The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway

Abstract

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

FIG. 1.

Export of thioredoxin fused to various signal sequences. (A) Western analysis of fractionated extracts from cells expressing wild-type thioredoxin (+) (DHB4), cells with the trxA gene deleted (ΔtrxA) (WP570), cells expressing thioredoxin fused to DsbAss (DsbAss-TrxA) (WP570/pCFS120), and cells expressing thioredoxin fused to PhoAss (PhoAss-TrxA) (WP570/pCFS122). β-Lactamase was included as a periplasmic control. (B) Western analysis of fractionated extracts from cells expressing either chromosomal thioredoxin or thioredoxin fused to MBPss (MBPss-TrxA) (WP570/pCFS118). IPTG was used to induce expression of the MBPss-TrxA construct, as indicated. β-Lactamase was included as a periplasmic control. T, total extract; P, periplasmic fraction; C, cytoplasmic fraction plus cytoplasmic membrane fraction.

FIG. 2.

Export of thioredoxin fused to DsbAss is impaired by a mutation in the ffh gene: Western analysis of fractionated cell extracts from either Ffh⁺ cells (+) (CFS456) or ffh mutant cells (ffh⁻) (CFS403) expressing thioredoxin fused to DsbAss (pCFS123). T, total extract; P, periplasmic fraction; C, cytoplasmic fraction plus cytoplasmic membrane fraction; p, precursor containing the signal sequence; m, mature protein.

FIG. 3.

DsbAss alleviates the SecB dependence of MBP export: Western analysis of fractionated extracts from secB mutant cells expressing wild-type MBP (MBP) (CFS406/pCFS127) or MBP with its signal sequence replaced by that of DsbA (DsbAss-MBP) (CFS406/pCFS128). DegP and β-lactamase were included as examples of SecB-dependent and SecB-independent protein export, respectively. T, total extract; P, periplasmic fraction; C, cytoplasmic fraction plus cytoplasmic membrane fraction.

FIG. 4.

Mutation in ffh restores the SecB dependence of export of MBP fused to DsbAss. (A) Western analysis of fractionated extracts of ffh mutant cells expressing wild-type MBP (MBP) (CFS401/pCFS127) or MBP with its signal sequence replaced by that of DsbA (DsbAss-MBP) (CFS401/pCFS128). (B) Western analysis of fractionated extracts of ffh secB double mutant cells expressing wild-type MBP (MBP) (CFS469 and CFS127) or MBP with its signal sequence replaced by that of DsbA (DsbAss-MBP) (CFS469 and CFS128). DegP and β-lactamase were included as examples of SecB-dependent and SecB-independent protein export, respectively. T, total extract; P, periplasmic fraction; C, cytoplasmic fraction plus cytoplasmic membrane fraction.

FIG. 5.

Replacement of MBPss with DsbAss results in cotranslational processing: two-dimensional analysis of the processing of MBP containing its native signal peptide (MBPss-MBP) (a and b) or DsbAss (DsbAss-MBP) (c and d). Exponentially growing cultures expressing either MBPss-MBP (pCFS127) or DsbAss-MBP (pCFS128) were radiolabeled for 15 s in the presence (a and c) or in the absence (b and d) of sodium azide, and extracts were subjected to immunoprecipitation with anti-MBP antibodies. The MBP immunoprecipitates were separated on SDS-12% PAGE gels (first dimension). The fractionated peptides were subjected to partial proteolysis by V8 protease in a second dimension and analyzed by using SDS-15% PAGE gels and fluorography. The arrows indicate the positions of the amino-terminal fragments derived from full-length unprocessed precursors (p′) and from mature processed MBP (m′). The positions of the standards used for identification of processed and unprocessed nascent polypeptide are indicated by asterisks. Only the relevant portions of the gels are shown.

FIG.6.

Export of thioredoxin by DsbAss is SecA dependent. DRH223 (secA⁺) and DRH226 [secA(Ts)] containing plasmid pCFS123 (DsbAss-TrxA) were grown at 30°C and subjected to fractionation. Western blot analysis with thioredoxin and β-lactamase was performed by using samples electrophoresed on an SDS-15% PAGE gel. The positions of the precursor of mature thioredoxin (DsbAss-TrxA) (p) and the mature thioredoxin (m) are indicated. T, total extract; P, periplasmic fraction; C, cytoplasmic fraction plus cytoplasmic membrane fraction.

FIG. 7.

Export of DsbA in a secA(Ts) strain. Cells were grown at 30°C and shifted to 37°C for 2 h. Extracts from secA(Ts) (secA51) cells expressing MBP (CF330/pCFS127), DsbAss-MBP (CFS330/pCFS128), and DsbA (CFS330/pCH3) were subjected to Western blot analysis. All strains expressed wild-type chromosomal DsbA. β-Lactamase was included as a control. T, total extract; P, periplasmic fraction; C, cytoplasmic fraction plus cytoplasmic membrane fraction.