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Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway
Author(s) -
Fernández Luis A.,
De Lorenzo Víctor
Publication year - 2001
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2001.02410.x
Subject(s) - periplasmic space , secretion , biology , biochemistry , cytoplasm , escherichia coli , signal peptide , secretory protein , transport protein , atp binding cassette transporter , bacterial outer membrane , extracellular , transporter , peptide sequence , gene
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.