FIG. 5.

Mitochondrial Prx1 regulates the redox state of Trx3. (A) Trx3::C58S is present in many disulfide-bonded forms in cells grown under respiratory conditions. Western blot analysis of Trx3 is shown for the glr1 trr2 trx3 mutant containing wild-type (TRX3) or Cys mutant versions of TRX3 (C55S, C58S, C55/58S) grown to an exponential phase under fermentative (SD) or respiratory (SGE) growth conditions. Proteins were separated using nonreducing SDS-PAGE. (B) Prx1 can be detected in a disulfide bonded form with Trx3::C58S. Western blot analysis of Prx1 is shown for the glr1 trr2 trx3 mutant containing wild-type (TRX3) or Cys mutant of TRX3 (C58S) grown to an exponential phase under respiratory (SGE) growth conditions. Proteins were separated using nonreducing (NR) or reducing (R) SDS-PAGE. The position of a Prx1-Trx3 dimer is indicated. (C) Prx1 is required to oxidize Trx3 in a glr1 trr2 mutant. The wild-type glr1 trr2, glr1 trr2 prx1, and prx1 mutant strains were grown to an exponential phase in SGE media. Proteins were precipitated with TCA and free thiols modified by reaction with AMS. Samples were separated using SDS-PAGE and Trx3 detected by Western blot analysis. Oxidized and reduced proteins are indicated. (D) Deletion of PRX1 restores the growth of a glr1 trr2 mutant under respiratory conditions. The wild-type, glr1 trr2, glr1 trr2 prx1, and glr1 trr2 trx3 mutant strains were grown to a stationary phase and spotted onto fermentative (YEPD) or respiratory (YEPGE) media. AMS, 4-acetamido-4′maleimidyldystilbene-2,2′-disulfonic acid; TCA, trichloracetic acid.