Research Data supporting "Doxorubicin recognition and transport by the MATE multidrug transporter NorM from Vibrio cholerae"

Data sets concerning activity measurements for multidrug transport protein NorM from Vibrio cholerae (NorM-VC) expressed in Lactococcus lactis or incorporated in proteoliposomes in an inside-out fashion. Data set for Figure 5b. Facilitated doxorubicin uptake by wildtype NorM-VC or single mutants in ATP-depleted cells. Data contain the initial uptake rates (based on fluorescence decrease over time) and the total and the free doxorubicin concentrations. Data set for Figure 6a-e. Uptake and active doxorubicin efflux in cells by wildtype NorM-VC (a) and single mutants (b-e). Experiments started with ATP-depleted cells in the presence of 5 µM doxorubicin. After a steady-state level of fluorescence was reached, 25 mM glucose was added and fluorescence was followed in the presence of proton motive force (Δp), transmembrane potential difference (Δψ) only, transmembrane chemical proton gradient (ΔpH) only, or in the absence of proton motive force (no Δp). Endpoint data refer to the mean (± SEM) fluorescence levels at 1000 s after the addition of the glucose. Data set for Figure 7. Effect of nanobody Nb17_4 on doxorubicin transport by NorM-VC proteins in proteoliposomes. (a) Doxorubicin transport activity was followed by the decrease in fluorescence following the start of the transport reaction at 200s. (b) Data on transport rates were analysed by calculating the initial rate of fluorescence decrease. Data on end levels were calculated by averaging the fluorescence levels at 990 - 1000 s after the transport reaction was initiated. Data set for Figure S5a. Facilitated ethidium uptake by NorM-VC or single mutants in ATP-depleted cells. Data contain the initial uptake rates (based on fluorescence increase over time) and the ethidium concentration. Data set for Figure S6. (a) Uptake and active acriflavine efflux in cells by wildtype NorM-VC and E255Q mutant compared to empty vector control. Experiments started with ATP-depleted cells in the presence of 10 µM acriflavine. After a steady-state level of fluorescence was reached, 25 mM glucose was added and fluorescence was followed in the presence of proton motive force (Δp), transmembrane potential difference (Δψ) only, transmembrane chemical proton gradient (ΔpH) only, or in the absence of proton motive force (no Δp). (b) End level data for fluorescence emission. Data set for Figure S7. Measurements of proton permeability of the phospholipid bilayer in liposomes containing nanobody Nb17_4 and 100 μM of the fluorescent pH-probe BCECF. The liposomes were incubated without a transmembrane chemical proton gradient (pHext 6.8/pHin 6.8) or in the presence of an outwardly-directed transmembrane chemical proton gradient (pHext 8.0/pHin 6.8). BCECF fluorescence was followed over time. Triton X-100 was added around 1000 s, leading to disintegration of the liposomes and release of the BCECF into the external buffer.