Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+-/H+ exchangers
Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+-/H+ exchangers
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DOI：
10.1042/0264-6021:3510241
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发表时间：
2000-10-01
影响因子：
4.1
通讯作者：
de Boer, AH
中科院分区：
文献类型：
--
作者：
Darley, CP;van Wuytswinkel, OCM;de Boer, AH
细胞质中高毫摩尔浓度的钠对植物和酵母细胞是有毒的。将Na+离子隔离到液泡中是赋予这些生物体Na+耐受性的一种机制。在本研究中，我们提供了直接的证据表明，拟南芥At-NHX 1基因和酵母NHX 1基因编码低亲和力电中性Na+/H+交换。我们利用异源表达的At-NHX 1的能力，在功能上补充酵母nhx 1无效突变体。从表达At-NHX 1或NHX 1的酵母中分离的液泡囊泡的实验为pH梯度激发的Na+积累到液泡中提供了直接证据。NHX 1和At-NHX 1之间的主要区别是前者基因中存在可切割的N-末端信号肽（SP）。SP与At-NHX 1的融合导致Na+/H+交换的幅度增加，表明SP在蛋白质靶向或调节中的作用。植物和酵母反向转运蛋白之间的另一个区别特征是它们对利尿化合物阿米洛利的敏感性。而At-NHX 1被阿米洛利完全抑制，NHX 1活性仅降低20- 40%。这些结果表明，酵母作为一种异源表达系统，提供了一个方便的模式来分析植物Na+/H+反向转运蛋白的结构和调控特性。
Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole is one mechanism to confer Na+-tolerance on these organisms. In the present study we provide direct evidence that the Arabidopsis thaliana At-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na+/H+ exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na+ accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na+/H+ exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20-40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na+/H+ antiporters.