Effect of block deletions in the C-terminus on the functional expression of human anion exchanger 1 (AE1)
Effect of block deletions in the C-terminus on the functional expression of human anion exchanger 1 (AE1)
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DOI：
10.1080/09687860600945263
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Fu, Guo-Hui
中科院分区：
文献类型：
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作者：
Wang, Yong;Wu, Shao-Fang;Fu, Guo-Hui
人阴离子交换蛋白1（AE 1）是红细胞中最丰富的膜蛋白，负责Cl-与HCO 3-的交换。然而，AE 1 C-末端区域在阴离子易位和膜运输过程中所起的详细作用仍不清楚。本论文通过对人AE 1基因C端Ala（891）-Phe（895）、AsP 896-Glu（899）、Asp（902）-Glu（906）和瓦尔（907）-瓦尔（911）四个氨基酸残基的缺失，构建了四个突变体，以研究这些序列在AE 1功能表达中的作用。WT AE 1及其缺失突变体构建体在HEK 293细胞中表达。Western blotting结果显示，Ala（891）-Phe（895），Asp（896）-Glu（899），瓦尔（907）-瓦尔（911）缺失可诱导AE 1的高表达，而Asp（902）-Glu（906）缺失则导致稳定的低表达。WT AE 1及其突变体的脉冲追踪试验表明，蛋白质的稳定性不受AE 1及其突变体的表达水平的影响。与WT AE 1相比，Ala（891）-Phe（895）、Asp（902）-Glu（906）和瓦尔（907）-瓦尔（911）突变体表现出更低水平的质膜转运，而Asp（896）-Glu（899）突变体在质膜上的表达更高。突变体介导转染细胞中Cl-/HCO 3-交换的能力降低，表明缺失序列在转运活性中具有重要作用。这些结果表明，在AE 1的C-末端的研究残基不同地影响表达，膜运输和功能折叠的AE 1。
The human anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is responsible for the exchange of Cl- for HCO3-. However, the detailed role played by the AE1 C-terminal region in the anion translocation and membrane trafficking process remains unclear. In this paper, we created four mutants in the human AE1 C-terminus by deletion of the residues Ala(891)-Phe(895), AsP896-Glu(899), Asp(902)-Glu(906) and Val(907)-Val(911), to investigate the role of these sequences in functional expression of AE1. WT AE1 and its deletion mutant constructs were expressed in HEK293 cells. Western blotting showed that deletions of Ala(891)-Phe(895), Asp(896)-Glu(899), and Val(907)-Val(911) induced high expression of AE1, whereas loss of Asp(902)-Glu(906) results in stable low expression. Pulse chase assays of WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala(891)-Phe(895), Asp(902)-Glu(906) and Val(907)-Val(911) mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp(896)-Glu(899) mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl-/HCO3- exchange in transfected cells revealed that the deletion sequences have an important role in transport activity. These results demonstrate that the studied residues in the AE1 C-terminus differently affect the expression, membrane trafficking and functional folding of AE1.