Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae

The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cell s against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins ( Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized , all of them located in the cytoplasm, We have identified and characterize d a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14 -kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CA VC) present in prokaryotic thioredoxin reductases and binding sites for NAD PH and FAD, We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay, Trx3 and Trr2 proteins have N-terminal domain extensions with chara cteristics of signals for import into mitochondria, By immunoblotting analy sis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria, We have also constructed S. cerevisia e strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide, The Delta trr2 mutant was more sensitive to H2O2, where as the Delta trx3 mutant was as sensitive as the wild type. These results s uggest an important role of the mitochondrial thioredoxin reductase in prot ection against oxidative stress in S. cerevisiae.