Dynamic action of the Sec machinery during initiation, protein translocation and termination
- University of Leeds, United Kingdom
- University of South Bohemia, Czech Republic
- University of Bristol, United Kingdom
Figures
Figure 1 with 2 supplements
Channel opening and helical plug motion illustrated by available high-resolution structures.
(A) Closed SecYEG (PDB: 5AWW, Tanaka et al., 2015). SecYEG (light red) is shown embedded in a modelled membrane (grey) with the plug helix highlighted (purple). Also identified are the transmembrane …
Figure 1—figure supplement 1
Modelling of fluorescent dye accessible volumes.
(A) Closed state (PDB: 1RHZ) (Van den Berg et al., 2004). Accessible volumes (orange for dye attached to the reference residue and cyan for the plug probe) obtained by modelling the dye positions …
Figure 1—figure supplement 2
Activity of dual labelled SecYMKEG in a translocation assay.
(A) Protease protection assay performed with wild-type WT SecYEG (unlabelled) and SecYMKEG mutant labelled with AlexaFluor dyes. A 233 amino acid N-terminal fragment of proOmpA was used as …
Figure 2 with 3 supplements
Monitoring plug movement by single molecule FRET.
(A) Expected changes in the FRET efficiency as a consequence of plug displacement during translocation. Pre-translocation, high FRET closed state (black dashed line) changes rapidly to a low FRET, …
Figure 2—figure supplement 1
1D FRET efficiency histograms for controls.
(A) SecYMKEG alone. All data were collected using the confocal setup and histograms are fitted to a sum (solid magenta) of three Gaussians (dashed lines), of which the two minor contributions …
Figure 2—figure supplement 2
Correction of 1D FRET efficiency histograms for unresponsive population of SecYMKEG in opposite orientation.
FRET distributions from confocal experiment were corrected for contribution from the 50% SecYMKEG in opposite orientation by subtracting appropriately scaled FRET distribution of SecYMKEG alone (Figu…
Figure 2—figure supplement 3
RASP workflow describing how to obtain two-dimensional FRET efficiency histograms and transition density plots from fluorescence bursts.
The top panel depicts a time trace of fluorescence counts with one train of recurring bursts belonging to the same vesicle. For detecting transitions within a set time window (e.g. 50 ms), an …
Figure 3 with 5 supplements
Monitoring fast plug movement during initiation.
(A) Two-dimensional FRET efficiency contour plots (transition density plots) were obtained from bursts collected for SecYMKEG:SecA:SecB:pOA (proOmpA 100 aa) in the presence of 0.1 mM ATP using the …
Figure 3—figure supplement 1
Burst duration and recurrence probability.
(A) Burst width distribution for diffusing proteoliposomes. (B) Recurrence probability was obtained from data in (A) as described in Materials and methods. The recurrence time of 80 ms, …
Figure 3—figure supplement 2
Two-dimensional transition density plots.
A detailed sampling of time resolved transition density plots shown in Figure 3A for SecYMKEG:SecA:SecB:pOA (proOmpA 100 aa) in the presence of 0.1 mM ATP. Note a wider scale (count contour level …
Figure 3—figure supplement 3
Spontaneous opening and closing in the absence of translocation substrate.
Opening (blue) and closing (red) transition times as a function of ATP concentration for SecYMKEG:SecA:SecB in the presence of 1 mM ATP (no proOmpA). Neither opening (average 24.3 ± 5.2 ms) nor …
Figure 3—figure supplement 4
RASP derived two-dimensional transition density plots in the presence of AMP-PNP.
Time evolution of states for SecYMKEG:SecA:SecB:pOA (proOmpA 100 aa) in the presence of 1 mM AMP-PNP. RASP time window is indicated in each panel. The scale bar on the lower right depicts count …
Figure 3—figure supplement 5
Activation energies for plug opening and closing.
Arrhenius plot of reciprocal transition times (rate constants) for the plug opening (blue) and closing (red) under saturating 2 mM ATP concentration. Activation energy of 61.2 ± 4.5 kJ/mol for …
Figure 4 with 2 supplements
Two-dimensional FRET efficiency histograms detect transitions between states.
