SummaryAn ABC-transporter of Arabidopsis thaliana exhibiting high sequence similarity to the human (MRP1) and yeast (YCF1) glutathione-conjugate transporters has been analysed and used to complement a cadmium-sensitive yeast mutant (DTY168) that also lacks glutathione-conjugate transport activity. Comparison of the hydrophobicity plots of this A. thaliana MRP-like protein with MRP1 and YCF1 demonstrates that the transmembrane domains are conserved, even at the N-terminus where sequence identity is low. Cadmium resistance is partially restored in the complemented ycf1 mutant, and glutathione-conjugate transport activity can be observed as well. The kinetic properties of the A. thaliana MRP-like protein (AtMRP3) are very similar to those previously described for the vacuolar glutathioneconjugate transporter of barley and mung bean. Furthermore, a hitherto undescribed ATP-dependent transport activity could be correlated with the gene product, i.e. vesicles isolated from the complemented yeast, but not from DTY168 or the wild type, take up the chlorophyll catabolite Bn-NCC-1. The results indicate that the product of the MRP-like gene of A. thaliana is capable of mediating the transport of the two different classes of compounds.

Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCFI) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione Sconjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.Members of the adenosine 5'-triphosphate (ATP)-binding cassette family (ABC) of transporter proteins play a central role in cellular detoxification processes in animals (1), fungi (2, 3), and plants (4,5). This is illustrated by multidrug resistant cancer cells, resistant against a range of drugs with different chemical structures and cellular targets following exposure to a single cytotoxic drug (1). At least two ABC transporters can contribute to multidrug resistance in human cancer cells: The MDR1 P-glycoprotein (for review see ref. 1) and the multidrug resistance-associated protein (MRP) (6). Both are plasma membrane glycoproteins that can lower the intracellular concentration of natural product drugs (1,7,8).Another cellular detoxification mechanism with broad substrate specificity is the enzymatic (transferase-mediated) or nonenzymatic association of compounds with the thiol group of glutathione (GSH 11). These transporters have not only been found in many mammalian cell types (9), but also in yeast (3) and plant cells (4, 12). They have a relatively broad substrate specificity, but prefer substrates containing a large hydrophobic moiety and two negative charges (9, 10). GS-X pumps are also involved in the detoxification of heavy metals (13-15).Several recent studies indicate that the human MRP is one of these elusive GS-X pumps: (i) Overexpression of MRP in human cancer cells resulted in an increased ATP-dependent transport of GSH S-conjugates into isolated plasma membrane vesicles (16,17). (ii) Transfection of an MRP cDNA in pig kidney epithelial cells increased the basolateral export of a GSH S-conjugate (18). (iii) The export of anti-cancer drugs by MRP requires intracellular GSH (19,20). (iv) Overexpression of MRP increased the efflux of GSH from human lung canc...

Under phosphorous deficiency, plants of white lupin (Lupinus albus L.) develop root clusters, which are also called proteoid roots due to their preferential presence in the Proteaceae. In their mature stage, these roots acidify the soil and excrete high amounts of carboxylates [up to 1.5 and 7 micromol (g FW)(-1) h(-1) of malate and citrate, respectively] enabling lupins to utilise sparingly available sources of phosphate. Using the amplified fragment length polymorphism (AFLP) technique, we identified genes predominantly expressed in juvenile and mature cluster roots. Transcripts for two enzymes involved in glycolysis, fructokinase and phosphoglucomutase, were identified in juvenile cluster roots and one, sucrose synthase, in mature cluster roots. In order to verify these observations we performed quantitative reverse transcription-polymerase chain reaction (RT-PCR) and could confirm the increased transcript level. Measurements of enzymatic activities showed that fructokinase and phosphoglucomutase activities increased in juvenile cluster roots, whereas sucrose synthase activity was maximal in mature cluster roots. These results indicate that formation of proteoid roots and citrate excretion increase sink strength locally. Production of citrate and inhibition of respiration are likely to result in an increased NADH/NAD+ ratio, which may be toxic for the plant. The fermentation pathway would allow oxidation of NADH by decarboxylation of pyruvate and subsequent reduction of the resulting acetaldehyde. Determination of alcohol dehydrogenase activity showed that this enzyme is strongly induced in mature proteoid roots. However, ethanol production was not increased, indicating that pyruvate is shunted to citrate synthesis and not to ethanol production.