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.

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

“…Processing of PrePulG* on the periplasmic side of the hydrophobic TM segment provided a direct kinetic assay for correct membrane insertion. However, pulse-chase analyses showed that PrePulG* is not processed posttranslationally, as observed for the DsbA protein, an SRP substrate with a natural cleavable signal sequence (52,54). Therefore, we measured the steady-state levels of PulG by fractionation and immunoblotting as sensitive means to probe PulG insertion, especially in rich media that lead to more pronounced protein translocation defects (34).…”

“…In vivo studies showed that SRP targets IM proteins to the SecYEGDF translocon for insertion (23,60), while in vitro cross-linking of nascent IM proteins revealed contacts with Ffh, Fts, SecY, and SecA (43,52). SecA is required for the insertion of IM proteins with periplasmic loops longer than 30 amino acids (1,19,43), while single-spanning IM proteins such as PulG always require SecA, regardless of the length of their periplasmic domain (15).…”

A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant

“…YaeL shares the same membrane topology as that predicted for its homologue MmpA (Fig. 1B) (Kanehara et al, 2001;Drew et al, 2002), while DsbA (a disulphide bond oxidoreductase) and MBP (maltose binding protein) are both soluble periplasmic proteins (Collier et al, 1988;Kumamoto and Gannon, 1988;Liu et al, 1989;Schierle et al, 2003). YaeL was fused to mRFP1 or to YFP, placed under the control of a LacI-regulated promoter on a relatively highcopy plasmid, and expressed in E. coli without induction.…”

Application of split‐green fluorescent protein for topology mapping membrane proteins in Escherichia coli

“…To traffic [1][2][3][4][5][6][7][8][9][10] ] to the periplasm, we cloned the region encoding the signal peptide from the periplasmic protein DsbA (DsbA 1-19 ) upstream of the region encoding [1-10 OPT ], so that the latter would be secreted to the periplasm via the SRP/SecYEG pathway. 17 Fig. 3(A)].…”

“…The [11 H7 ] tag was created by site-directed mutagenesis of the [11 M3 ] tag. The regions encoding MBP and DsbA [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] were amplified by PCR from the MG1655 strain and cloned into the pGFPe vector, upstream of the region encoding [1-10 OPT ]. The region-encoding TorA 1-39 was amplified by PCR from the MG1655 strain and cloned into the pBAD24 vector, upstream of the region-encoding [1-10 OPT ].…”