Department of Biological Sciences, Graduate School of Science, Osaka University, Machikaneyama-cho 1-1, Toyonaka City, Osaka, Japan.
Am J Physiol Cell Physiol. 2011 Dec;301(6):C1431-44. doi: 10.1152/ajpcell.00154.2011. Epub 2011 Aug 31.
In mammalian cells, nine conserved isoforms of the Na(+)/H(+) exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1-5 are localized to the plasma membrane, whereas NHE6-9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ∼15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrin-dependent endocytosis of transferrin via pH regulation.
在哺乳动物细胞中，已知有 9 种保守的 Na(+)/H(+)交换器 (NHE) 同工型对于细胞质和细胞器腔的 pH 调节很重要。NHE1-5 定位于质膜，而 NHE6-9 则定位于不同的细胞器。NHE6 主要定位于内体隔室，但也存在于质膜中。为了研究 NHE6 在胞吞作用中的作用，我们建立了 NHE6 敲低的 HeLa 细胞，并分析了这种敲低对胞吞作用的影响。敲低细胞中 NHE6 的表达水平下降到对照细胞的约 15%。转铁蛋白的摄取也减少了。对表皮生长因子或霍乱毒素 B 亚基的内吞作用没有影响。此外，在 NHE6 敲低的细胞中，发现转铁蛋白的摄取在胞吞作用的早期阶段受到影响。显微镜分析显示，在胞吞作用开始后 2 分钟，观察到 NHE6、网格蛋白和转铁蛋白的共定位，这表明 NHE6 定位于胞吞作用的网格蛋白包被小泡。此外，在敲低细胞中，转铁蛋白阳性内体被酸化，但细胞质 pH 没有受到影响。在过表达野生型 NHE6 的细胞中，观察到转铁蛋白摄取增加，但在过表达缺乏离子转运功能的突变型 NHE6 的细胞中没有观察到这种增加。在过表达野生型 NHE6 的细胞中，转铁蛋白阳性内体的腔内 pH 被碱化，但在过表达突变型 NHE6 的细胞中，腔内 pH 正常。这些观察结果表明，NHE6 通过 pH 调节来调节网格蛋白依赖性的转铁蛋白内吞作用。
