Quantifying secondary transport at single-molecule resolution

An Author Correction to this article was published on 20 February 2020

This article has been updated

Abstract

Secondary active transporters, which are vital for a multitude of physiological processes, use the energy of electrochemical ion gradients to power substrate transport across cell membranes1,2. Efforts to investigate their mechanisms of action have been hampered by their slow transport rates and the inherent limitations of ensemble methods. Here we quantify the activity of individual MhsT transporters, which are representative of the neurotransmitter:sodium symporter family of secondary transporters3, by imaging the transport of individual substrate molecules across lipid bilayers at both single- and multi-turnover resolution. We show that MhsT is active only when physiologically oriented and that the rate-limiting step of the transport cycle varies with the nature of the transported substrate. These findings are consistent with an extracellular allosteric substrate-binding site that modulates the rate-limiting aspects of the transport mechanism4,5, including the rate at which the transporter returns to an outward-facing state after the transported substrate is released.

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Fig. 1: Design and characterization of a hydrophobic amino acid sensor.
Fig. 2: Single-molecule, multi-turnover MhsT transport assay.
Fig. 3: Single-molecule, single-turnover measurements of first half-cycle MhsT transport rates.
Fig. 4: Substrate identity affects the return rate of MhsT after substrate release to the proteoliposome lumen.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Code availability

The full source code of SPARTAN37, which was used for all analysis of smFRET data, is publicly available at http://www.scottcblanchardlab.com/software.

Change history

  • 20 February 2020

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank R. B. Altman for the preparation of microscope slides and other reagents for single-molecule experiments, and M. Saper for providing the pET29a His-tagged LIV-BP plasmid. This work was supported by National Institutes of Health grants 1R21MH099491 and R01GM098859 (to S.C.B), DA041510 and U54 GM087519 (to J.A.J) and the National Institute on Drug Abuse of the National Institutes of Health grant F31DA044688-01 (to G.A.F).

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Authors

Contributions

S.C.B. and D.S.T. conceptualized the single-molecule transport assay. G.A.F. prepared and labelled LIV-BP and carried out the single-molecule experiments. G.A.F., D.S.T. and S.C.B. analysed and interpreted the single-molecule data. J.A.J. and M.Q. designed the cysteine accessibility and vesicle topology studies and interpreted the data. M.Q. and A.L.W. prepared purified MhsT and MhsT-containing bacterial membranes. M.Q. carried out radioactive uptake assays and cysteine accessibility assays. All authors contributed to overall experimental design and the writing of the manuscript.

Corresponding authors

Correspondence to Matthias Quick or Jonathan A. Javitch or Scott C. Blanchard.

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Competing interests

S.C.B. has an equity interest in Lumidyne Technologies.

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Peer review information Nature thanks Antoine van Oijen, Robert Vandenberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Salt and pH dependence of LIV-BPWT affinity for leucine and single-molecule calibration curve of LIV-BPSS.

a, Bulk acceptor fluorescence of LIV-BPWT measured in the presence of varying leucine concentrations and buffer conditions, demonstrating a lack of dependence on salt and pH conditions. b, Surface-immobilized LIV-BPSS incubated in the presence of varying concentrations of leucine. For each concentration, FRET efficiencies were summed across time to generate a histogram (symbols) and fit to Gaussian functions (lines) to obtain estimates of the mean FRET value. Data were collected at least three times with similar results. c, Mean FRET values for leucine (black circles), obtained as shown in a, fit with equation (1) in Supplementary Methods (black line) to obtain the Kd. Analogous data with mean FRET values are shown for each concentration for isoleucine (red), valine (blue) and leucine (black) were fit with equation (1) to determine the Kd. The grey data points and line (LeuL) indicate LIV-BPSS encapsulated within the liposome with leucine allowed to equilibrate at each concentration for at least 10 min into the liposome lumen. This indicates that the binding activity of LIV-BP is unaffected by encapsulation within the liposome lumen. Mean ± s.e.m. (n = 3 separate days with 2 protein preparations).

Extended Data Fig. 2 Single-molecule imaging of LIV-BPWT and LIV-BPSS reveals kinetic determinants of analogue and digital responses.

