PMID:11309117
| Citation |
Fernández, LA and de Lorenzo, V (2001) Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway. Mol. Microbiol. 40:332-46 |
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| Abstract |
In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit. |
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| Keywords |
Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Blotting, Western; Carrier Proteins/metabolism; Disulfides/chemistry; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli/metabolism; Escherichia coli Proteins; Hemolysin Proteins/genetics; Hemolysin Proteins/metabolism; Immunoglobulin Fragments/genetics; Immunoglobulin Fragments/metabolism; Immunoglobulin Variable Region/genetics; Immunoglobulin Variable Region/metabolism; Periplasm/metabolism; Protein Disulfide-Isomerases/metabolism; Protein Transport; Recombinant Proteins/metabolism; Thioredoxin-Disulfide Reductase/metabolism; Thioredoxins/metabolism |
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