Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli - PubMed (original) (raw)
Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli
Christina Wilson Bowers et al. J Bacteriol. 2003 Oct.
Abstract
LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli. Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway. Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm. Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion. Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E. coli and argue against a role for trigger factor in pathway discrimination.
Figures
FIG. 1.
Structure of the diploid fusion strains. A λ specialized transducing phage carrying the _H*lamB_′_-′_lacZ gene fusion integrated at the chromosomal malB locus of strain CWB210 to produce a lysogen (CWB299) that carried both H*lamB and the _H*lamB_′_-′_lacZ fusion.
FIG. 2.
Increasing signal sequence hydrophobicity reroutes LamB to the SRP pathway. (A) SecB dependence. E. coli cells were pulse-labeled and chased for 30 s, and this was followed by immunoprecipitation. The precursor LamB and MalE bands are indicated by arrows. (B) SRP dependence. E. coli with H*lamB and either wild-type or mutant SRP alleles (ffh87 or ffs69) was pulse-labeled for 30 s and chased for 10 s. (C) Reduced SecA requirement. E. coli with lamB+ or H*lamB in the presence or absence of secA51(Ts) was grown overnight at 30°C, subcultured, and then induced with 0.4% maltose. A pulse-chase analysis was performed at 30°C. The precursor band is indicated by an asterisk. The arrow indicates the position of the presumed H*LamB precursor. WT, wild type.
FIG. 3.
H*LamB-LacZ does not jam the secretion machinery. Strains were pulse-labeled and chased for 30 s. The arrows indicate the positions of the precursor LamB and MalE. WT, wild type.
FIG. 4.
H*LamB-LacZ localizes to the periplasm. (A) Maltose sensitivity disk assay. Overnight cultures of _lamB_′_-′_lacZ and _H*lamB_′_-′_lacZ strains, with and without DegP overexpression (pCLC11), were washed and concentrated 2.5-fold. Fifty microliters of cell suspension was added to 3 ml of molten F-top agar at 42°C and spread onto glycerol minimal plates. Fifteen microliters of 20% maltose was spotted in the center of a disk, and inhibition zones were measured after 15 h of incubation at 37°C. The results of one representative experiment are shown. (B) Disulfide bond formation in H*LamB-LacZ. Extracts of the _lamB_′_-′_lacZ and _H*lamB_′_-′_lacZ strains were prepared as described in Materials and Methods under reducing (R) and nonreducing (NR) conditions for SDS-PAGE and immunoblot analysis. Blots were probed with antibody against LamB. The position of the fusion band is indicated by an arrow. WT, wild type.
FIG. 5.
A 4.5S RNA lesion impairs H*LamB-LacZ secretion in vivo. _H*lamB_′_-′_lacZ strains with wild-type ffs or ffs69 were streaked onto LB agar containing X-Gal and incubated overnight at 37°C and then for 2 days at room temperature.
FIG. 6.
secY40 impairs H*LamB-LacZ secretion in vivo. _H*lamB_′_-′_lacZ strains with either secY+, secY39, or secY40 were streaked onto LB agar containing X-Gal and incubated overnight at 37°C and then for 2 days at room temperature.
FIG. 7.
Secretion of LamB-LacZ in a tig strain is SecA dependent. Strains were streaked onto LB agar plates containing X-Gal and incubated for 15 h at 30°C. (A) _lamB_′_-′_lacZ (wild-type) strains CWB281 and CWB294 [secA(Ts)]. (B) _H*lamB_′_-′_lacZ strains CWB299 and CWB309 [secA(Ts)]. (C) _lamB_′_-′_lacZ strains CWB289 (tig<>kan) and CWB341 [tig<>kan secA(Ts)]. (D) _H*lamB_′_-′_lacZ strains CWB305 (tig<>kan) and CWB 342 [tig<>kan secA(Ts)].
FIG. 8.
LamB-LacZ localizes to the periplasm in a tig strain. Strains were processed as described in the legend to Fig. 3. In the presence of pCLC11 at 42°C, the maltose sensitivity of a _lamB_′_-′_lacZ strain in the tig strain background was partially suppressed.
FIG. 9.
Fusion-induced jamming is reduced but not eliminated in tig fusion strains. Strains were pulse-labeled and chased (each for 30 s), immunoprecipitated with anti-LamB and anti-MalE antisera, and prepared for SDS-PAGE as described in Materials and Methods. The position of the precursor is indicated by arrows. WT, wild type.
FIG. 10.
Secretion of LamB is SecA dependent and SRP independent in tig strains. Pulse-labeling was performed at 30°C for 30 s with a 10-s chase. Samples were immunoprecipitated with anti-MalE and anti-LamB antisera.
FIG. 11.
Possible secretion mechanisms: comparison of secretion modes for LamB-LacZ variants. In mechanism I SecA-dependent, posttranslational secretion of LamB-LacZ jams the translocation channel. In mechanism II cotranslational, SRP-dependent secretion of H*LamB-LacZ relieves jamming and results in localization of the fusion to the periplasm. The ribosome is shown in loose association with the translocation channel. SecA may or may not be required. In mechanism III cotranslational, SecA-dependent secretion of LamB-LacZ in the absence of Tig occurs. Secretion commences prior to completion of translation, reducing the potential for cytoplasmic, folding-based toxicity. SecA is clearly required.
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