Functional analysis of plant magnesium transport proteins

1.分析了植物叶绿体中游离镁离子浓度的变化机理。测定了植物叶绿体内部的镁离子浓度，表征了叶绿体膜上的镁离子转运系统。我们指出，间质碱化可以在没有光照的情况下诱导间质[mg^&lt；2+&gt；]的增加。膜质子转运ATPase活性的一些参与H^+外流的抑制剂可抑制碱化引起的[mg^&lt；2+&gt；]的增加。2.膜上镁离子转运活性的测定。我们测定了镁离子转运蛋白重组到蛋白脂质体后的转运活性。由于放射性镁很难使用，因此使用荧光指示剂MAG-Fura-2来测量镁离子在磷脂膜上的转运活性。在MAG-Fura-2存在下制备脂质体。脂质体中MAG-Fura-2的保留率达30%以上。这使得利用MAG-Fura-2.3测量镁离子转运活性成为可能。重组植物镁离子转运蛋白我们表达了拟南芥的一种蛋白AtMRS2-10。拟南芥MRS2基因家族属于转运蛋白CORA超家族的一部分。我们已经在大肠杆菌细胞中表达了AtMRS2-10，并附加了6个His标签。重组蛋白经0.3%的肌糖溶解后纯化。这使得通过将纯化的蛋白重组到脂质体中并进行鉴定，对镁离子转运蛋白进行分子分析成为可能。

1.Analysis of changing mechanisms of free Mg^<2+> concentration ([Mg^<2+>]) in plant chloroplasts.We measured internal chloroplast [Mg^<2+>] and characterized Mg^<2+> transport systems in chloroplast membranes. We indicated that stromal alkalinization can induce an increase in stromal [Mg^<2+>] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H^+ efflux inhibited the alkalinization -induced increase in [Mg^<2+>].2.Measurement of Mg^<2+> transport activity across membrane.We have measured Mg^<2+> transport activity following the reconstitution of the Mg^<2+> transport protein into proteoliposomes. Since radioactive ^<28>Mg is difficult to use, Mg^<2+> transport activity across phospholipid membrane is measured using a fluorescent indicator, mag-fura-2. Liposomes were constituted in the presence of mag-fura-2. More than 30% of mag-fura-2 was retained into liposome. This makes measurement of Mg^<2+> transport activity possible using mag-fura-2.3.Recombinant plant Mg^<2+> transport proteinsWe have expressed an Arabidopsis thaliana protein, AtMRS2-10. The Arabidopsis thaliana MRS2 gene family belongs to a subset of the CorA super-family of Mg^<2+> transport proteins. We have expressed AtMRS2-10 with an additional 6 x His tag in Escherichia coli cells. The recombinant protein was solubilized from the E.coli membrane fraction by 0.3% sarkosyl and then purified. This makes molecular analysis of Mg^<2+> transport proteins possible by reconstitution of the purified proteins into liposomes and characterization.