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

植物和人类需要适量的微量元素，如铁和锌，但积累过量或吸收化学相关的非必需金属，如镉或铅，可能是极其有害的。CDF（阳离子扩散促进者）家族的蛋白质参与了细菌、酵母、动物和（很可能）植物中Cd2+、Co2+、Fe2+和Zn2+的稳态。我们的目的是阐明至少9个CDF蛋白在拟南芥中的作用。互补dna将在酿酒酵母的适当突变体中功能性表达，以测试其功能。将选择3 ~ 4个具有代表性的拟南芥CDFs进行敲除突变体的鉴定和RNA干扰技术的应用。这些CDFs的调控和定位将通过在拟南芥中表达启动子：GUS融合和表位标记融合蛋白，以及使用特异性抗体来研究。有实验证据表明，哺乳动物CDF与一种未知蛋白质之间存在相互作用（Murgia et al. 1999）。这种相互作用可能对CDF的功能至关重要，因为它们的底物金属阳离子可能与细胞质中的金属伴侣蛋白结合。选择的CDF家族成员或其片段将在经典酵母双杂交和酵母分裂泛素筛选中作为诱饵，后者特别适合膜蛋白。

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.