Ullers et al. 10.1073/pnas.0608232104. |
Fig. 6. The tig mutation partially suppresses the Cs and Ts phenotypes of secA and secY mutants. The tig::Tn10-13(KanR) mutant allele, the result of a miniTn10(KanR) insertion 86 nucleotides upstream of the tig initiation codon, was introduced by P1 transduction into the following sec mutant backgrounds: secA51 Ts (1), secAcsR11 Cs (2), and secY40cs Cs (3). Transductants were tested for their ability to grow on LB agar plates at several temperatures. The results show that the absence of TF exerts a positive effect on bacterial growth with variously compromised sec alleles, albeit considerably less than observed with the secB null mutant.
Fig. 7. Representation of the proteins identified in the aggregates described in SI Table 1. Note that only the aggregation profiles of lanes 1 and 3 from Fig. 4C are compared. Outer membrane proteins are shown in blue.
Fig. 8. TF modulates the membrane association of SecA and ribosomes at 16°C. Western blot analysis of either whole-cell or IM preparations obtained at 16°C, showing SecA, L4, L6, S3, YidC, and SecE levels in the MC4100 wild type and its Dtig mutant derivative.
Fig. 9. Quantification of SecA and ribosomes in cytoplasmic and total membrane preparations. MC4100 Dtig was transformed with either p29SEN vector (-) or p29SENTF (+) and grown in 200 ml of LB medium at 37°C to mid-log phase. TF expression was subsequently induced for 1 h with 100 mM IPTG. At an OD600 of about 1.0, cells were collected by centrifugation for 10 min at 7,000 rpm (4°C), and pellets were resuspended in 1/10 volume of buffer K (50 mM triethanolamine, 250 mM sucrose, 1 mM EDTA, 1 mM DTT, pH 7.5). Cells were lysed by passing twice through a French press (12,000 psi), and suspensions were cleared by two short centrifugation steps. The supernatant fractions were then ultracentrifuged (40,000 rpm, 90 min, 4°C) using a Beckman 60 Ti rotor. The 20-ml supernatants (total cytoplasmic fractions) were collected, and the pellets (total membrane fractions) were resuspended in 1 ml buffer K. Next, ~20 mg of the cytoplasmic fractions and ~7 mg of the membrane fractions were loaded and analyzed by SDS/PAGE followed by Coomassie blue staining (A) or Western blot analysis with anti-SecA, anti-S3, and anti-YidC antibodies (B). Western blots were further quantified using the ChemiDoc Bio-Rad chemiluminescent detection system and the estimated values, normalized to the whole membrane fraction found without TF (-), are shown under each blot of panel (B). After adjustment of the values to compensate for the differences in quantity loaded between the cytoplasmic and the whole membrane fractions, we estimated that without TF about 20% of the total SecA and about 4% of the total S3 was found in the membrane fraction. Remarkably, this percentage was reduced by a factor of 2 when plasmid-encoded TF was present.
Fig. 10. Proposed model for Dtig suppression in the absence of downstream chaperones. In the wild-type situation most, if not all, nascent polypeptide chains interact cotranslationally with ribosome-bound TF (7, 8). In the course of the elongation process, TF leaves the ribosome, in complex with the polypeptide chain, toward the cytoplasmic space (9). Polypeptides can subsequently fold or interact with downstream chaperones and targeting factors [e.g., SecB with presecretory proteins (A)] (10). Because both TF and the SecYEG protein-conducting channel have overlapping ribosomal docking sites (11-14), the presence of TF bound at the polypeptide exit may prevent unproductive association of the ribosome with the Sec translocon and, hence, facilitate the release of polypeptides into the cytoplasm. In the absence of downstream chaperones (B), newly synthesized presecretory proteins released into the cytoplasmic space can form partially folded intermediates ("I"), which are not competent for translocation. Such cytoplasmically trapped presecretory proteins may eventually be targeted for proteolysis. Following temperature downshift, the detrimental effect of TF on protein export may be further exacerbated by the loss of membrane fluidity and/or by the intrinsic Cs property of the export process (15). The absence of TF (C) may partially compensate by facilitating the association of translating ribosomes with IMs and/or the early cotranslational interaction of SecA [note that in this cartoon SecA is represented as a dimer, although the oligomeric state of functional SecA is still under debate (16, 17)] with emerging polypeptide chains, thus bypassing the need for cytoplasmic chaperone holdases such as SecB.
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SI Materials and Methods
Plasmid Constructs.
