Enhanced Multispecificity of Arabidopsis Vacuolar Multidrug Resistance-associated Protein-type ATP-binding Cassette Transporter, AtMRP2

Abstract

Recent investigations have established thatArabidopsis thaliana contains a family of genes encoding ATPbinding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (At-MRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17␤-estradiol 17-(␤-D-glucuronide) (E 2 17␤G) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E 2 17␤G uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E 2 17␤G uptake, neither competes with DNP-GS for uptake nor is subject to E 2 17␤G-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2. ATP-binding cassette (ABC)1 transporters are starting to assume prominence in considerations of energy-dependent transport in plants. Constituted of one or two copies each of two core structural elements: a hydrophobic, membrane spanning domain (MSD) containing multiple (usually four or six) transmembrane spans and a cytosolically oriented ATP-binding domain (nucleotide binding fold, NBF) containing Walker A, Walker B, and ABC signature sequence motifs, ABC transporters are MgATP-energized pumps that as a superfamily are active in the transport of a broad range of substances including alkaloids, amino acids, sugars and sugar conjugates, peptides and peptide conjugates, heavy metal chelates, and lipids across membranes (1).Two classes of findings were instrumental in prompting studies of ABC transporters in plants. The first was the molecular cloning of a multidrug resistance (MDR)-like gene from Arabidopsis thaliana (2) and the subsequent independent isolation of other MDR homologs from the same and other plant species (3, 4). Because all of these genes encode ABC transporters bearing...

Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis

“…The Arabidopsis ABC subfamily C (ABCC) transporters AtABCC1 and AtABCC2 were previously demonstrated to localize to the vacuolar membrane (Liu et al, 2001;Geisler et al, 2004) and have been shown to transport organic anion conjugates (Lu et al, 1998;Liu et al, 2001). AtABCC14 is also localized to the tonoplast, as shown by several proteomic analyses (Carter et al, 2004;Shimaoka et al, 2004;Jaquinod et al, 2007).…”

“…Several MATE and ABCC-type proteins implicated in vacuolar conjugate transport have been shown to be multispecific (i.e. they transport structurally unrelated compounds, whereby the affinities toward the individual substrates vary considerably; Liu et al, 2001;Martinoia et al, 2012). The low affinity of the ABA-GE transport further suggests that multispecific transporters involved in the vacuolar sequestration of different metabolites also mediate the uptake of ABA-GE.…”

“…The Arabidopsis AtABCC1 and especially AtABCC2 mediate the transport of structurally diverse metabolites, such as phytochelatins, folates, and conjugates of chlorophyll catabolite and xenobiotics (Liu et al, 2001;Frelet-Barrand et al, 2008;Raichaudhuri et al, 2009;Song et al, 2010). We expressed AtABCC2 in yeast and observed a distinct MgATP-dependent ABA-GE transport activity of isolated membrane vesicles (Fig.…”

A Multidrug Resistance–Associated Protein Involved in Anthocyanin Transport in Zea mays

“…In A. thaliana, 8 of the 15 MRPs are found in homologous pairs that can have >85% sequence identity Kolukisaoglu et al, 2002). Interestingly, the highly homologous pair AtMRP1 and AtMRP2 shows both significant overlap of, and fundamental differences in, their in vitro substrate preferences Liu et al, 2001). Therefore, both functional redundancy and unique biological roles likely exist for closely related MRPs.…”

“…Substrates are not limited to glutathionated compounds but include a range of heterocyclic organic anions and heavy metals . Such compounds may or may not be conjugated to sugars or amino acids and can be transported alone or in conjunction with free GSH (Liu et al, 2001). In humans, MRP substrates include anticancer drugs and other xenobiotics, including plant flavonoids, and endogenous molecules, such as bile salts and leukotrienes (Borst et al, 1999).…”