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 K m of 0.93 ± 0.08 μM and a V max of 0.83 ± 0.02 substrate molecules per second. MhsT(S3C) labelled with a Cy7 fluorophore had a K m of 0.94 ± 0.11 μM and a V max of 0.82 ± 0.03 substrate molecules per second. MhsT(N452C) labelled with a Cy7 fluorophore had a K m of 0.90 ± 0.08 μM and a V max 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 V max and K m 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 K m values (the highest V max of 1.04 ± 0.02 s−1 and lowest K m 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 V max and K m in radiolabelled uptake measurements, a sampling time of 3 s was chosen. Mean ± s.e.m. (n = 3 replicates of 2 protein preparations).