Brown, Donna Anne; (2001) The cloning and characterisation of a multidrug resistance associated protein (MRP) from wheat. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Multidrug resistance associated protein (MRP) is the prototype of an ATP Binding Cassette (ABC) transporter subfamily first identified in a drug resistant lung cancer cell line. In plants, MRP homologues mediate ATP-dependent transport of glutathione conjugates and are thought to play a role in the metabolism of herbicides and endogenously-generated toxicants, by sequestering conjugates in the vacuole. MRP homologues have been cloned from the model plant Arabidopsis but little is known about the MRP family in cereals. The aim of this thesis therefore, was to isolate MRP homologues from wheat.Since MRP is induced in Arabidopsis by a range of xenobiotics, selective inducibility was employed as a tool to facilitate cloning of wheat MRP isoforms. The amino acid sequences of Arabidopsis MRPs were aligned and degenerate primers corresponding to conserved regions were designed. These primers were used in RT-PCR reactions containing total RNA from xenobiotic-treated wheat tissue as a template. A 791 bp fragment obtained was used to screen a cDNA library from xenobiotic-treated wheat shoots. A full-length cDNA containing an ORF of 4416 bp, encoding a protein with 60 % amino acid identity to AtMRP3 was obtained and designated TaMRPl. Hydropathy analysis indicated a similar gross topology to other members of the MRP subfamily, with a hydrophobic N-terminal extension and two transmembrane domains separated by two nucleotide binding folds. Northern analysis indicated that transcript levels of TaMRP2 were induced up to 3- fold by aminotriazole, menadione and phenobarbital. TaMRP2 was expressed in the Saccharomyces cerevisiae mutant, [delta]ycfl, which lacks the tonoplast MRP homologue, YCFl, and was shown to restore resistance to cadmium. It was also shown that expression of TaMRP2 in [delta]ycfl alleviated the toxic effects of the glutathione S-transferase substrate l-chloro-2,4-dinitrobenzene. These data suggest that TaMRP2 is a glutathione conjugate transporter which can also transport cadmium.

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