AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: Isolation and functional definition of a plant ATP-binding cassette transporter gene (anthocyaninsyherbicidesyheterologous expressionyvacuolar membraneyxenobiotic detoxification)

Because plants produce cytotoxic compounds to which they, themselves, are susceptible and are exposed to exogenous toxins (microbial products, allelochemicals, and agrochemicals), cell survival is contingent on mechanisms for detoxifying these agents. One detoxification mechanism is the glutathione S-transferase-catalyzed glutathionation of the toxin, or an activated derivative, and transport of the conjugate out of the cytosol. We show here that a transporter responsible for the removal of glutathione S-conjugates from the cytosol, a specific Mg21-ATPase, is encoded by the AtMRP1 gene of Arabidopsis thaliana. The sequence of AtMRP1 and the transport capabilities of membranes prepared from yeast cells transformed with plasmid-borne AtMRP1 demonstrate that this gene encodes an ATP-binding cassette transporter competent in the transport of glutathione S-conjugates of xenobiotics and endogenous substances, including herbicides and anthocyanins. The recent finding that intact vacuoles and vacuolar membrane vesicles isolated from vascular plants mediate MgATPdependent accumulation of glutathione S-conjugates (GSconjugates) in the absence of a transmembrane H1electrochemical potential difference (1, 2) is seminal in two respects. Not only does it implicate MgATP as a direct energy source for organic solute transport across plant membranes but, because the compounds transported include glutathionated herbicides (1, 2), isoflavonoid phytoalexins (3), and possibly anthocyanins (4), a mechanism for the vacuolar sequestration of GS-conjugable xenobiotics and endogenous substances appears to have been discovered. Despite its strategic value for manipulating and investigating toxin compartmentation in plants, neither the protein nor the gene encoding the plant vacuolar GS-conjugate pump have been identified. Significant therefore are three clues as to its possible identity. (i) The resemblance of plant vacuolar GSconjugate transport to that mediated by the GS-conjugate transporting Mg21-ATPase, GS-X pump (5), of mammalian cells. In both cases, transport is selective for GS-conjugates and oxidized glutathione (GSSG), but not reduced glutathione (GSH), directly energized by MgATP and potently inhibited by the phosphoryl transition state analog, vanadate (1, 2). (ii) The ability of the human multidrug resistance-associated protein gene (HmMRP1), an ATP-binding cassette transport protein (ABC transporter) gene isolated from drug-resistant small cell lung carcinoma cell lines (6), to confer GS-X pump activity, specifically MgATP-energized transport of leukotriene C4 and related GS-conjugates, on transfected cells (7, 8). (iii) The functional and structural equivalence of the Saccharomyces cerevisiae (yeast) cadmium factor (ScYCF1) gene product, identified according to its ability to confer cadmium resistance (9), to HmMRP1, its localization to the vacuolar membrane (10) and the capacity of HmMRP1 for alleviating the cadmium-hypersensitive phenotype and restoring GS-conjugate transport in membranes prepared from yeast strains deleted for the YCF1 gene (11, 12). Here we describe the isolation of a gene from Arabidopsis thaliana (AtMRP1), encoding a protein belonging to the same subclass of ABC transporters as HmMRP1 and ScYCF1, whose heterologous expression in yeast confers MgATP-energized GS-conjugate transport. In so doing, we provide a functional definition of an ABC transporter from vascular plants and identify an element involved in the removal of GS-conjugable compounds from the cytosol in a manner independent of the transmembrane H1 gradient. MATERIALS AND METHODS Isolation of AtMRP1. On the basis of the functional resemblance between plant vacuolar GS-conjugate transport and that mediated by ScYCF1 and HmMRP1, and the 44.6% sequence identity (63.9% similarity) between ScYCF1 and HmMRP1, degenerate oligonucleotide primers corresponding to the second ATP-binding cassette of ScYCF1 and HmMRP1 (positions 1,300–1,321 and 1,474–1,494, respectively)—two of the most ScYCF1and HmMRP1-specific sequences common to both—were used for the isolation of plant genes likely involved in GS-conjugate transport by PCR amplification of A. thaliana genomic DNA. The sequences of the two primers yielding a 0.6-kb HmMRP1and ScYCF1-hybridizing amplification product by this approach were: 59-GARAARGTIGGIATHGTIGGIMGIACIGGIGC-39 (MRP2) and 59-TCCATDATIGTRTTIARICKTGIGC-39 (MRP4), where I 5 inosine, K 5 T or G, M 5 C or A, and R 5 A or G. MRP2 corresponds to amino acid residues 1,300–1,310 and 1,321– 1,331 of ScYCF1 and HmMRP1, respectively; MRP4 corresponds to residues 1,466–1,474 and 1,486–1,494 (6, 9). After determining that the 0.6-kb PCR product exhibited greatest similarity to ScYCF1 and HmMRP1 plus the putative translation product of an unidentified 1.6-kb A. thaliana expressed sequence tag (EST) (ATTS1246) (13), a mixed probe consistThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y948243-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: ABC transporter, ATP-binding cassette transport protein; GST, glutathione S-transferase; C3G, cyanidin 3-glucoside; DNP-GS, S-(2,4-dinitrophenyl)glutathione; GS-conjugate, glutathione S-conjugate; GSH, glutathione; GSSG, oxidized glutathione; MRP1, multidrug resistance-associated protein; HmMRP1, human MRP1; YCF1, yeast cadmium factor protein; ScYCF1, Saccharomyces cerevisiae (yeast) cadmium factor; EST, expressed sequence tag; BAC, bacterial artificial chromosome; CFTR, cystic fibrosis transmembrane conductance regulator; NBF, nucleotide-binding fold. Data deposition: The cDNA and genomic sequences reported in this paper have been deposited in the GenBank database (accession nos. AF008124 and AF008125, respectively). *To whom reprint requests should be addressed. e-mail: parea@sas. upenn.edu.