Identification and Functional Characterization of a Novel Mitochondrial Thioredoxin System in Saccharomyces cerevisiae

Abstract

The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells 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 characterized 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 (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH 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 characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The ⌬trr2 mutant was more sensitive to H 2 O 2 , whereas the ⌬trx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae. Thioredoxin (Trx)1 is a small protein (M r 12,000) with a conserved sequence (Trp-Cys-Gly-Pro-Cys) in its active site. When thioredoxin is in a reduced state, Trx-(SH) 2 , the two active-site cysteines form a dithiol group that is able to catalyze the reduction of disulfides in a number of proteins. Oxidized thioredoxin (Trx-S 2 ) can be reduced by NADPH through the catalytic action of the flavoenzyme thioredoxin reductase (Trr). Thus, Trx, Trr, and NADPH form a system (the thioredoxin system) that operates as a general disulfide reductase system by the following sequence of reactions (Reactions 1 and 2).

One Single In-frame AUG Codon Is Responsible for a Diversity of Subcellular Localizations of Glutaredoxin 2 in Saccharomyces cerevisiae

“…The functions of both systems overlap to a certain extent, although the major difference is that Grxs have a glutathione binding site and are capable of reducing GSH protein-mixed disulfides (6,13,14). Mitochondrial isoforms of each member of both systems have been characterized in yeast (15)(16)(17) and mammals (18 -20). Three Grx subfamilies have been distinguished (21).…”

Cytochrome c Maturation

“…If formation of a disulfide must be avoided, a thioredoxin-like protein could be required in the intermembrane space (30). A thioredoxin (Trx3) and its reductase (Trr2) are known to be present in yeast mitochondria, but submitochondrial localization to the matrix is suspected rather than to the intermembrane space (31). These proteins can in any case be deduced to be dispensable for cytochrome c biogenesis in yeast, as mutants carrying specific disruptions in the corresponding genes were able to grow normally under aerobic, respiratory conditions, which require participation of cytochrome c in the mitochondrial respiratory chain.…”

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

“…The superoxide anion can be converted to hydrogen peroxide by superoxide dismutases, and hydrogen peroxide can be converted to water either by cytosolic or peroxisomal catalases or by thiol-based peroxidases [2]. These peroxidases are present in various subcellular compartments [3][4][5], and their redox-active glutathione or thioredoxin cofactors are maintained in reduced states by NADPH-dependent reductases [5][6][7][8].…”