Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli  cells

Abstract

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30-Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA-DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone͞ menaquinone deprivation. Disulfide bond formation of ␤-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Impact of Membrane Fusion and Proteolysis on SpoIIQ Dynamics and Interaction with SpoIIIAH

“…The major signal from the amino acid sequencing analysis was GKSMEN (corresponding to amino acids 73-78 of SpoIIQ), suggesting that extracellular cleavage occurs between Val-72 and Gly-73. Second, we constructed a series of SpoIIQ derivatives with individual cysteines introduced between codons 62 and 74 and used AMS modification (35) to determine if the cysteine residue was present in the C-terminal degradation product, which confirmed that in intact cells proteolysis occurred between Val-72 and Gly-73 (supplemental Fig. S1).…”

DHEA attenuates PDGF-induced phenotypic proliferation of vascular smooth muscle A7r5 cells through redox regulation

“…The redox states of proteins were assessed by modifying free thiol with AMS [11]. Briefly, after incubation with or without PDGF-BB, cell lysates or proteins were treated with trichloroacetic acid at a final concentration of 7.5% to denature and precipitate the proteins as well as to avoid any subsequent redox reactions.…”

Characterization of the Menaquinone-dependent Disulfide Bond Formation Pathway of Escherichia coli

“…After overnight shaking, cells were diluted into the same medium for an additional 3 h of growth at 37°C. Cultures were directly treated with trichloroacetic acid (5%) to "freeze" the redox states of the proteins, which were dissolved in SDS solution containing AMS and subjected to SDS-PAGE and anti-DsbA immunoblotting (9). In this assay, reduced DsbA is mobility-shifted due to the AMS modification.…”

“…States of DsbA-E. coli strains CU141 (MC4100/FЈ, lacI q lacPL8 lacZ ϩ Y ϩ A ϩ pro ϩ ) (22), SS141 (CU141, dsbB::kan5) (3,23), TA36 (KS272, ubiA::Cm) (9), and AN384 (ubiA420 menA401) (21) were grown at 37°C in L-broth supplemented with 90 mM sodium phosphate (pH 7.5), 0.1 mM UQ1 (Sigma), and appropriate antibiotics. Exponentially growing cells were harvested by centrifugation, washed with the same medium without UQ1, and then re-inoculated into the UQ1-free medium.…”

“…DsbB, a plasma membrane protein, re-oxidizes the DsbA cysteines to enable catalytic functioning of DsbA (3)(4)(5)(6). The ultimate source of an oxidizing equivalent for this system is oxygen under aerobic conditions (7,8), in which respiratory chain components mediate electron transport (9). Most importantly, ubiquinone-8 (UQ8) 1 interacts directly with DsbB to receive electrons from the DsbA-DsbB oxidative system (7,10).…”