The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner - PubMed (original) (raw)

The disulphide isomerase DsbC cooperates with the oxidase DsbA in a DsbD-independent manner

Didier Vertommen et al. Mol Microbiol. 2008 Jan.

Abstract

In Escherichia coli, DsbA introduces disulphide bonds into secreted proteins. DsbA is recycled by DsbB, which generates disulphides from quinone reduction. DsbA is not known to have any proofreading activity and can form incorrect disulphides in proteins with multiple cysteines. These incorrect disulphides are thought to be corrected by a protein disulphide isomerase, DsbC, which is kept in the reduced and active configuration by DsbD. The DsbC/DsbD isomerization pathway is considered to be isolated from the DsbA/DsbB pathway. We show that the DsbC and DsbA pathways are more intimately connected than previously thought. dsbA(-)dsbC(-) mutants have a number of phenotypes not exhibited by either dsbA(-), dsbC(-) or dsbA(-)dsbD(-) mutations: they exhibit an increased permeability of the outer membrane, are resistant to the lambdoid phage Phi80, and are unable to assemble the maltoporin LamB. Using differential two-dimensional liquid chromatographic tandem mass spectrometry/mass spectrometry analysis, we estimated the abundance of about 130 secreted proteins in various dsb(-) strains. dsbA(-)dsbC(-) mutants exhibit unique changes at the protein level that are not exhibited by dsbA(-)dsbD(-) mutants. Our data indicate that DsbC can assist DsbA in a DsbD-independent manner to oxidatively fold envelope proteins. The view that DsbC's function is limited to the disulphide isomerization pathway should therefore be reinterpreted.

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Figures

Fig. 1

Fig. 1. The absence of dsbA and dsbC has phenotypical consequences

A. Growth curves of wild-type (□), _dsbC_− (▴), _dsbA_− (*), _dsbA_−_dsbD_− (formula image) and _dsbA_−_dsbC_− (●) strains in M63 minimal media at 37°C. Growth was monitored at A600. B. SDS sensitivity of wild-type (lane 1), _dsbA_− (lane 2), _dsbA_−_dsbC_− (lane 3) and _dsbA_−_dsbD_− (lane 4) strains. Strains were grown in LB at 37°C to an A600 of 0.5. The cultures were then serially diluted 107-fold in 10-fold increments. 10 μl of each dilution were then spotted on LB plates containing 2.5% SDS and grown overnight. C. Western blot showing protein expression levels. The upper bands correspond to the LamB protein. Outer membrane proteins prepared from wild-type (lane 1), _dsbA_− (lane 2), _dsbC_− (lane 3), _dsbA_−_dsbC_− (lane 4), and _LamB_− (lane 5) strains. The lower bands correspond to an unknown protein recognized by the anti-LamB antibody, which was used as an internal standard.

Fig. 2

Fig. 2

In vivo redox state of DsbC. Exponentially growing cells (in LB) were TCA-precipitated, free cysteines were modified by AMS, and DsbC was detected by Western blot analysis. Lanes: 1, wild-type; 2, _dsb_A−; 3, _dsbA_−_dsbD_−; 4, _dsbD_−.

Fig. 3

Fig. 3

The number of spectral counts correlates with the abundance of a protein. Varying amounts (2–60 pmoles) of two eukaryotic proteins, ovalbumin and carbonic anhydrase, were added to 300 μg of periplasmic proteins. The SC values obtained for these two proteins in the various samples were then plotted against the corresponding protein amounts. After linear regression, we found that the R2 values obtained for the spectral counts were 0.94 and 0.92 for ovalbumin and carbonic anhydrase, respectively. This indicates that the number of spectral counts reliably reflects protein abundance in the sample.

Fig. 4

Fig. 4. The overall protein content of a _dsbA_−_dsbC_− mutant is different compared to _dsbA_− and _dsbA_−_dsbD_− strains

A. The logarithms of the SC values reported for the _dsbA_− strain were plotted against those reported for the _dsbA_−_dsbD_− mutant. Most of the SC values are similar in both strains, which is reflected by a quasi-linear distribution. B. The logarithms of the SC values reported for the _dsbA_− strain were plotted against those reported for the _dsbA_−_dsbC_− mutant. The distribution is more dispersed, which indicates that the overall protein content of this double mutant is different.

Fig. 4

Fig. 4. The overall protein content of a _dsbA_−_dsbC_− mutant is different compared to _dsbA_− and _dsbA_−_dsbD_− strains

A. The logarithms of the SC values reported for the _dsbA_− strain were plotted against those reported for the _dsbA_−_dsbD_− mutant. Most of the SC values are similar in both strains, which is reflected by a quasi-linear distribution. B. The logarithms of the SC values reported for the _dsbA_− strain were plotted against those reported for the _dsbA_−_dsbC_− mutant. The distribution is more dispersed, which indicates that the overall protein content of this double mutant is different.

Fig. 5

Fig. 5. A revised model for the formation of disulfide bonds in the E. coli periplasm

Disulfide bonds are introduced by the DsbA/DsbB pathway. Non-native disulfides are corrected by DsbC, which is recycled by DsbD. Both pathways are kinetically isolated. Our results indicate that DsbC is also able to function on the other side of the barrier where it assists DsbA in a DsbD-independent manner. DsbC may be acting as a stand-alone protein folding catalyst that cycles from the reduced to the oxidized state upon substrate oxidation and substrate reduction, respectively. Although kinetics data showed that DsbC is not a good substrate for DsbB, we cannot exclude that a slow oxidation of DsbC by DsbB may play a more significant role in the absence of DsbA. The Western blot data presented in Figure 2 also suggest that in the absence of DsbD, DsbA may be responsible for the oxidation of DsbC. The redox potentials of DsbA and DsbC are −125 mV and −130 mV, respectively.

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