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. 2002 Jun;1(3):391–400. doi: 10.1128/EC.1.3.391-400.2002

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Copyright © 2002, American Society for Microbiology

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FIG. 4. — MgATP-dependent uptake of [³H]DNP-GS, [³H]E₂17βG, and [³H]GSH by vacuolar membrane vesicles from WT and mutant strains. For the large graphs in panels A, B, and C, MgATP-dependent uptake of the indicated transport substrates was measured for 10 min, at the concentrations of substrate indicated, in the presence or absence of 3 mM ATP. The level of uptake at each substrate concentration was calculated by subtracting the amount of radioactivity detected in the absence of ATP from that detected when ATP was present. The data were fitted to the Michaelis-Menten equation by nonlinear least-squares analysis. The values obtained for each substrate and each strain are as follows. (A) For the WT, K_m_(DNP-GS) = 5.8 ± 2.3 μM and V_max(DNP-GS) = 30.7 ± 2.3 nmol/mg/10 min; for the bpt1Δ strain, K_m_(DNP-GS) = 6.3 ± 3.8 μM and V_max(DNP-GS) = 25.3 ± 3.0 nmol/mg/10 min; for the ycf1Δ strain, K_m_(DNP-GS) = 268.4 ± 157 μM and V_max(DNP-GS) = 20.3 ± 7.8 nmol/mg/10 min. (B) For the WT, K_m_(E217βG) = 386.0 ± 33.0 μM and V_max(E217βG) = 35.3 ± 13.5 nmol/mg/10 min; for the bpt1Δ strain, K_m_(E217βG) = 580.0 ± 38.0 μM and V_max(E217βG) = 23.2 ± 8.0 nmol/mg/10 min. (C) For the WT, K_m_(GSH) = 3.8 ± 0.4 mM and V_max(GSH) = 149.4 ± 76.3 nmol/mg/10 min; for the bpt1Δ strain, K_m_(GSH) = 3.8 ± 0.4 mM and V_max(GSH) = 87.5 ± 43.3 nmol/mg/10 min. Kinetic parameters were not calculated for DNP-GS uptake by the ycf1Δ bpt1Δ strain or for E₂17βG and GSH uptake by the ycf1Δ and ycf1Δ bpt1Δ mutants, because the data did not approximate the Michaelis-Menten equation. Individual data points are means ± standard errors (n = 3 to 6). The insets in panels A and B show the time courses for DNP-GS and E₂17βG uptake, respectively. The strains used for the preparation of vacuolar vesicles are ABC154 (wild type), ABC470 (ycf1Δ), ABC791 (bpt1Δ), and ABC794 (ycf1Δ bpt1Δ).

FIG. 4. — MgATP-dependent uptake of [³H]DNP-GS, [³H]E₂17βG, and [³H]GSH by vacuolar membrane vesicles from WT and mutant strains. For the large graphs in panels A, B, and C, MgATP-dependent uptake of the indicated transport substrates was measured for 10 min, at the concentrations of substrate indicated, in the presence or absence of 3 mM ATP. The level of uptake at each substrate concentration was calculated by subtracting the amount of radioactivity detected in the absence of ATP from that detected when ATP was present. The data were fitted to the Michaelis-Menten equation by nonlinear least-squares analysis. The values obtained for each substrate and each strain are as follows. (A) For the WT, K_m_(DNP-GS) = 5.8 ± 2.3 μM and V_max(DNP-GS) = 30.7 ± 2.3 nmol/mg/10 min; for the bpt1Δ strain, K_m_(DNP-GS) = 6.3 ± 3.8 μM and V_max(DNP-GS) = 25.3 ± 3.0 nmol/mg/10 min; for the ycf1Δ strain, K_m_(DNP-GS) = 268.4 ± 157 μM and V_max(DNP-GS) = 20.3 ± 7.8 nmol/mg/10 min. (B) For the WT, K_m_(E217βG) = 386.0 ± 33.0 μM and V_max(E217βG) = 35.3 ± 13.5 nmol/mg/10 min; for the bpt1Δ strain, K_m_(E217βG) = 580.0 ± 38.0 μM and V_max(E217βG) = 23.2 ± 8.0 nmol/mg/10 min. (C) For the WT, K_m_(GSH) = 3.8 ± 0.4 mM and V_max(GSH) = 149.4 ± 76.3 nmol/mg/10 min; for the bpt1Δ strain, K_m_(GSH) = 3.8 ± 0.4 mM and V_max(GSH) = 87.5 ± 43.3 nmol/mg/10 min. Kinetic parameters were not calculated for DNP-GS uptake by the ycf1Δ bpt1Δ strain or for E₂17βG and GSH uptake by the ycf1Δ and ycf1Δ bpt1Δ mutants, because the data did not approximate the Michaelis-Menten equation. Individual data points are means ± standard errors (n = 3 to 6). The insets in panels A and B show the time courses for DNP-GS and E₂17βG uptake, respectively. The strains used for the preparation of vacuolar vesicles are ABC154 (wild type), ABC470 (ycf1Δ), ABC791 (bpt1Δ), and ABC794 (ycf1Δ bpt1Δ).