Reconstitution of surface lipoprotein translocation through the Slam translocon
- Department of Biochemistry, University of Toronto, Canada
- MRC Laboratory of Molecular Biology, University of Cambridge, United Kingdom
Figures
Figure 1 with 7 supplements
Slam1 is necessary for translocation of unfolded TbpB.
(a) Model of a defined in vitro assay for TbpB translocation. M. catarrhalis TbpB (folded and urea-unfolded) is translocated inside Slam1-containing proteoliposomes. SM2 biobeads were used to remove …
-
Figure 1—source data 1
Quantification of in vitro translocation of purified TbpB.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig1-data1-v2.zip
Figure 1—figure supplement 1
Purification of M. catarrhalis Slam1.
(a) Sequence identity of N. meningitidis Slam1 and M. catarrhalis Slam1. (b) Overall membrane protein expression and purification pipeline. (c) Sodium dodecyl sulfate–polyacrylamide gel …
Figure 1—figure supplement 2
Translocation of Mcat TbpB to the surface of E. coli cells.
(a) Plate reader assay was used to examine the function of Slam homologs. Slams and TbpB were coexpressed in E. coli strain C43(DE3) and probed with α-flag antibodies followed by labeling with the …
-
Figure 1—figure supplement 2—source data 1
Fluorescent signal of TbpB on the surface of E.coli.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig1-figsupp2-data1-v2.zip
Figure 1—figure supplement 3
Generation of Slam1 and Bam proteoliposomes.
(a) Protocol used for insertion of outer membrane proteins (OMPs) into liposomes. OMP-DDM protein–detergent complexes were diluted (below the critical micellar concentration [CMC] of DDM) into …
Figure 1—figure supplement 4
Purification and characterization of E. coli Bam complex.
(a) BamABCDE fractions obtained from a S200 gel filtration column. The BamABCDE complex was obtained using previously described protocols (Hagan et al., 2010). Some non-Bam complex bands (marked in …
Figure 1—figure supplement 5
Characterization of Mcat Slam1 and BamABCDE containing proteoliposomes.
(a) Sucrose floatation assay for Slam1 and Bam proteoliposomes. Proteoliposomes were resuspended to a final concentration of 60% sucrose and subsequently layered with 30% sucrose and buffer B (50 mM …
Figure 1—figure supplement 6
Purification and functional characterization of M. catarrhalis TbpB.
(a) Purified TbpB–DDM complexes obtained from a S200 size chromatography column. The sample was subsequently used for the in vitro translocation assay. (b) Dot blot assay with biotinylated human …
Figure 1—figure supplement 7
Protection of urea-unfolded TbpB (flag-tag) and negative control urea-unfolded AfuA (his-tag) by liposomes, proteoliposomes, and purified Slam1–DDM complex.
Mixture of urea-unfolded TbpB and urea-unfolded AfuA (1:1 molar ratio) was incubated with empty liposomes, proteoliposomes, or Slam1–DDM complex (1:20 volume ratio), followed by proteinase K …
Figure 2 with 2 supplements
In vitro translocation assay for reconstitution of Slam-dependent SLP translocation.
(a) Model of the proposed in vitro proteoliposomes translocation assay for TbpB secreted directly from E. coli spheroplast. As E. coli cell expressing TbpB, the cells were converted into spheroplast …
-
Figure 2—source data 1
Quantification of in vitro translocation of spheroplast-secreted TbpB.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig2-data1-v2.csv
-
Figure 2—source data 2
Quantification of time-dependent translocation of spheroplast-secreted TbpB.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig2-data2-v2.csv
Figure 2—figure supplement 1
Purification and functional characterization of LolA from E. coli.
(a) Model of the release of SLPs from spheroplasts upon addition of purified E. coli LolA. SLPs are synthesized in the cytoplasm and transported to the periplasm via the Sec translocon. After the …
Figure 2—figure supplement 2
Characterization of spheroplast-secreted TbpB.
(a) Western blot accessing the protection of secreted TbpB in the absence of liposomes. Similar background protection was observed in no liposomes and liposome negative control (8.5% and 9.3%, …
Figure 3
Periplasmic chaperone Skp interacts with surface lipoproteins TbpB and HpuA after being released from the inner membrane.
