Numata M, Petrecca K, Lake N, Orlowski J
Department of Physiology, McGill University, Montréal, Québec H3G 1Y6, Canada.
J Biol Chem. 1998 Mar 20;273(12):6951-9. doi: 10.1074/jbc.273.12.6951.
The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.
质子与Na⁺和K⁺在线粒体内膜上进行的电中性交换有助于细胞器体积和Ca²⁺稳态。这些转运蛋白的分子特性仍不清楚。在本报告中，我们鉴定了酿酒酵母中的一个新基因（YDR456w；重命名为NHA2），其推导的蛋白质序列与哺乳动物Na⁺/H⁺交换器基因家族的成员同源。荧光显微镜显示，Nha2-绿色荧光蛋白嵌合体与线粒体DNA的4',6-二脒基-2-苯基吲哚染色共定位。为了评估Nha2的功能，我们通过同源破坏删除了NHA2基因，发现突变株中线粒体对苯甲咪抑制的、酸激活的²²Na⁺摄取被消除。与亲本菌株相比，它在非发酵碳源上的生长也受到抑制，并且在细胞周期的静止期存活率严重降低，这与有氧代谢缺陷一致。序列比较显示，Nha2与人类中一个假定的Na⁺/H⁺交换器同源物（KIAA0267；重命名为NHE6）具有最高的同源性。Northern印迹分析表明，NHE6在各处均有表达，但在富含线粒体的组织如脑、骨骼肌和心脏中最为丰富。荧光显微镜显示，NHE6-绿色荧光蛋白嵌合体也在转染的HeLa细胞的线粒体中积累。这些数据表明，NHA2和NHE6编码同源的Na⁺/H⁺交换器，并表明它们可能分别对低等和高等真核生物的线粒体功能很重要。
