On the functional interchangeability, oxidant versus reductant, of members of the thioredoxin superfamily

Abstract

Escherichia coli thioredoxin 1 has been characterized in vivo and in vitro as one of the most efficient reductants of disulfide bonds. Nevertheless, under some conditions, thioredoxin 1 can also act in vivo as an oxidant, promoting formation of disulfide bonds in the cytoplasm (E. J. Stewart, F. Aslund, and J. Beckwith, EMBO J. 17:5543-5550, 1998). We recently showed that when a signal sequence is attached to thioredoxin 1 it is exported to the periplasm, where it can also act as an oxidant, replacing the normal periplasmic catalyst of disulfide bond formation, DsbA, in oxidizing cell envelope proteins (L. Debarbieux and J. Beckwith, Proc. Natl. Acad. Sci. USA 95:10751-10756, 1998). Here we report pulse-chase studies of the efficiency of disulfide bond formation in strains exporting thioredoxin 1 and more-oxidizing variants of it. While the exported thioredoxin 1 itself substantially speeds up the kinetics of disulfide bond formation, a version of this protein containing the DsbA active site exhibits kinetics that are indistinguishable from those of the DsbA protein itself. Further, we confirm the findings of Jonda et al. (S. Jonda, M. Huber-Wunderlich, R. Glockshuber, and E. Mössner, EMBO J. 18:3271-3281, 1999), who found that DsbB is responsible for the oxidation of exported thioredoxin 1, and we report the detection of a disulfide-bonded DsbB-thioredoxin 1 complex. Finally, we have found that under conditions of high-level expression of exported thioredoxin 1, the protein can act as both an oxidant and a reductant.

FIG. 1

Rate of oxidation of OmpA catalyzed by exported thioredoxin 1. After labeling of cells with [³⁵S]methionine (200 μCi/ml) for 1 min, chase point samples were obtained and incubated in the presence of 100 mM iodoacetamide in order to alkylate free thiol residues (0.1% final concentration of cold methionine was used for the chase). An OmpA antiserum was used to immunoprecipitate this protein (see reference for details), and the samples were separated on a nonreducing sodium dodecyl sulfate-polyacrylamide gel. The OmpA oxidation rate was determined in a wild-type strain (A), a dsbA strain (B to D), and a dsbA dsbC dsbD triple-mutant strain (E to G) expressing cytoplasmic thioredoxin 1 (A, B, and E), exported thioredoxin 1 (C and F), or exported thioredoxin 1 with a DsbA active site (D and G). Values for 50% oxidation time (t_1/2ox) were determined after the quantitation of reduced (red) and oxidized (ox) forms of OmpA by phosphorimager analysis (Bio-Rad).

FIG. 2

Detection of a mixed disulfide bond between exported thioredoxin 1 and DsbB. Equal amounts of proteins from a wild-type (lanes 1 and 4), a dsbA (lanes 2 and 5), and a dsbB (lanes 3 and 6) strain expressing cytoplasmic thioredoxin 1-Cys35⧫Tyr (lanes 1 to 3) or exported thioredoxin 1-Cys35⧫Tyr (lanes 4 to 6) were separated on a nonreducing (minus dithiothreitol [−DTT]) and a reducing (plus dithiothreitol [+DTT]) sodium dodecyl sulfate-polyacrylamide gel and transferred to two nitrocellulose membranes. The left part of each of the membranes was incubated with TrxA antiserum (anti-TrxA Ab.), and the right part was incubated with DsbB antiserum (anti-DsbB Ab.). Prestained molecular mass markers were loaded between the left and the right panels so that the two pieces of membrane could be aligned. Band A is the cytoplasmic thioredoxin 1, band B is the 6 amino acids-thioredoxin 1 processed form, band C is the precursor APss-6 amino acids-thioredoxin 1, band D is DsbB, and band E is the mixed disulfide complex of exported thioredoxin 1 Cys35⧫Tyr and DsbB.