Thiol-Independent Action of Mitochondrial Thioredoxin To Support the Urea Cycle of Arginine Biosynthesis in Schizosaccharomyces pombe

Thioredoxins usually perform a role as a thiol-disulfide oxidoreductase using their active-site cysteines. The fission yeastSchizosaccharomyces pombe contains two thioredoxins: Trx1 for general stress protection and Trx2 for mitochondrial functions. TheΔtrx2 mutant grows as well as the wild type on complex media containing glucose. However, on nonfermentable carbon source such as glycerol, the mutant did not grow, indicating a defect in mitochondrial function. The mutant also exhibited auxotrophy for arginine and cysteine on minimal medium. In order to find the reason for the unexpected arginine auxotrophy, we searched for multicopy suppressors and found that thearg3 + gene encoding ornithine carbamoyltransferase (OCTase) in the urea cycle of the arginine biosynthetic pathway rescued the arginine auxotrophy. The levels ofarg3 + transcript, Arg3 protein, and OCTase activity were all decreased inΔtrx2 . Through immunocoprecipitation, we observed a direct interaction between Trx2 and Arg3 in cell extracts. The mutant forms of Trx2 lacking either one or both of the active site cysteines through substitution to serines also rescued the arginine auxotrophy and restored the decreased OCTase activity. They also rescued the growth defect ofΔtrx2 on glycerol medium. This contrasts with the thiol-dependent action of overproduced Trx2 in complementing glutathione reductase. Therefore, Trx2 serves multiple functions in mitochondria, protecting mitochondrial components against thiol-oxidative damage as a thiol-disulfide oxidoreductase, and supporting urea cycle and respiration in mitochondria in a manner independent of active site thiols.