a, b, Representative traces of LIV-BPWT (a) and LIV-BPSS (b) imaged at 100-ms time resolution in the absence of substrate. c, d, LIV-BPWT (c) and LIV-BPSS (d) variants imaged in the presence of leucine at the Kd of each variant (40 nM and 5.6 μM, respectively) and at the indicated time resolution in milliseconds. FRET values from all selected traces (count at top right of each panel) summed into time-dependent population FRET histograms, represented as contour plots. Scale bar is shown at the right. Two distinct FRET peaks are apparent at all time resolutions for LIV-BPWT; LIV-BPSS displayed only a single peak at low time resolution (≥100 ms) that resolved into two district populations in the millisecond regime. eh, LIV-BPWT and LIV-BPSS imaged at time resolutions that most-completely sampled the FRET transitions (25 ms and 0.25 ms, respectively) and idealized using the segmental k-means algorithm (Supplementary Methods). Dwell-time distributions of LIV-BPWT in the low- (e) and high-FRET (f) states, and LIV-BPSS in the low- (g) and high-FRET (h) states, in the presence of the indicated concentrations of leucine. All experiments were performed at least three times with similar results. i, j, Rate constants derived from maximum likelihood analysis of dwell times in the low- (black squares) and high-FRET (red circles) states, fit to lines to determine ligand association (kon) (black lines) and ligand dissociation (koff) (red lines) rate constants for LIV-BPWT (i) and LIV-BPSS (j). As expected for a bimolecular interaction, the ligand-binding rate increased linearly with ligand concentrations, whereas the ligand-dissociation rate remained constant. kon values were similar in LIV-BPWT and LIV-BPSS at 77 and 30 μM−1 s−1, respectively, whereas koff values differed by nearly two orders of magnitude at 4.0 and 212 s−1, respectively. Together, these results are consistent with the observed 100-fold difference in binding affinity in the two sensor variants (Fig. 1c). All experiments were performed at least three times with similar results.

Extended Data Fig. 3 LIV-BP rapidly binds and responds to ligand.

a, Schematic of His-tagged LIV-BPSS directly surface-immobilized. b, Representative fluorescence (donor in green and acceptor in red) and FRET (blue) traces from a single LIV-BPSS sensor imaged at 15-ms time resolution during the rapid delivery (vertical dashed line) of 10 μM (subsaturating) leucine. c, Ensemble-average FRET efficiency (symbols), fit to a single exponential function with a time-constant of approximately 16 ms (red line). d, FRET values of an ensemble of surface-immobilized LIV-BPSS molecules summed into a contour plot (scale at right), demonstrating rapid and uniform response to leucine addition. e, Schematic of His-tagged LIV-BPWT directly surface-immobilized. f, Representative fluorescence (donor in green and acceptor in red) and FRET (blue) traces from a single LIV-BPWT sensor imaged at 10-ms time resolution during the rapid delivery (vertical dashed line) of 3 μM (saturating) leucine. g, Ensemble-average FRET efficiency of leucine (grey squares), isoleucine (red circles) or valine (blue triangles). Data are fit to a single exponential function with a time-constant of approximately 27 ms for leucine (grey line), 25 ms for isoleucine (red line) and 32 ms for valine (blue line). h, FRET histogram of an ensemble of surface-immobilized LIV-BPWT molecules responding to leucine summed into a contour plot (scale at right), demonstrating rapid and uniform response to leucine addition. i, Schematic of LIV-BPWT encapsulated within the lumen of liposomes in identical conditions to those used for transport assays (Supplementary Methods). Liposomes were pre-incubated in 100 μg ml−1 α-haemolysin for 15 min at room temperature. j, Encapsulated LIV-BPWT FRET response to injection of 3 μM leucine, in which the time of mixing is marked (dashed grey line). The time constant of the observed FRET response (about 23 ms), fit as above (green line), was identical to LIV-BPWT directly immobilized to the surface. All experiments were performed at least three times with similar results.

Extended Data Fig. 4 Quantifying the timing of transport initiation.

a, The precise timing of ligand injection was estimated by co-injecting a low concentration (0.5 nM) of Cy5-labelled 21-mer DNA duplex (Supplementary Methods) to the injected solution of interest, and measuring the increase in background fluorescence on the Cy5 channel in regions far from immobilized particles. The mean background fluorescence is shown, from a representative experiment in which LIV-BPWT-containing liposomes that lack MhsT were imaged before and after (vertical dashed line) the injection of Cy5-labelled DNA tracer, with the exact time of injection determined by the midpoint of the step-like increase in fluorescence. Inset, zoomed-in view of the period immediately before and after injection. b, c, Representative single-molecule fluorescence (donor in green and acceptor in red) and FRET (blue) traces (b) and FRET contour plot (c) of encapsulated LIV-BPWT from these experiments, demonstrating minimal changes in apparent FRET efficiency with co-injection of low concentrations of the Cy5 fluorophore. Experiments were performed at least three times with similar results.

