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Direct visualization of the E. coli Sec translocase engaging precursor proteins in lipid bilayers

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Science Advances  12 Jun 2019:
Vol. 5, no. 6, eaav9404
DOI: 10.1126/sciadv.aav9404
  • Fig. 1 Translocase activity evaluated in solution and on mica.

    (A) Translocation activity of radiolabeled, oxidized pOmpA (red squares, n = 3) or oxidized pGBP (black circles, n = 3) assayed in vitro by protection from proteinase K. Error bars are SDs, and lines are fits to an exponential rise to maximum. (B) Image of radioactivity from mica disks with pOmpA ± ATP. (C) Data (mean ± SEM) showing mol precursor pOmpA or pGBP protected on mica in the presence or absence of ATP. (D) Visualization of translocase dynamics in the absence of precursor. SecA dissociates from the membrane to reveal SecYEG beneath, and then SecA is seen to associate. Note that SecYEG in the bottom left remains unoccupied for all images. The time of each frame is listed (bottom right, units: seconds); the lateral scale bar is 50 Å, and the vertical false color scale is 50 Å.

  • Fig. 2 Sec translocase topography varies as a function of precursor species and translocation stage.

    (A) AFM images of Sec translocases: (i) with pOmpA at the initial stage (30 s) of translocation, the arrow identifies a void in an otherwise continuous lipid bilayer; (ii) with pOmpA at the plateau stage of activity (3 min); (iii) with pGBP at 30 s; and (iv) with pGBP at plateau (4.5 min). Lateral scale bars are 1000 Å and the false color vertical scale spans 160 Å. Individual features are shown as insets with scale bars: 100 Å (lateral) and 60 Å (vertical). (B) Height and (D) volume distributions of active Sec translocases at 30 s with either pOmpA (red curve, number of features included, n = 12,387) or pGBP (black curve, n = 10,063). Data for translocases SecYEG·SecA with no precursor (green dashed curve, n = 587) as well as SecYEG alone (magenta dotted curve, n = 1875) are shown for reference. After reaching the plateau stage of translocation activity, the (C) height and (E) volume distributions of translocases engaged with pOmpA (red curve, n = 9565) or with pGBP (black curve, n = 6592) are shown. In all cases, translocation was halted by adding ADPAlF at the prescribed times. Note that the vertical scales for SecYEG alone data were compressed two- or fivefold in the height or volume plots, respectively. (F) Arithmetic means of the height distributions. Error bars show SEM.

  • Fig. 3 SecA readily unbinds from SecYEG in the presence of pOmpA but not with pGBP.

    (A) Height distribution of Sec translocase engaging pOmpA and different nucleotides: ADP only (black curve, n = 2975), ATP only (red curve, n = 4010), or ATP followed by ADPAlF (blue dotted curve, n = 9565). (B) Height distribution of Sec translocase engaging pGBP and different nucleotides: ADP only (black curve, n = 3962), ATP only (red curve, n = 9277), or ATP followed by ADPAlF (blue dotted curve, n = 6592). Data with no precursor are overlaid for reference (green dashed curve, n = 587). Gray and blue regions in (A) and (B) indicate the approximate height range of the translocon SecYEG alone and the translocase complex, respectively. (C) Bar graphs show the arithmetic mean of the height distributions. Error bars are SEM.

  • Fig. 4 Oligomeric state of SecA changes with nucleotide.

    (A) Volume distribution of translocase engaging pOmpA with different nucleotides: ADP only (black curve, n = 1708), ATP only (red curve, n = 2518), or ATP followed by ADPAlF (blue dotted curve, n = 5088). (B) Volume distribution of translocase engaging pGBP with different nucleotides: ADP only (n = 2814), ATP only (n = 8441), or ATP followed by ADPAlF (n = 4973). The approximate monomer and dimer volume ranges of SecA are indicated by the gray shaded region and blue shaded region, respectively.

  • Fig. 5 Active translocase conformations differ with precursor species.

    Height distributions are shown for translocases engaging pGBP when exposed to (A) ATP (n = 3431), (B) ADP (n = 1128), or (C) ADPAlF (n = 1860), as well as translocases engaging pOmpA when exposed to (D) ATP (n = 1047), (E) ADP (n = 661), or (F) ADPAlF (n = 2112). Only translocases exhibiting volumes in the range corresponding to SecA2 or SecA monomer were included, as indicated. Bayesian information criterion was used to determine the optimal number of gamma distributions for each fit. The analysis prescribed one gamma distribution for (A) and (B), two for (C) and (F), and three for (D) and (E).

  • Fig. 6 Precursor-dependent model of translocase activity.

    We present a summary of results in cartoon form (not to scale) for pOmpA (A, C, D, F) or for pGBP (B, E, G) under the conditions listed. Individual components of the system include the following: precursor (red) with an N-terminal signal sequence and a disulfide-stabilized loop, SecA2 (orange), and SecYEG (yellow) in a lipid bilayer (light blue). The dashed lines indicate distinct stable conformations on the time scale of AFM imaging. AFM measurements of the active Sec translocase do not provide direct visualization of SecYEG or the oligomeric state of SecYEG because it is buried underneath SecA. In the absence of this information, monomer SecYEG is drawn in all conditions. In addition, cartoons are not meant to accurately convey the orientation of SecA or SecA2.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaav9404/DC1

    Fig. S1. Traditional translocation assays.

    Fig. S2. Calibration of surface translocation assays.

    Fig. S3. Surface translocation assays.

    Fig. S4. AFM images of Sec translocases engaging precursors at the initial stage of activity.

    Fig. S5. AFM Images of Sec translocases engaging precursors at the plateau stage of activity.

    Fig. S6. Liposomes alone and liposomes containing just SecYEG.

    Fig. S7. Translocase topography at lower protein-to-lipid (P:L) ratio.

    Fig. S8. Representative Sec translocase monomers and dimers.

    Fig. S9. SecA monomers and dimers without SecYEG.

    Fig. S10. Simulated images of monomeric SecA and comparison to experiment.

    Fig. S11. Simulated AFM images of SecA monomers in different conformations and dimeric SecA.

    Fig. S12. Representative AFM images of Sec translocases with ATP.

    Fig. S13. Representative AFM images of Sec translocases with ADP.

    Fig. S14. Integrated volume histograms.

    Fig. S15. Model optimization using Bayesian information criterion.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Traditional translocation assays.
    • Fig. S2. Calibration of surface translocation assays.
    • Fig. S3. Surface translocation assays.
    • Fig. S4. AFM images of Sec translocases engaging precursors at the initial stage of activity.
    • Fig. S5. AFM Images of Sec translocases engaging precursors at the plateau stage of activity.
    • Fig. S6. Liposomes alone and liposomes containing just SecYEG.
    • Fig. S7. Translocase topography at lower protein-to-lipid (P:L) ratio.
    • Fig. S8. Representative Sec translocase monomers and dimers.
    • Fig. S9. SecA monomers and dimers without SecYEG.
    • Fig. S10. Simulated images of monomeric SecA and comparison to experiment.
    • Fig. S11. Simulated AFM images of SecA monomers in different conformations and dimeric SecA.
    • Fig. S12. Representative AFM images of Sec translocases with ATP.
    • Fig. S13. Representative AFM images of Sec translocases with ADP.
    • Fig. S14. Integrated volume histograms.
    • Fig. S15. Model optimization using Bayesian information criterion.

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