(A) SecYMKEG alone. Transition density for delays up to 21 ms were obtained from RASP analysis of 10,000 events. In all panels, the crosshair symbols indicate positions of the open and closed state …
Figure 4—figure supplement 1
FRET characterization of SecYMK, R357EEG ‘closed’ plug mutant.
(A) Two-dimensional FRET efficiency histograms for SecYMK,R357EEG alone. Transition density for delays up to 21 ms were obtained from RASP analysis of 10,000 events. In all panels, the crosshair …
Figure 4—figure supplement 2
Burst variance analysis for selected states.
BVA can discriminate between static and dynamic heterogeneity. Blue dots show measured standard deviations (SE) for all individual bursts. Due to statistical nature of these estimates, the variance …
Figure 5 with 4 supplements
Substrate length dependence of dwell times and determination of translocation rates.
(A) Dwell time dependence for open state (EFRET ~0.2) on the substrate length (schematically shown in the inset, SS depicted as orange bar) in the presence (black) or absence (red) of SecB. Ordinary …
Figure 5—figure supplement 1
Translocation of proOmpA constructs with different lengths.
Reactions were performed in the presence or absence of SecB. Gel lane: 10% of starting proOmpA loaded without protease treatment (positive and normalization control); control without ATP, …
Figure 5—figure supplement 2
Ensemble ATPase activity stimulated by proOmpA constructs with different lengths.
Ensemble ATPase activity stimulated by proOmpA constructs with different length in the presence (black) or absence (red) of SecB (10 µM). The rates were obtained under saturating ATP/proOmpA …
Figure 5—figure supplement 3
Deconvolution of photobleaching effect from the dwell time distributions for the longest 683 aa substrate.
(A) Without SecB, uncorrected distributions (shown as probability density functions) are shown in green, corrected distribution shown in red. Solid lines represent fitted gamma distribution …
Figure 5—figure supplement 4
Dwell time of the closed EFRET ~ 0.4 state as a function of the translocating substrate length.
(A) Without SecB. Error bars represent the standard deviation (s.d.) computed from the distribution of the dwell times. (B) As A), but in the presence of SecB (10 μM).
Figure 6
Summary of detected plug states and initiation and translocation stages.
Colour coding: SecYEG – red, SecA – blue, proOmpA/OmpA – thick green line, SS –green rectangle, stationary plug – purple, plug in intermediate or transient state – magenta, lipid bilayer – grey. …
Videos
Video 1
Detailed sampling of time resolved transition density plots shown in Figure 3A.
Conditions: SecYMKEG:SecA:SecB:pOA in the presence of 0.1 mM ATP.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Software, algorithm | iSMS software | (Preus et al., 2015) - doi:10.1038/nmeth.3435 | ||
| Software, algorithm | LabView | (Lee et al., 2005) - doi: 10.1529/biophysj.104.054114 | ||
| Software, algorithm | FRETbursts | (Ingargiola et al., 2016b) - doi: 10.1371/journal.pone.0160716 | ||
| Software, algorithm | photon-hdf5 | (Ingargiola et al., 2016a) - doi: 10.1016/j.bpj.2015.11.013 | ||
| Software, algorithm | dual-channel burst search | (Nir et al., 2006) - DOI: - 10.1021/jp063483n | ||
| Software, algorithm | regularized inverse transform | (Provencher, 1982) - DOI10.1016/0010-4655(82)90173-4 | ||
| Software, algorithm | graphics: library Seaborn, based on Matplotlib | (Hunter, 2007) - DOI10.1109/MCSE.2007.55 |
Additional files
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Source code 1
Python script source code.
- https://doi.org/10.7554/eLife.35112.025
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Source data 1
Primary single molecule fluorescence data file for use with the Python script.
- https://doi.org/10.7554/eLife.35112.026
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Transparent reporting form
- https://doi.org/10.7554/eLife.35112.027
