Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai, 980-8579, Japan.
RIKEN Center for Sustainable Resource Science, Wako, Saitama, 351-0198, Japan.
Sci Rep. 2019 Jul 11;9(1):10040. doi: 10.1038/s41598-019-46463-7.
Arabidopsis thaliana contains the putative K efflux transporters KEA1-KEA6, similar to KefB and KefC of Escherichia coli. KEA1-KEA3 are involved in the regulation of photosynthetic electron transport and chloroplast development. KEA4-KEA6 mediate pH regulation of the endomembrane network during salinity stress. However, the ion transport activities of KEA1-KEA6 have not been directly characterized. In this study, we used an E. coli expression system to examine KEA activity. KEA1-KEA3 and KEA5 showed bi-directional K transport activity, whereas KEA4 and KEA6 functioned as a K uptake system. The thylakoid membrane-localized Na/H antiporter NhaS3 from the model cyanobacterium Synechocystis is the closest homolog of KEA3. Changing the putative Na/H selective site of KEA3 (Gln-Asp) to that of NhaS3 (Asp-Asp) did not alter the ion selectivity without loss of K transport activity. The first residue in the conserved motif was not a determinant for K or Na selectivity. Deletion of the possible nucleotide-binding KTN domain from KEA3 lowered K transport activity, indicating that the KTN domain was important for this function. The KEA3-G422R mutation discovered in the Arabidopsis dpgr mutant increased K transport activity, consistent with the mutant phenotype. These results indicate that Arabidopsis KEA1-KEA6 act as K transport systems, and support the interpretation that KEA3 promotes dissipation of ΔpH in the thylakoid membrane.
拟南芥中含有 K 外排转运蛋白 KEA1-KEA6，与大肠杆菌的 KefB 和 KefC 相似。KEA1-KEA3 参与光合作用电子传递和叶绿体发育的调节。KEA4-KEA6 介导盐胁迫下内质网网络的 pH 调节。然而，KEA1-KEA6 的离子转运活性尚未直接表征。在这项研究中，我们使用大肠杆菌表达系统来研究 KEA 的活性。KEA1-KEA3 和 KEA5 表现出双向 K 转运活性，而 KEA4 和 KEA6 作为 K 摄取系统发挥作用。模式蓝藻集胞藻的类 thylakoid 膜定位的 Na/H 反向转运蛋白 NhaS3 是 KEA3 的最接近同源物。改变 KEA3 的假定 Na/H 选择性位点（Gln-Asp）为 NhaS3 的 Asp-Asp 不会改变离子选择性而不丧失 K 转运活性。保守基序中的第一个残基不是 K 或 Na 选择性的决定因素。从 KEA3 中缺失可能的核苷酸结合 KTN 结构域会降低 K 转运活性，表明 KTN 结构域对该功能很重要。在拟南芥 dpgr 突变体中发现的 KEA3-G422R 突变增加了 K 转运活性，与突变体表型一致。这些结果表明，拟南芥 KEA1-KEA6 作为 K 转运系统发挥作用，并支持 KEA3 促进类囊体膜中 ΔpH 耗散的解释。
