Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor.
Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor.
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DOI：
10.1038/s41598-017-18146-8
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发表时间：
2017-12-21
影响因子：
4.6
通讯作者：
Chang G
中科院分区：
文献类型：
--
作者：
Doshi R;McGrath AP;Piñeros M;Szewczyk P;Garza DM;Kochian LV;Chang G
世界上约50%的耕地是强酸性的(pH ≤ 5)。低pH值使粘土矿物中的根毒性离子铝(Al3+)物种溶解，推动了栽培作物中反作用适应的进化。粮食作物高粱上调其根部嵌入膜的转运蛋白SbMATE。SbMATE介导有机酸的阴离子形式柠檬酸外流到土壤根际，螯合Al3+离子，从而通过将Al+3排除在生长中的根尖而增强抗铝性。在这里，我们在两个异源表达系统中使用电生理、放射性标记和基于荧光的转运分析来建立SbMATE的广泛底物识别图谱，显示了质子和/或钠驱动的14C-柠檬酸阴离子以及有机单价阳离子乙锭的转运，但不显示其二价类似物丙啶。我们通过测量底物与洗涤剂纯化的SbMATE蛋白的结合来进一步补充我们的转运试验。最后，我们使用纯化的膜蛋白作为抗原，利用一种无动物的mRNA/cDNA展示技术来发现天然的构象结合和运输功能改变的纳米体。我们的结果证明了利用巴斯德毕赤酵母作为有效的真核宿主来表达大量的植物转运蛋白。纳米体发现方法也适用于其他非免疫原性植物蛋白。
About 50% of the world’s arable land is strongly acidic (pH ≤ 5). The low pH solubilizes root-toxic ionic aluminium (Al3+) species from clay minerals, driving the evolution of counteractive adaptations in cultivated crops. The food crop Sorghum bicolor upregulates the membrane-embedded transporter protein SbMATE in its roots. SbMATE mediates efflux of the anionic form of the organic acid, citrate, into the soil rhizosphere, chelating Al3+ ions and thereby imparting Al-resistance based on excluding Al+3 from the growing root tip. Here, we use electrophysiological, radiolabeled, and fluorescence-based transport assays in two heterologous expression systems to establish a broad substrate recognition profile of SbMATE, showing the proton and/or sodium-driven transport of 14C-citrate anion, as well as the organic monovalent cation, ethidium, but not its divalent analog, propidium. We further complement our transport assays by measuring substrate binding to detergent-purified SbMATE protein. Finally, we use the purified membrane protein as an antigen to discover native conformation-binding and transport function-altering nanobodies using an animal-free, mRNA/cDNA display technology. Our results demonstrate the utility of using Pichia pastoris as an efficient eukaryotic host to express large quantities of functional plant transporter proteins. The nanobody discovery approach is applicable to other non-immunogenic plant proteins.
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影响因子：
4.6
作者：
Doshi R;Chen BR;Vibat CR;Huang N;Lee CW;Chang G
通讯作者：
Chang G
影响因子：
4.9
作者：
VONUEXKULL, HR;MUTERT, E
通讯作者：
MUTERT, E
影响因子：
3.2
作者：
He, GX;Kuroda, T;Tsuchiya, T
通讯作者：
Tsuchiya, T
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3.2
作者：
Otsuka, M;Yasuda, M;Moriyama, Y
通讯作者：
Moriyama, Y
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16.8
作者：
Lu, Min;Radchenko, Martha;Symersky, Jindrich;Nie, Rongxin;Guo, Yi
通讯作者：
Guo, Yi