Functional analysis of plant magnesium transport proteins

1.植物叶绿体内游离Mg^<2+>浓度（[Mg^<2+]）变化机理的分析：测定了叶绿体内[Mg^<2+]浓度，并对叶绿体膜上Mg^<2+]转运系统进行了表征。我们发现基质碱化可以在无光照条件下诱导基质[Mg^<2+>]增加。一些参与H^+外排的包膜质子转运ATP酶活性抑制剂可抑制碱化诱导的[Mg^<2+>]增加。2. Mg^<2+>跨膜转运活性的测定我们测定了Mg ^<2 +>转运蛋白重组为脂蛋白体后的Mg^<2 +>转运活性。由于放射性<28>Mg很难使用，所以Mg^2+穿过磷脂膜的转运活性是用荧光指示剂mag-fura-2来测量的。脂质体在mag-fura-2的存在下构成。30%以上的mag-fura-2保留在脂质体中。3.重组植物Mg^2+转运蛋白我们表达了拟南芥AtMRS 2 -10蛋白。拟南芥MRS 2基因家族属于Mg^&lt;2+&gt;转运蛋白CorA超家族的一个子集。我们已经在大肠杆菌细胞中表达了带有额外6 x His标签的AtMRS 2 -10。用0.3%的肌氨酰从大肠杆菌膜组分中溶解重组蛋白，然后纯化。这使得Mg^2+转运蛋白的分子分析成为可能，通过将纯化的蛋白质重组到脂质体中并进行表征。

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.