A general method for determining secondary active transporter substrate stoichiometry
A general method for determining secondary active transporter substrate stoichiometry
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DOI：
10.7554/elife.21016.001
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发表时间：
2017-01-25
期刊：
影响因子：
7.7
通讯作者：
Mindell, Joseph A.
中科院分区：
文献类型：
--
作者：
Fitzgerald, Gabriel A.;Mulligan, Christopher;Mindell, Joseph A.
驱动底物转运通过次级活性转运蛋白所需的离子数量决定了蛋白质产生底物梯度的能力，这是其生理功能所必需的特征，并对转运蛋白机制施加了基本限制。化学计量学对于多种哺乳动物转运蛋白是已知的，但是，由于缺乏容易获得的工具，对于大多数可获得高分辨率结构的原核转运蛋白不是已知的。在这里，我们描述了一个一般的方法，使用放射性标记的底物通量测定，以确定耦合化学计量的产电二级活性转运蛋白重组蛋白脂质体通过测量转运平衡电位。我们证明了这种方法的实用性，通过确定耦合化学计量的VcINDY，细菌Na+-偶联琥珀酸转运蛋白，并进一步验证它通过确认的耦合化学计量的vSGLT，细菌糖转运蛋白。这种稳健的热力学方法在探测具有可用结构的转运蛋白的机制时特别有用。
The number of ions required to drive substrate transport through a secondary active transporter determines the proteins ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporters mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na+-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.