An optogenetic assay method for electrogenic transporters using Escherichia coli co‐expressing light‐driven proton pump
An optogenetic assay method for electrogenic transporters using Escherichia coli co‐expressing light‐driven proton pump
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DOI：
10.1002/pro.4154
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发表时间：
2021-07
期刊：
影响因子：
8
通讯作者：
M. Hayashi;Keiichi Kojima;Y. Sudo;A. Yamashita
中科院分区：
文献类型：
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作者：
M. Hayashi;Keiichi Kojima;Y. Sudo;A. Yamashita
在生物体中，营养物质和废物在细胞膜上移动，在细胞膜上嵌入的转运蛋白促进和抑制这种移动。尽管具有生理意义，但目前使用的转运蛋白活性测定方法需要繁琐的准备和分析过程。在这项研究中，我们报道了无同位素和无标签的电致转运体运输活动测量系统。在该系统中，在大肠杆菌细胞中共表达了两种分子，一种是光驱动向内质子泵紫红质XeR，另一种是代表电致转运蛋白草酸转运蛋白OxlT。在共表达XeR和OxlT的细胞的光照下，体溶液的pH值增加，并且草酸盐的加入显著增强了pH值变化的程度，这表明XeR的光诱导质子向内传输与OxlT的负电传输耦合。这种pH值的增加与草酸浓度有关，而与XeR表达水平无关。值得注意的是，OxlT、R272A和K355Q的无功能突变体没有观察到pH升高，这支持了该系统的有效性。因此，我们成功地开发了一种利用大肠杆菌共表达光驱动质子泵检测电致转运体的光遗传学方法。
In organisms, nutrients and wastes move across the cellular membrane, in which membrane‐embedded transporters facilitate and inhibit the movement. Despite the physiological significances, the currently used assay methods for transporter activities require tedious preparation and analytical processes. In this study, we report the isotope‐free and label‐free measurement system for the transport activities of electrogenic transporters. In the system, two molecules, a light‐driven inward proton pump rhodopsin, xenorhodopsin (XeR), and a representative of an electrogenic transporter, an oxalate transporter (OxlT), were co‐expressed in Escherichia coli cells. The light illumination of the cells co‐expressing XeR and OxlT showed an increase in the pH of the bulk solution and that the extent of the pH change is significantly enhanced by adding the oxalate, suggesting the light‐induced inward proton transport by XeR coupled to the negative electrogenic transport by OxlT. Such a pH increase was dependent on the oxalate concentration, but not on the XeR expression level. Of note, pH increase was not observed for the nonfunctional mutants of OxlT, R272A, and K355Q, supporting the validity of the system. Thus, we successfully developed an optogenetic assay method for electrogenic transporters using E. coli co‐expressing light‐driven proton pump.