The plasmids p29SEN vector (pSC101ori, ptrc, lacIq, AmpR), p29SEN-TF (1), and p29SEN-SecB (2) have been described. To construct the p29SEN-SecA plasmid, the secA gene was amplified E. coli MG1655 using primers secA-for(5'-atgctaatcaaattgttaactaaag-3') and secA-rev(5'-cgcggatccttattgcaggcggccatggcac-3'), and digested with BamHI. The p29SEN vector was digested with EcoRI and its overhangs were filled-in to form blunt ends. The blunt open p29SEN vector was then digested with BamHI and ligated with the BamHI-digested secA fragment. The p29SEN-DnaJ plasmid was obtained by subcloning dnaJ from pWKG90 as an EcoRI/BglII fragment (3) into EcoRI/BamHI-digested p29SEN vector. The plasmid p29SEN-TF(F198A) was constructed by site-directed mutagenesis using the appropriate primers and p29SEN-TF as template. The plasmids p29SEN-TF(1-148), p29SEN-TF(149-432), and p29SEN-TF[D(148-251)] were obtained as follows: the StyI/HindIII TF fragments from both pSE380-based TF(1-148) and TF[D(148-251)] constructs (1) were subcloned into p29SEN-TF digested with the same enzymes. For p29SENTF(149-432), the StyI/HindIII TF fragment from the pGPTFP-based TF(149-432) construct (1) was subcloned into p29SEN-TF digested with the same enzymes. The plasmid constructs obtained by PCR were sequence verified using the appropriate primers.Bacterial Viability Assay.
Bacteria were grown to mid-log phase in LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7), supplemented when necessary with the appropriate antibiotics (ampicillin 100 mg/ml; chloramphenicol 10 mg/ml; kanamycin 50 mg/ml; tetracycline 12 mg/ml), serially diluted, spotted on LB agar plates, and incubated at the indicated temperatures.Pulse-Chase Analysis.
The MC4100 DsecB mutant harboring pSEN29 vector or p29SEN-SecB was grown overnight at 30°C in M9 minimal medium supplemented with 0.2% glycerol and ampicillin, diluted to 1:100 in fresh medium, and grown at 30°C to an OD600 of 0.1. Expression of SecB and MBP was induced with 10 mM IPTG and 0.2% maltose, respectively, for 2 h. Cells were spun down and resuspended in medium containing a cysteine- and methionine-free amino acid mix. After recovery for 30 min at 30°C, cells were pulse-labeled for 1 min with 10 mCi/ml [35S]methionine/cysteine and chased with 2 mM cold methionine/cysteine mix for the times indicated. After the chase, cells were precipitated with 10% (wt/vol) trichloroacetic acid on ice. Samples were analyzed following immunoprecipitation either with anti-MBP or with anti-OmpA antibodies as described by Jong and colleagues (4).Fresh transformants of the MC4100 Dtig DsecB double mutant harboring the various p29SEN-based TF mutants were grown overnight at 37°C in M9 minimal medium supplemented with 0.4% glucose and ampicillin, diluted to an OD600 of 0.05 in fresh medium and grown at 23°C to an OD600 of 0.1. Expression of the various TF mutants was induced with 100 mM IPTG for 2 h and the pulse chase analysis was carried out as described above.
MC4100 and MC4100 DsecB transformed with either p29SEN vector or p29SEN-SecA were grown overnight at 37°C in M9 minimal medium supplemented with 0.2% glycerol and ampicillin, diluted to an OD600 of 0.1 in fresh medium and grown at 23°C to an OD600 of 0.2. IPTG was added (0.1 mM) for 1 h and the pulse-chase analysis of OppA, using rabbit polyclonal antibody raised against OppA (a gift of Kazuei Igarashi, Chiba University, Chiba, Japan), was carried out essentially as described above, except that cells were pulse-labeled for 15 sec instead of 1 min.
Steady-State Protein Expression Levels.
Whole-cell extracts were prepared and probed with anti-TF, anti-SecB (both kind gifts of Joen Luirink, Vrije Universiteit, Amsterdam, The Netherlands), anti-DnaK or anti-DnaJ (laboratory collection) antibodies, as described (5). To monitor expression levels of the p29SEN-based TF constructs, fresh transformants were first grown at 37°C in LB ampicillin medium to an OD600 of 0.4. At this stage, 100 mM IPTG was added and cultures were then grown to an OD600 of 1.2. Next, 1 ml of each culture was precipitated with 10% (wt/vol) trichloroacetic acid, washed with acetone, and resuspended in SDS/PAGE loading buffer as described (6).1. Genevaux P, Keppel F, Schwager F, Langendijk-Genevaux PS, Hartl FU, Georgopoulos C (2004) EMBO Rep 5:195-200.
2. Ullers RS, Luirink J, Harms N, Schwager F, Georgopoulos C, Genevaux P (2004) Proc Natl Acad Sci USA 101:7583-7588.
3. Kelley WL, Georgopoulos C (1997) Proc Natl Acad Sci USA 94:3679-3684.
4. Jong WS, ten Hagen-Jongman CM, Genevaux P, Brunner J, Oudega B, Luirink J (2004) Eur J Biochem 271: 4779-4787.
5. Kelley WL, Georgopoulos C (1997) Proc Natl Acad Sci USA 94:3679-3684.
6. Genevaux P, Schwager F, Georgopoulos C, Kelley WL (2002) Genetics 162:1045-1053.