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集胞藻PCC 6803类囊体膜上Na+/H+逆向转运蛋白Nhas3的鉴定与特性分析

Identification and characterization of the Na+/H+ antiporter Nhas3 from the thylakoid membrane of Synechocystis sp. PCC 6803.

作者信息

Tsunekawa Kenta, Shijuku Toshiaki, Hayashimoto Mitsuo, Kojima Yoichi, Onai Kiyoshi, Morishita Megumi, Ishiura Masahiro, Kuroda Teruo, Nakamura Tatsunosuke, Kobayashi Hiroshi, Sato Mayuko, Toyooka Kiminori, Matsuoka Ken, Omata Tatsuo, Uozumi Nobuyuki

机构信息

Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601.

From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579.

出版信息

J Biol Chem. 2009 Jun 12;284(24):16513-16521. doi: 10.1074/jbc.M109.001875. Epub 2009 Apr 16.

Abstract

Na+/H+ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na+/H+ antiporters, nhaS1-5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na+/H+ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na+ concentrations. NhaS3 had no K+/H+ exchange activity itself but enhanced K+ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO2 to high CO2 conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO2 consumption caused by the ceasing of O2-evolving photosynthesis. This is the first report of a Na+/H+ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H+, Na+, and K+ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.

摘要

钠/氢反向转运蛋白影响跨膜质子或钠动力势。集胞藻PCC 6803有六个编码钠/氢反向转运蛋白的基因,即nhaS1 - 5和sll0556。在本研究中,对NhaS3的功能进行了检测。NhaS3对集胞藻的生长至关重要,大肠杆菌钠/氢反向转运蛋白NhaA的表达不能弥补nhaS3的缺失。通过免疫印迹和免疫金标记进行膜分级分离显示,NhaS3定位于集胞藻的类囊体膜中。当在大肠杆菌中表达时,NhaS3在pH 6.5至9.0的范围内具有功能。集胞藻基因组中nhaS3拷贝数的减少使细胞对高钠浓度更敏感。NhaS3本身没有钾/氢交换活性,但在大肠杆菌钾吸收突变体中表达时可增强从培养基中吸收钾的能力。从低二氧化碳条件转变为高二氧化碳条件后,nhaS3的表达增加。还发现nhaS3的表达受昼夜节律控制。基因表达在主观夜间开始时达到峰值。这与由于放氧光合作用停止导致二氧化碳消耗速率最低的时间相吻合。这是关于定位于类囊体膜中的钠/氢反向转运蛋白的首次报道。我们的结果表明,NhaS3在维持H⁺、Na⁺和K⁺的离子稳态以支持光合产物的转化以及在黑暗中提供能量方面发挥作用。

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