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 K_m of 0.06 ± 0.01 μM and a V_max 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 K_m of 0.19 ± 0.02 μM and a V_max 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 K_m of 69.9 ± 4.25 mM and a V_max 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 K_m of 63 ± 15 mM, V_max 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).