Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters
Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na+/K+ translocating AtHKT1 protein, a member of the superfamily of K+ transporters
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DOI：
10.1073/pnas.101556598
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发表时间：
2001-05-22
影响因子：
11.1
通讯作者：
Uozumi, N
中科院分区：
文献类型：
--
作者：
Kato, Y;Sakaguchi, M;Uozumi, N
拟南芥 AtHKT1 蛋白是一种 Na+/K+ 转运蛋白，能够介导非洲爪蟾卵母细胞中的内向 Na+ 电流和大肠杆菌中的 K+ 吸收。 HKT1 蛋白是 K+ 转运蛋白超家族的成员。这些蛋白质被认为含有八个跨膜片段和四个成孔区域，其排列方式类似于 K+ 通道四聚体。然而，对 AtHKT1 序列的计算机分析发现了 11 个潜在的跨膜片段。我们使用三种不同的技术研究了 AtHKT1 的膜拓扑结构。首先，在大肠杆菌中进行的基因融合碱性磷酸酶研究清楚地定义了 AtHKT1 N 端和中间区域的拓扑结构，但必须完善 C 端区域的膜折叠模型。其次，利用补充有狗胰腺微粒体的网织红细胞裂解物，我们证明 N-糖基化发生在 AtHKT1 的 429 位。工程化的非糖基化蛋白变体 N429Q 在非洲虎卵母细胞中介导 Na+ 电流，具有与野生型蛋白相同的特征，表明 N-糖基化对于 AtHKT1 的功能表达和膜靶向不是必需的。N429Q 中引入了五个潜在的糖基化位点。他们的糖基化模式支持基于大肠杆菌碱性磷酸酶数据的模型。第三，在HEK293细胞中用FLAG标记的AtHKT1进行的免疫细胞化学实验表明，AtHKT1的N和C末端以及含有残基135-142和377-384的区域面向胞质溶胶，而残基55-62的区域暴露于外部。综上所述，我们的结果表明 AtHKT1 包含八个跨膜片段。
The Arabidopsis thaliana AtHKT1 protein, a Na+/K+ transporter, is capable of mediating inward Na+ currents in Xenopus laevis oocytes and K+ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K+ transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K+ channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na+ currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1, Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135-142 and 377-384, face the cytosol, whereas the region of residues 55-62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.