Magnesium Adenosine 5[prime]-Triphosphate-Energized Transport of Glutathione-S-Conjugates by Plant Vacuolar Membrane Vesicles

Abstract

By characterization of the uptake of glutathione-S-conjugates, principally dinitrophenyl-S-glutathione (DNP-CS), by vacuolar membrane vesicles, we demonstrate that a subset of energy-dependent transport processes in plants are not H+-coupled but instead are directly energized by MgATP. The most salient features of this transport pathway are: (a) its specific, obligate requirement for MgATP as energy source; (b) the necessity for hydrolysis of the y-phosphate of MgATP for uptake; (c) the insensitivity of uptake to uncouplers of the transtonoplast H+ gradient (carbonylcyanide 4-trifluoromethoxyphenylhydrazone, gramicidin-D, and NH,CI); (d) its pronounced sensitivity to vanadate and partia1 inhibition by vinblastine and verapamil; (e) the lack of chemical modification of DNP-CS either during or after transport; (0 the capacity of S conjugates of chloroacetanilide herbicides, such as metolachlor-CS, but not free herbicide, to inhibit uptake; and (g) the ability of vacuolar membrane vesicles purified from a broad range of plant species, including Arabidopsis, Befa, Vigna, and Zea, to mediate MgATP-dependent, H+-electrochemical potential difference-independent DNP-CS uptake. O n the basis of these findings it is proposed that the transport of DNP-CS across the vacuolar membrane of plant cells is catalyzed by a glutathione-conjugate transporter that directly employs MgATP rather than the energy contained i n the transtonoplast H+-electrochemical potential difference to drive uptake. The broad distribution of the vacuolar DNP-CS transporter and its inhibition by metolachlor-CS are consistent with the notion that it plays a general role in the vacuolar sequestration of glutathione-conjugable cytotoxic agents.The prevailing working model for energy-dependent solute transport by plant cells is chemiosmosis (Mitchell, 1976;Spanswick, 1981). According to this scheme, the primary energizers for solute transport are pumps that mediate electrogenic H+-translocation across the membranes concerned to generate a gradient of electrical potential (AI)) and chemical potential (ApH). Depending on the net charge on the solute-porter complex, the motive force for transport is believed to be derived from the ApH and/or AI) components of the H+-electrochemical potential difference (A,%"+). For with or in exchange for H+, transport is driven by ApH; for solute-porter complexes that carry a net charge, transport is driven by AI).The notion of phosphoanhydride-energized H+-translocation across the principal membranes of plant cells is irrefutable. Primary ATP-energized Hf pumps and a PPienergized H+ pump in the case of the vacuolar membrane (Rea and Poole, 1993) have been demonstrated in the membranes bounding most of the compartments of plant cells, and in a11 cases the pumps concerned have been shown to be critica1 for establishment of the resting membrane potential and transmembrane pH gradients (Spanswick, 1981;Sze, 1985;Rea and Poole, 1993). What is not so clear, however, is whether essentially a11 energy-dependent secondary transport...

Alternate Energy-Dependent Pathways for the Vacuolar Uptake of Glucose and Glutathione Conjugates

“…2A) amounts to an accumulation ratio of 60. This value is similar to that computed for S-(2,4-dinitrophenyl) glutathione (DNP-GS), a model MRPtype ABC transporter substrate, for the same membrane preparation (Li et al, 1995).…”

“…It has been reported that red beet vacuolar membrane vesicles, purified by the same procedure (Li et al, 1995), accumulate a glutathione conjugate of pHCA, a key intermediate in the phenylpropanoid pathway, and that conjugation is necessary for uptake (Walczak and Dean, 2000). In light of these findings, the likely involvement of an MRP-type transporter in uptake of the glutathione conjugate, and the fact that plants primarily glucosylate pHCA, we synthesized the phenolic glucoside of pHCA (pHCAG) to see if it also undergoes transport and, if so, whether it is subject to MRP-mediated or H ϩ -coupled transport.…”

“…First defined in the context of glutathione conjugate transport (Martinoia et al, 1993;Li et al, 1995), it is now clear that many other xenobiotics and secondary metabolites are amenable to accumulation by vacuolar ABC transporters Rea, 1999;Martinoia et al, 2001). The salient properties of ABC transporter-catalyzed vacuolar uptake are: (a) direct energization by ATP hydrolysis-other nucleoside triphosphates can substitute, but nonhydrolyzable analogs and inorganic pyrophosphate cannot; (b) exquisite sensitivity to vanadate-micromolar concentrations of vanadate, a phosphoryl transition-state analog, strongly inhibit uptake (Li et al, 1995); (c) little or no reliance on the transmem-brane H ϩ -electrochemical gradient-abolition of the H ϩ gradient can partially inhibit transport but seldom, if ever, to the extent that vanadate does (Li et al, 1995;Lu et al, 1998;Klein et al, 2000).…”

“…Here, we attempt to address both issues-the first through the development of a liquid chromatography (LC)-mass spectrometry (MS)-based protocol for quantitative analysis of the transport of unlabeled compounds by vacuolar membrane vesicles, and the second by direct comparisons of the energetics and kinetics of uptake of closely related compounds conjugated to different ligands. Using vacuolar membrane vesicles isolated from the red beet (Beta vulgaris) storage root as a model system (Rea and Turner, 1990;Li et al, 1995), we have examined the transport properties of two structurally related sulfonylurea herbicides, chlorsulfuron (CS) and chlorimuron-ethyl (CE), before and after conjugation with Glc and glutathione, respectively. The results demonstrate that the glucosylated herbicide is transported by a carrier that is energetically coupled to the transmembrane H ϩ gradient, whereas the glutathionated herbicide is transported by an ABC transporter-like carrier that is directly energized by ATP.…”

Barley Aleurone Cells Contain Two Types of Vacuoles: Characterization of Lytic Organelles by Use of Fluorescent Probes

“…Taurocholate may act as a competitive inhibitor to BCECF in these experiments. Vanadate also inhibited the directly energized transporter that pumps these organic anions into barley aleurone vacuoles in a manner similar to that found for these transporters in other types of plant cells Li et al, 1995b;Coleman et al, 1997a). Consistent with its ability to inhibit anion transport, probenecid decreased the ATP-dependent uptake of BCECF into isolated vacuoles.…”