Extended Data Fig. 5 Representative traces of multi-round and single turnover assays with simulations of multi-round activity.

a, b, Single-molecule transport traces with 50 mM Na+ and leucine (5 μM and 0.1 μM for a and b, respectively) at external pH 8 and internal pH 6, with the time of injection indicated by a dashed grey vertical line. a, Representative single-molecule fluorescence (top) (donor in green and acceptor in red) and FRET (bottom) (blue) traces and fits to exponential functions (red) from experiments imaging LIV-BPSS encapsulated within proteoliposomes that contain MhsT. b, As in a, but for the single-turnover assay using LIV-BPWT and state assignments in red (bottom panels). Traces shown are representative of experiments performed at least three times. c, Representative simulated FRET traces generated by a model in which distinct states correspond to the distinct FRET values that would be reported by the sensor, using the calibration curve according to the number of substrate molecules in the liposome (Extended Data Fig. 1). We assume transport occurs at a rate of one event per second and is irreversible, so transitions to lower-FRET states (state 1 to state 0) are not allowed. d, Noise that mimics experimental noise was added to representative FRET traces (different individual traces to those shown in c), which masks the individual steps observable in a. This indicates that, while in ideal circumstances we should be able to monitor individual transport events in this assay, in practice such an analysis would be difficult. e, Experimental FRET traces with the same apparent transport rate as the simulated data demonstrate a notable likeness to the simulated traces. Over 1,000 traces were simulated with similar results and representative traces were taken from experiments repeated at least 3 times.

Extended Data Fig. 6 3H-leucine uptake activity of wild-type MhsT, and immobilization mutants and membranes with and without MhsT.

a, Turnover rates of purified and reconstituted wild-type MhsT (grey) and Cy7-labelled MhsT(S3C) (red) and MhsT(N452C) (blue) assessed at a series of external leucine concentrations. Leucine uptake by wild-type MhsT exhibited a Km of 0.93 ± 0.08 μM and a Vmax of 0.83 ± 0.02 substrate molecules per second. MhsT(S3C) labelled with a Cy7 fluorophore had a Km of 0.94 ± 0.11 μM and a Vmax of 0.82 ± 0.03 substrate molecules per second. MhsT(N452C) labelled with a Cy7 fluorophore had a Km of 0.90 ± 0.08 μM and a Vmax of 0.84 ± 0.02 substrate molecules per second. b, Leucine uptake by MQ614 cells expressing wild-type MhsT (grey), MhsT(P86C) or MhsT(G87C) in the presence of 150 mM Na+ at pH 8.5. Mean ± s.e.m. (n = 3 experiments). c, Inside-out and outside-out vesicles prepared (Supplementary Methods) and assayed for uptake activity in the presence and absence of the MhsT transporter as indicated. Transport is observed only in the presence of MhsT in the outside-out orientation. d, Radioactive glycine uptake by the native CycA glycine:H+ symporters in inside-out or outside-out prepared vesicles in the presence of an inwardly directed proton gradient. Both preparations show robust activity, indicating that both vesicle preparations are intact and contain functional transporters. Lactic acid was added (arrow) to vesicles during the glycine-uptake time course to establish a proton gradient relative to the vesicle orientation. This creates an inwardly directed proton gradient in outside-out vesicles, and the opposite in inside-out vesicles. We observe an increase in the rate of glycine transport in outside-out vesicles and a marked decrease in inside-out vesicles, as expected, which demonstrates that we have prepared the vesicles in the indicated orientation. Mean ± s.e.m. (n = 3 experiments). e, The Vmax and Km of leucine uptake by wild-type MhsT were measured at a series of external leucine concentrations for the indicated periods of time. Assays were performed with proteoliposomes that contain known amounts of MhsT prepared at protein-to-lipid reconstitution ratios of 1:150 (w/w) (solid symbols) or 1:300 (w/w) (open symbols) for time periods of 2, 3, 5 or 10 s (total decays per minute at the corresponding time points were background-corrected for decays per minute determined at 0 s). The partially filled square indicates the virtual overlap of data points. The specific radioactivity–decays per minute correlation was verified using known amounts of 3H-leucine. Shorter sampling times yielded higher turnover rates and lower Km values (the highest Vmax of 1.04 ± 0.02 s−1 and lowest Km of 0.21 ± 0.01 μM were determined at the 2-s sampling time), but the technically challenging nature of these experiments precluded further shortening of the sampling time. To ensure the reliable determination of the Vmax and Km in radiolabelled uptake measurements, a sampling time of 3 s was chosen. Mean ± s.e.m. (n = 3 replicates of 2 protein preparations).

Extended Data Fig. 7 Determination of the number of transporters per liposome.

a, The number of Cy7-labelled transporters observed in each liposome. Reported values were corrected for a labelling efficiency of 75% to determine the final estimate of the number of transporters per liposome. Black symbols represent individual data points; bars represent mean ± s.e.m. (n = 3 experiments). b, c, Representative Cy7 fluorescence (left) and FRET (right) traces of a liposome with a single transporter (b) or two transporters (c). Traces are representatives of experiments performed at least three times.

