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Lipopolysaccharide is transported to the cell surface by a membrane-to-membrane protein bridge

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Science  16 Feb 2018:
Vol. 359, Issue 6377, pp. 798-801
DOI: 10.1126/science.aar1886
  • Fig. 1 Energy-dependent LPS transport to LptA is stimulated by LptC.

    (A) Bridge model of LPS biogenesis and chemical structure of Escherichia coli LPS. The E. coli lipopolysaccharide transport proteins and E. coli LPS were used for all experiments presented here. LptBFG extracts LPS from the inner membrane and transports it to LptC using energy from ATP hydrolysis. Additional energy from ATP hydrolysis is harnessed to push LPS from LptC to LptA. Kdo, 3-deoxy-d-manno-octo-2-ulosonic acid; Hep, l-glycero-d-manno-heptose; Glu, d-glucose; Gal, d-galactose. (B) LPS photocrosslinks to LptC in an ATP- and time-dependent manner. Assays were initiated by adding 5 mM ATP or buffer (“- ATP”) to proteoliposomes containing LPS and LptBFGC-T47pBPA. (C) Time dependence of LPS release to LptA. Assays were initiated by adding 5 mM ATP to proteoliposomes containing LPS and LptBFG, LptB-E163Q-LptFGC, or LptBFGC mixed with soluble LptA-I36pBPA. In (B) and (C), aliquots were taken at the indicated time points and UV-irradiated. Cross-linking was detected by immunoblotting. Cartoons show experimental designs of the reconstituted systems. Proteins and LPS can be inserted into liposomes in either orientation, but only the productive orientation is shown for simplicity. The yellow star denotes the photocrosslinking amino acid.

  • Fig. 2 Reconstitution of membrane-to-membrane LPS transport.

    (A) Seven Lpt proteins and ATP are necessary and sufficient to observe LPS cross-linking to LptD. Proteoliposomes containing LptD-Y112pBPA/LptE and associated LptA were incubated with LPS-containing liposomes with or without LptBFGC. Assays were initiated with 5 mM ATP (or buffer). (B) LPS transport to LptD depends on time and ATP concentration. Assays were conducted as in (A), initiating with either 0.5 mM or 5 mM ATP. (C) LPS simultaneously cross-links to LptC and LptD. Proteoliposomes containing purified LptD-Y112pBPA/LptE with LptA were incubated with proteoliposomes containing LPS and LptBFGC-T47pBPA. In (A) to (C), aliquots were taken at the indicated time points and UV-irradiated. Cross-linking was detected by immunoblotting. Cartoons show experimental designs of the reconstituted systems. Proteins and LPS can be inserted into liposomes in either orientation, but only the productive orientation is shown for simplicity. The yellow star denotes the photocrosslinking amino acid.

  • Fig. 3 LptA induces the physical association of inner membrane (IM) and outer membrane (OM) proteoliposomes.

    (A) Schematic of predicted proteoliposome states in the presence or absence of LptA. Proteoliposomes containing LptD-Y112pBPA/LptE were labeled with Atto-488 fluorophore, and proteoliposomes containing LptBFGC and LPS (not shown for simplicity) were labeled with Atto-565 fluorophore. (B) Flow cytometric analysis of reaction mixtures containing fluorescent proteoliposomes. Atto-488–labeled proteoliposomes containing LptD-Y112pBPA/LptE with or without preincubation with LptA were incubated with Atto-565–labeled proteoliposomes containing LptBFGC and LPS. An N-terminal truncated variant of LptD and an N-terminal blocked LptA were used in separate experiments as controls to substitute the corresponding Lpt components in the reaction mixtures. Samples were incubated as described for cross-linking experiments, initiating with buffer instead of ATP. After incubation, samples were diluted 10-fold and analyzed on a BD FACSAria flow cytometer. Equivalent particle distributions were observed in the presence of ATP. (C) Distribution of particle counts in gated populations shown in (B). Data were normalized such that percentages of counts represent the portion of the total number of counts in all gated subpopulations. Data represent the mean and SD of triplicate experiments.

  • Fig. 4 Observation of a long-lived, protein-mediated bridge by confocal microscopy.

    (A) Representative confocal microscope images of population IM and population OM sorted particles. Atto-488–labeled proteoliposomes containing LptDE with associated LptA were incubated with Atto-565–labeled proteoliposomes containing LptBFGC and LPS and were sorted by gating based on fluorescence thresholds with a BD FACSAria flow cytometer and imaged at 100× magnification. Scale bar: 10 μm. (B) Representative confocal microscope images of population A and population B. Atto-488–labeled proteoliposomes containing LptDE with associated LptA were incubated with Atto-565–labeled proteoliposomes containing LptBFGC and LPS and sorted by gating based on fluorescence thresholds using a BD FACSAria flow cytometer and imaged at 100× magnification. Scale bar: 10 μm.

Supplementary Materials

  • Lipopolysaccharide is transported to the cell surface by a membrane-to-membrane protein bridge

    David J. Sherman, Ran Xie, Rebecca J. Taylor, Alexander H. George, Suguru Okuda, Peter J. Foster, Daniel J. Needleman, Daniel Kahne

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figs. S1 to S13
    • Tables S1 to S4
    • References 

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