Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway

Abstract

SummaryIn 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 disulphidecontaining proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.

Conjugal type IV macromolecular transfer systems of Gram-negative bacteria: organismal distribution, structural constraints and evolutionary conclusions

“…The only exceptions are the proteins of clusters 2, 9 and 10 (see Table 2). The same general conclusion has been reached regarding other macromolecular export systems including the type I secretory pathway (ABC) (Fernandez & de Lorenzo, 2001) and type III secretory pathway (Vir) (Plano et al, 2001) systems (see Kuan et al, 1995 ;Nguyen et al, 2000 ;Paulsen et al, 1997). In all three cases, the lack of shuffling indicates the occurrence of extensive proteinprotein interactions, allowing construction of transenvelope exporters that can secrete their substrates directly from the cell cytoplasm to the extracellular medium without allowing accumulation of a periplasmic intermediate.…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Premature folding, as judged by cytoplasmic disulfide bond formation, prevents translocation (152). Furthermore, folding of the same disulfide-containing protein was not affected by the addition of competing sulfhydryl alkylating agents outside the cell, suggesting that folding may commence as the protein traverses the periplasm inside TolC (152). The dimensions of the TolC tunnel (140 Å by 30 Å) are similar to those of other protein folding chambers, such as GroEL and GroES (202,254).…”

“…The glycine-rich motifs bind Ca 2ϩ , and the scarcity of Ca 2ϩ in the interior of the bacterial cell may prevent the folding of the repeat, facilitating the initiation of the translocation process. Premature folding, as judged by cytoplasmic disulfide bond formation, prevents translocation (152). Furthermore, folding of the same disulfide-containing protein was not affected by the addition of competing sulfhydryl alkylating agents outside the cell, suggesting that folding may commence as the protein traverses the periplasm inside TolC (152).…”

Novel GFP Expression Using a Short N-Terminal Polypeptide through the Defined Twin-Arginine Translocation (Tat) Pathway

“…E. coli has two types of secretion mechanisms that can be used to secrete a number of native proteins (Mergulhão et al, 2005). The type-I mechanisms export high molecularweight toxins and exoenzymes to the culture medium (Fernandez and de Lorenzo, 2001), whereas the type-II mechanisms utilize a two-step process for extracellular secretion in which periplasmic translocation (Koster et al, 2000) is followed by outer membrane translocation.…”