Funktionelle Charakterisierung der CDF ("cation diffusion facilitator")-Familie von Metalltransportern in A. thaliana

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Plants and humans require adequate amounts of micronutrients like iron and zinc, but accumulation of an excess or uptake of chemically related non-essential metals like cadmium or lead can be extremely harmful. Proteins of the CDF (cation diffusion facilitator) family are involved in the homeostasis Cd2+, Co2+, Fe2+ and Zn2+ in bacteria, yeasts, animals, and - most probably - plants. Our aim is to elucidate the role of the at least nine CDF proteins in Arabidopsis thaliana. Complementary DNAs will be functionally expressed in appropriate mutants of Saccharomyces cerevisiae to test their function. Three to four representative Arabidopsis CDFs will be selected for the identification of knockout mutants and the use of RNA interference technology. Regulation and localization of these CDFs will be investigated by expressing promoter:GUS fusions and epitope-tagged fusion proteins in A. thaliana, and by using specific antibodies. There is experimental evidence for an interaction between a mammalian CDF and an unknown protein (Murgia et al. 1999). Such interactions might be vital for CDF function because their substrate metal cations are presumably bound to metallochaperone proteins in the cytoplasm. The selected CDF family members or fragments thereof will be used as a bait in both classical yeast two-hybrid and yeast split-ubiquitin screens, the latter specially suited for membrane proteins.