Characterization of determinants for the specificity of Arabidopsis thioredoxins h in yeast complementation

The disruption of the two thioredoxin genes in Saccharomyces cerevisiae lea ds to a complex phenotype, including the inability to use methionine sulfox ide as sulfur source, modified cell cycle parameters, reduced H2O2 toleranc e, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements onl y some aspects of the phenotype, depending on the expressed thioredoxin: At TRX2 or AtTRX3 induce methionine sulfoxide assimilation and restore a norma l cell cycle. In addition AtTRX2 also confers growth on sulfate but no H2O2 tolerance, In contrast, AtTRX3 does not confer growth on sulfate but induc es H2O2 tolerance. We have constructed hybrid proteins between these two th ioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information need ed for H2O2 tolerance is located in the N-terminal part of AtTRX3. In addit ion, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the mul tiple Arabidopsis thioredoxins h originate from a totipotent ancestor with all the determinants necessary for interaction with the different thioredox in target proteins. After duplications each member evolved by losing or mas king some of the determinants.