(a) Model of pulldown assay using the flag-tag on the C-terminus of TbpB. Samples were analyzed using mass spectrometry (summarized in Table 1) and examined on western blots. (b) Representative …
-
Figure 3—source data 1
Mass spectrometry of pulldown samples.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig3-data1-v2.rar
Figure 4
Periplasmic chaperone Skp is required for Slam1–TbpB translocation in the reconstitution systems.
(a) Translocation of TbpB via Slam1 to the surface of E. coli K12 mutants. Depletion of Skp significantly reduces the translocation of TbpB to the surface (by 50% – detecting by using α-flag …
-
Figure 4—source data 1
Fluorescence of TbpB on surface of E. coli.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig4-data1-v2.csv
-
Figure 4—source data 2
Quantification of in vitro translocatio.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig4-data2-v2.csv
-
Figure 4—source data 3
Quantification of in vitro translocation following sucrose floatation assay.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig4-data3-v2.csv
Figure 5 with 3 supplements
Periplasmic chaperone Skp is important for translocation of TbpB to the surface of N. meningitidis.
(a) Relative fluorescence intensity accessing the presence of TbpB (65 kDa) on the surface of N. meningitidis mutants using α-TbpB antibody (exposure – top) and biotinylated human transferrin …
-
Figure 5—source data 1
Fluorescent signal of TbpB on the surface of N. meningitidis.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig5-data1-v2.csv
Figure 5—figure supplement 1
Deletion of Skp in N. meningitidis.
(a) The complete knockout process using spot transformation. Gene skp in N. meningitidis B16B6 is replaced by kan cassette. (b) Agarose gel of single colony PCR using external primers (targeting DNA …
Figure 5—figure supplement 2
Purification of Nme Skp for antibody production and antibody test.
Coomassie blue stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of Nme Skp after S75 gel filtration to access its purity prior to antibody production (left panel) and …
Figure 5—figure supplement 3
Growth of B16B6 N. meningitidis strains in BHI media.
(a) OD600 was recorded every 30 min over a 24-hr period. Δskp and Δslam1 mutants were lagging behind but reached OD600 of 0.7 eventually. (b) Representative western blots detecting the expression of …
-
Figure 5—figure supplement 3—source data 1
Growth of N. meningitidis.
- https://cdn.elifesciences.org/articles/72822/elife-72822-fig5-figsupp3-data1-v2.csv
Figure 6
Proposed model of localization for Slam-dependent surface lipoproteins in Gram-negative bacteria.
Once the surface lipoproteins emerged from the Sec complex, periplasmic chaperone Skp binds to the SLPs to prevent early folding while their N-terminus is modified and lipidated by Lgt, Lsp, and Lnt …
Tables
Table 1
Summary of mass spectrometry for flag-tagged TbpB pulldown.
| AfuA | TbpB | |||||||
|---|---|---|---|---|---|---|---|---|
| Proteins | Total peptides | % Coverage | Location | Proteins | Total peptides | % Coverage | Location | |
| 1 | AfuA | 380 | 78% | P | TbpB | 265 | 31% | – |
| 2 | TufA | 119 | 70% | IM | LolA | 146 | 73% | P |
| 3 | LolA | 36 | 72% | P | TufA | 57 | 59% | IM |
| 4 | OmpF | 12 | 34% | OM | OmpF | 21 | 38% | OM |
| 5 | DegP | 10 | 30% | P | DegP | 15 | 32% | P |
| 6 | FlgH | 12 | 34% | P | OmpA | 15 | 34% | OM |
| 7 | DegQ | 6 | 14% | P | Skp | 9 | 27% | P |
| 8 | OmpA | 5 | 22% | OM | FlgH | 8 | 26% | P |
| 9 | DegQ | 5 | 13% | P | ||||
Additional files
-
Supplementary file 1
Strains, plasmids, and antibodies used in this study.
Raw images for figures and figure supplements.
- https://cdn.elifesciences.org/articles/72822/elife-72822-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/72822/elife-72822-transrepform1-v2.pdf
-
Source data 1
Mass spectrometry results.
- https://cdn.elifesciences.org/articles/72822/elife-72822-data1-v2.zip
-
Source data 2
Original gels and blots used for the figures.
- https://cdn.elifesciences.org/articles/72822/elife-72822-data2-v2.zip
