Roberto Tommasini

Higher plants are equipped with a remarkably versatile system that protects them from the potentially phytotoxic actions of xenobiotics, i.e. synthetic chemicals present in the plant's environment. Particularly striking is the natural tolerance of certain plants toward herbicides that profoundly affect closely related species. This phenomenon of herbicide selectivity is widely exploited in agriculture to control competing weeds in a field of crop plants that are tolerant to the particular herbicide. Herbicide selectivity is, in most cases, based primarily on the differential ability of plant species to metabolically detoxify the herbicide (Lamoureux et al., 1991; Cole, 1994). Metabolic herbicide inactivation has been employed in the genetic engineering of crops for herbicide tolerance (Hinchee et al., 1993). Finally, enhanced herbicide detoxification is one of the mechanisms of herbicide resistance, in addition to altered target site susceptibility and yet-unknown mechanisms, that may emerge within formerly susceptible weed species upon continuous use of the same herbicide or herbicide class (Holt et al., 1993). It is well documented that plants are able to metabolize and detoxify herbicides by a variety of enzymatic reactions and with extraordinary diversity among species. Furthermore, recent research has revealed that transporters in the vacuolar membrane mediate the energydependent export of herbicide metabolites into the large central vacuole (Martinoia et al., 1993).

An 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 glutathione-conjugate 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.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) 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 S-conjugates 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.