Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin
Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin
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DOI：
10.1152/ajpcell.00154.2011
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发表时间：
2011-12-01
影响因子：
5.5
通讯作者：
Kanazawa, Hiroshi
中科院分区：
文献类型：
--
作者：
Lou Xinhan;Matsushita, Masafumi;Kanazawa, Hiroshi
张文，张文. Na +/H+交换器亚型6（NHE6/SLC9A6）参与转铁蛋白的网格蛋白依赖性内吞作用。美国生理学杂志细胞生理学301：C1431-C1444，2011年。首次发表于2011年8月31日; doi：10.1152/ajpcell.00154.2011。在哺乳动物细胞中，已知Na +/H+交换器（NHE）的九种保守亚型对于细胞质和细胞器腔的pH调节是重要的。NHE 1 - 5定位于质膜，而NHE 6 - 9定位于不同的细胞器。NHE6主要定位于内体区室中，但也存在于质膜中。为了研究NHE6在内吞作用中的作用，我们建立了NHE6敲低的HeLa细胞，并分析了这种敲低对内吞事件的影响。敲低细胞中NHE6的表达水平降低至与对照细胞中所见水平的15%相似。转铁蛋白的摄取也减少。未发现对表皮生长因子的内吞作用或对霍乱毒素B亚单位的影响。此外，在NHE6敲低细胞中，发现转铁蛋白摄取在胞吞作用的早期阶段受到影响。显微镜分析显示，在2分钟后开始的内吞作用，共定位的NHE6，网格蛋白，和转铁蛋白被观察到，这表明NHE6被定位到内吞，网格蛋白包被的囊泡。此外，在敲除细胞，转铁蛋白阳性内体被酸化，但没有发现对细胞质pH值的影响。在细胞过度表达野生型NHE6，增加转铁蛋白摄取观察到，但没有这样的增加被认为是在细胞过度表达突变NHE6缺乏离子转运。转铁蛋白阳性内体的管腔pH值在过表达野生型NHE6的细胞中碱化，但在过表达突变型NHE6的细胞中正常。这些观察结果表明，NHE6通过pH调节调节转铁蛋白的网格蛋白依赖性内吞作用。
Xinhan L, Matsushita M, Numaza M, Taguchi A, Mitsui K, Kanazawa H. Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin. Am J Physiol Cell Physiol 301: C1431-C1444, 2011. First published August 31, 2011; doi:10.1152/ajpcell.00154.2011.-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 similar 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.