Typically, Na+/H+ antiporters (NHXs) possess a conserved N-terminus for cation binding and exchange and a hydrophilic C-terminus for regulating the antiporter activity. Plant endosomal-type NHXs play important roles in protein trafficking, as well as K+ and vesicle pH homeostasis, however the role of the C-terminal tail remains unclear. Here, the function of MnNHX6, an endosomal-type NHX in mulberry, was investigated using heterologous expression in yeast. Functional and localization analyses of C-terminal truncation and mutations in MnNHX6 revealed that the C-terminal conserved region was responsible for the function and stability of the protein and its hydrophobicity, which is a key domain requirement. Nuclear magnetic resonance spectroscopy provided direct structural evidence and yeast two-hybrid screening indicated that this functional domain was also necessary for interaction with sorting nexin 1. Our findings demonstrate that although the C-terminal tail of MnNHX6 is intrinsically disordered, the C-terminal conserved region may be an important part of the external mouth of this transporter, which controls protein function and stability by serving as an inter-molecular cork with a chain mechanism. These findings improve our understanding of the roles of the C-terminal tail of endosomal-type NHXs in plants and the ion transport mechanism of NHX-like antiporters.

中文翻译：

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植物内体型 NHXs 的 C 端尾部在其功能和稳定性中起关键作用

通常，Na+/H+ 反向转运蛋白 (NHX) 具有用于阳离子结合和交换的保守 N 端和用于调节反向转运蛋白活性的亲水性 C 端。植物内体型 NHX 在蛋白质运输以及 K+ 和囊泡 pH 稳态中发挥重要作用，但 C 端尾部的作用尚不清楚。在这里，使用酵母中的异源表达研究了桑椹中的内体型 NHX MnNHX6 的功能。MnNHX6 中 C 端截断和突变的功能和定位分析表明，C 端保守区域负责蛋白质的功能和稳定性及其疏水性，这是一个关键的域要求。核磁共振波谱提供了直接的结构证据，酵母双杂交筛选表明该功能域也是与分选 nexin 1 相互作用所必需的。我们的研究结果表明，虽然 MnNHX6 的 C 端尾部本质上是无序的，但 C 端是保守的该区域可能是该转运蛋白外口的重要组成部分，通过作为具有链式机制的分子间软木塞来控制蛋白质功能和稳定性。这些发现提高了我们对植物中内体型 NHX 的 C 末端尾部的作用和 NHX 样逆向转运蛋白的离子转运机制的理解。C端保守区可能是该转运蛋白外口的重要组成部分，通过作为具有链式机制的分子间软木塞来控制蛋白质的功能和稳定性。这些发现提高了我们对植物中内体型 NHX 的 C 末端尾部的作用和 NHX 样逆向转运蛋白的离子转运机制的理解。C端保守区可能是这种转运蛋白外口的重要组成部分，它通过作为具有链式机制的分子间软木塞来控制蛋白质的功能和稳定性。这些发现提高了我们对植物中内体型 NHX 的 C 末端尾部的作用和 NHX 样逆向转运蛋白的离子转运机制的理解。