have been identified (14). Such diversity raises the problem of the function and specificity of each TRX. To answer these questions, much recent work has been devoted to large-scale searches for their putative targets. Various improved in vitro proteomic approaches [such as affinity chromatography (16-21), labeled gel electrophore-sis in which target proteins are revealed after reduction of protein extract by a TRX͞NADPH thioredoxin reductase (NTR) system (22-24), or coimmunoprecipitation (25)] suggested Ͼ100 putative TRX targets, but for which further investigations will be required for target validation regarding TRX specificity. Other methods devoted to the large-scale isolation of TRX͞target complexes established in vivo have also been developed, including affinity chromatography of TRX͞target complexes established in vivo during cell culture or the yeast͞mammalian two-hybrid methods (26-31). However, such in vivo methods have led to the identification of only a few true TRX-interacting proteins. Their low efficiency to reveal TRX targets is directly correlated with the presence of functional endogenous TRX proteins in organisms in which large-scale search of TRX-interacting proteins is performed. These endogenous TRXs either compete with the foreign TRXs used as bait or most probably reduce the disulphide bond established in vivo between the TRX bait and its target protein (26). In the present work, we have developed a yeast two-hybrid (Y2H) system to allow high throughput detection of TRX͞target interactions in vivo. The main characteristic of this genetic selection is to bypass the disulfide bond reduction by endogenous yeast TRX1 and TRX2 cytosolic proteins, while establishing in vivo interaction between TRX introduced as bait and its putative targets. For this purpose, we created a yeast strain, termed CY306, that is depleted in both TRX1 and TRX2 genes but bears a functional Gal4 Y2H system. We show that CY306 exhibits the same phenotypes as those previously described for other trx1⌬ trx2⌬ yeast mutants. Thanks to strain CY306, we demonstrate in vivo Y2H interactions between TRXs and known or putative TRX targets that could not be revealed to date by classical Y2H systems or other in vivo systems. We specially show here that the yeast TRX2 interacts specifically with yeast AHP1 and TSA1 peroxiredoxins involved in oxidative stress response, whereas TRX1 preferentially interacts with PAPS reductase (MET16), an enzyme involved in sulfate assimilation. Regarding MET16, the use of CY306 was of great help in revealing the cysteine residue required for TRX binding. Finally, the high level of specificity in TRX͞target recognition obtained by the CY306 Y2H system contrasted with that observed by a His-tag affinity chromatography (His-TAC) approach with the same TRX baits. The use of the CY306 strain will be of great help first in analyzing by Y2H the numerous potential targets already isolated Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office.