Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking
Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking
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DOI：
10.1042/bj20060388
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发表时间：
2006-08-15
影响因子：
4.1
通讯作者：
Rao, Rajini
中科院分区：
文献类型：
--
作者：
Mukherjee, Sanchita;Kallay, Laura;Rao, Rajini
酵母Nhx1 [Na+(K+)/H+交换器1]是细胞内Na+(K+)/H+交换器，定位于内核体晚期，对离子平衡和囊泡运输至关重要。对NHE （Na+/H+交换器）序列进行系统发育分析，鉴定出生理作用未知的同源蛋白，包括HsNHE6（人NHE6）、HsNHE7和HsNHE9。这些似乎不同于经过充分研究的哺乳动物质膜异构体（NHE1-NHE5）。为了探索质膜和细胞内NHEs之间的差异，并了解离子平衡与囊泡运输之间的联系，我们研究了在跨膜段9和10之间替换Nhx1膜内H10环中的残基的后果。Glu（355）的羧基在离子传输中的关键作用与该残基在所有NHEs中的不变性一致。令人惊讶的是，在细胞内同种异构体中特异性保守的残基（如Phe（357）和Tyr(361)）不能被质膜同种异构体中发现的密切相似的残基（亮氨酸和苯丙氨酸）所取代而不丧失功能，揭示出意想不到的侧链特异性。所有Nhx1突变体的转运表型，包括湿热霉素敏感性和羧基肽酶Y的错误分类，都被发现与pH稳态缺陷直接相关，可以通过弱碱滴定来按比例纠正。本研究证明了NHE家族的H10环的重要性，强调了质膜和细胞内同种异构体之间的差异，并表明运输缺陷与pH平衡紧密耦合。
Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu(355) in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe(357) and Tyr(361)) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.