Ishijima Sumio, Shigemi Zenpei, Adachi Hiroaki, Makinouchi Nana, Sagami Ikuko
Kyoto Prefectural University, Shimogamo, Kyoto, Japan.
Biochim Biophys Acta. 2012 Sep;1818(9):2202-8. doi: 10.1016/j.bbamem.2012.04.015. Epub 2012 Apr 26.
Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.
镁离子（Mg(2+)）在许多生理过程中发挥着关键作用。镁离子转运机制在细菌中已有充分记载；然而，真核生物中镁离子转运体的相关信息却知之甚少。拟南芥AtMRS2家族由10个基因组成，属于CorA超家族蛋白的真核生物亚组。该超家族中的蛋白质通过一个普遍保守的甘氨酸-甲硫氨酸-天冬酰胺基序得以鉴定，并被表征为镁离子转运体。AtMRS2家族的一些成员，包括AtMRS2-10，可能会补充缺乏镁离子转运能力的细菌突变体或酵母突变体。在此，我们报道了AtMRS2-10的纯化及其在脂质体中的功能重建。含有N端组氨酸标签的AtMRS2-10在大肠杆菌中表达，并用肌氨酸钠溶解。纯化后的AtMRS2-10蛋白被重建到脂质体中。AtMRS2-10以单向方向插入脂质体。对蛋白脂质体中镁离子摄取的直接测量表明，重建后的AtMRS2-10在没有任何辅助蛋白的情况下转运镁离子。GMN基序中的M400突变为I使镁离子摄取失活。AtMRS2-10介导的镁离子内流被六胺钴（III）阻断，且在pH值5至9的外部环境中不受影响。AtMRS2-10的活性受到钴离子（Co(2+)）和镍离子（Ni(2+)）的抑制；然而，它不受钙离子（Ca(2+)）、亚铁离子（Fe(2+)）或铁离子（Fe(3+)）的抑制。虽然这些结果表明AtMRS2-10与细菌CorA蛋白具有相似的特性，但与细菌CorA蛋白不同的是，AtMRS2-10受到铝离子（Al(3+)）的强烈抑制。这些研究证明了AtMRS2蛋白在蛋白脂质体中研究结构-功能关系的功能能力。