Extended Data Fig. 8 Orientation-controlled single-turnover transport.

a, Schematics and corresponding contour plots of the single-turnover assay with liposomes immobilized by the S3C (inside-out) or N452C (outside-out) residues of MhsT. The third panel from the left shows the passive diffusion of leucine in the absence of Na+. The right panel shows transport data from proteoliposomes that contain a single (mixed orientation) MhsT transporter immobilized by His-tagged lipids. Occupancy of the high-FRET state following injection of leucine (grey dashed line) represents the proportion of vesicles in which transport has occurred. The N in top right corner indicates the total number of traces recorded over three separate experiments. b, Left, schematic of proteoliposome immobilization by biotin tags added at position P86C or G87C of MhsT, which result in inside-out orientations of the MhsT transporter (as for S3C). Right, contour plots of the single-turnover assay when immobilizing via biotin–P86C or biotin–G87C. The N in top right corner indicates the total number of traces over three separate experiments. c, Cumulative distributions of representative movies demonstrating the low translocation rates of the S3C (inside-out) immobilized liposomes with all three substrates, which match the leak rate in the absence of Na+. Experiments were repeated at least three times with similar results.

Extended Data Fig. 9 Michaelis–Menten kinetic parameters of MhsT transport.

a, FRET values were transformed into units of proteoliposome luminal leucine concentration (Supplementary Methods) and fit to linear functions (lines) to determine transport initial rates, from experiments in the presence of the indicated concentrations of external leucine. Unless otherwise stated, experiments were performed with external 150 mM Na+, 1 μM leucine and pH 8 outside with pH 6 inside, and with choline chloride used to maintain osmotic balance. b, Substrate accumulation rates from a (black squares) were fit to a Michaelis–Menten function (black line) with a Km of 0.06 ± 0.01 μM and a Vmax of 1.18 ± 0.04 s−1 with a Hill coefficient of 0.69 ± 0.08. Analogous radioactive uptake experiment (red circles) fit with a Michaelis–Menten function (red line) with a Km of 0.19 ± 0.02 μM and a Vmax of 0.99 ± 0.03 s−1 with a Hill coefficient of 1.85 ± 0.28. c, As in b, but varying the external concentration of Na+. Black line is a fit to a Hill equation with a Km of 69.9 ± 4.25 mM and a Vmax of 1.14 ± 0.05 s−1 with a Hill coefficient of 2.03 ± 0.17. Analogous radioactive uptake experiment (red circles) fit with a Hill equation with a Km of 63 ± 15 mM, Vmax of 0.74 ± 0.11 μM s−1 and Hill coefficient of 1.61 ± 0.40. d, Substrate accumulation rates in the presence of the indicated external pH (dark grey, pH 6 inside) and internal pH (light grey, pH 8 outside) and 150 mM Na+ and 1 μM leucine. The dark grey pH 8 bar and the light grey pH 6 bars are duplicated for comparison purposes. e, Substrate accumulation rates with 0.1 μM leucine pH 8 and 50 mM Na+ external solution and in the presence and absence of 50 mM Na+ at pH 6 and pH 8 of the internal solution. Robust transport is observed at both internal pH 6 and pH 8 in the absence of internal Na+ (red). Addition of internal Na+ completely abolishes transport (purple), with changes in internal pH having little to no further effect (cyan). Mean ± s.e.m. (n = 3+ experiments). f, Multi-round transport assay with 150 mM external Na+, 1 μM leucine and pH 8 with (red) and without (black) internal 50 μM tryptophan at pH 6. There is no significant difference between the two conditions when tested using a two-sided Student’s t-test that was not corrected for multiple hypotheses at a 95% confidence interval. Black diamonds represent individual data points, and bars represent mean ± s.e.m. (n = 3 experiments).

Extended Data Fig. 10 Proton leak into proteoliposomes monitored by lipid-linked pHrodo fluorophores.

Buffer was exchanged from pH 6 to pH 8 (outside) and a step-like approximately 50% decrease in fluorescence was observed, which we interpret as the quenching of lipid-linked pHrodo on the liposome exterior, followed by a slow decay in fluorescence (black squares). The data were fit with a single exponential function (red line) with a time constant of about 33 s, which we interpret as the slow leak of protons into the liposome. The gaps in the data are periods in the absence of illumination, to rule out photobleaching as a cause of the fluorescence decay we observed. These experiments confirm that the pH gradient was maintained during the full time-frame in which we measure transport. Experiments were performed three times with similar results.

Supplementary information

Supplementary Information

This Supplementary information includes four notes that provide explanations that inform on experiments and methodologies, one table containing the observed binding affinities of a panel of amino acid ligands for both LIV-BPWT and LIV-BPSS proteins and the full methods.

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Fitzgerald, G.A., Terry, D.S., Warren, A.L. et al. Quantifying secondary transport at single-molecule resolution. Nature 575, 528–534 (2019). https://doi.org/10.1038/s41586-019-1747-5

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