Alternate energy-dependent pathways for the vacuolar uptake of glucose and glutathione conjugates

Abstract

Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H(+) gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuron-ethyl, is incompletely inhibited by agents that dissipate the transmembrane H(+) gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H(+) gradient dependence of glucoside uptake. It is concluded that H(+) gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H(+) antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and/or prohibitively expensive.

Figure 1

Chemical structures of three herbicide conjugates that undergo vacuolar uptake. A, CSG, a Glc conjugate of 5-hydroxychlorsulfuron. B, CE-GS, a glutathione conjugate of chlorimuron-ethyl. C, HPSG, a Glc conjugate of hydroxyprimisulfuron.

Figure 2

ATP-dependent uptake of two structurally related herbicides by vacuolar membrane vesicles. Red beet vacuolar membrane vesicles were incubated at 25°C in the standard uptake medium containing 50 μm CSG (A) or 50 μm CE-GS (B). ATP was added or omitted as indicated. Uptake was terminated by rapid filtration and the amount of compound taken up by the vesicles was determined by LC-MS. The results of a representative experiment are shown. Each data point is the mean of duplicate measurements, all of which were within 15% of each other. The insets show the original LC-MS traces from the 60-min time points for vesicles incubated in the presence (top) or absence (bottom) of ATP. The traces are slightly offset from each other to emphasize the high signal-to-noise ratio for energized uptake.

Figure 3

Kinetics of ATP-energized uptake of CSG and CE-GS by vacuolar membrane vesicles. Uptake of CSG (A) or CE-GS (B) was measured at 25°C in standard uptake medium in the presence or absence of ATP. Uptake was allowed to proceed for 20 min (CSG) or 15 min (CE-GS) before rapid filtration and determination of the amount of the compound taken up by LC-MS. The values shown are the means of duplicate measurements minus the amount of compound that was taken up in the absence of ATP. Insets, Eadie-Hofstee (v/[S] versus v) plots, where v = velocity of uptake and [S] = substrate concentration.

Figure 4

Effects of inhibitors and ionophores on the uptake of CE-GS, DNP-GS, and CSG. The conditions for ATP-dependent uptake were as in Figure 2 except uptake was terminated after 30 min. All of the conjugates were present at a concentration of 50 μm. Where indicated, bafilomycin A₁, nigericin, valinomycin, and vanadate were added at concentrations of 0.4, 2, 2, and 200 μm, respectively. The bafilomycin A₁ stock solution was made up in dimethyl sulfoxide; the valinomycin and nigericin stock solutions were made up in ethanol. Control experiments confirmed that neither dimethyl sulfoxide nor ethanol at the concentrations at which they were added to the uptake medium (0.004% [v/v] and 0.04% [v/v], respectively) affected uptake. Uptake is expressed as a percentage of the uptake by controls to which no inhibitors or ionophores had been added. The control activities were 52.5, 7.7, and 13.4 nmol mg⁻¹ protein for CE-GS, DNP-GS, and CSG, respectively. The values shown are the means of duplicate measurements from a representative experiment.

Figure 5

Uptake of the phenolic glucoside of pHCA, from media containing (●) or lacking ATP (▪) plus no inhibitors (●), 200 μm vanadate (○), or 0.4 μm bafilomycin A₁ (□). The conditions for ATP-dependent uptake were as in Figure 2, but the transport substrate was 50 μm pHCAG. Inset, Eadie-Hofstee (v versus v/[S]) plot of the concentration dependence of ATP-energized pHCAG uptake. Uptake was measured at 25°C in standard uptake medium containing 50 μm pHCAG for the times indicated or for 20 min in uptake medium containing 12.5 to 400 μm pHCAG. pHCAG uptake was determined by LC-MS.

Figure 6

Effects of glutathione conjugates on Glc conjugate uptake and vice versa by vacuolar membrane vesicles. ATP-dependent uptake of 25 μm CSG (A) or CE-GS (B) was measured in standard uptake medium containing the other compounds indicated at concentrations of 400 μm. Reactions were terminated after 60 min and the amount of CSG or CE-GS taken up by membrane vesicles was determined by LC-MS. Uptake is expressed as a percentage of ATP-energized controls to which no other compounds were added. The compounds added were pHCAG, the phenolic glucoside of pHCA (β-anomer); pNP(α)G, pNP α-d-glucopyranoside; and pNP(β)GA, pNP β-d-GlcUA. Values shown are means ± se for triplicate measurements.

Figure 7

Four other glucosides whose energy-dependent accumulation by vacuolar membrane vesicles was examined. I, pHBA phenolic glucoside. II, pHBA Glc ester. III, pNP glucoside. IV, Methylparaben glucoside. All four compounds are 1-O-β-d-glucopyranosides.