Vagin Olga, Turdikulova Shahlo, Sachs George
Department of Physiology, School of Medicine at UCLA, Los Angeles, California 90073, USA.
J Biol Chem. 2005 Dec 30;280(52):43159-67. doi: 10.1074/jbc.M508262200. Epub 2005 Oct 17.
In most polarized cells, the Na,K-ATPase is localized on the basolateral plasma membrane. However, an unusual location of the Na,K-ATPase was detected in polarized HGT-1 cells (a human gastric adenocarcinoma cell line). The Na,K-ATPase alpha1 subunit was detected along with the beta2 subunit predominantly on the apical membrane, whereas the Na,K-ATPase beta1 subunit was not found in HGT-1 cells. However, when expressed in the same cell line, a yellow fluorescent protein-linked Na,K-ATPase beta1 subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous alpha1 subunit to the basolateral membrane. The human beta2 subunit has eight N-glycosylation sites, whereas the beta1 isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the beta2 subunit were successively introduced in the beta1 subunit by site-directed mutagenesis. The mutated beta1 subunits were detected on both apical and basolateral membranes. The fraction of a mutant beta1 subunit present on the apical membrane increased in proportion to the number of glycosylation sites inserted and reached 80% of the total surface amount for the beta1 mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous beta2 subunit and the beta1 mutant with additional glycosylation sites but not for the wild type beta1 subunit. Hence, the N-glycans linked to the beta2 subunit of the Na,K-ATPase contain apical sorting information, and the high abundance of the beta2 subunit isoform, which is rich in N-glycans, along with the absence of the beta1 subunit, is responsible for the unusual apical location of the Na,K-ATPase in HGT-1 cells.
在大多数极化细胞中，钠钾ATP酶定位于基底外侧质膜。然而，在极化的HGT-1细胞（一种人胃腺癌细胞系）中检测到钠钾ATP酶的位置异常。钠钾ATP酶α1亚基与β2亚基一起主要在顶端膜上被检测到，而钠钾ATP酶β1亚基在HGT-1细胞中未被发现。然而，当在同一细胞系中表达时，一种与黄色荧光蛋白相连的钠钾ATP酶β1亚基仅定位于基底外侧表面，并导致内源性α1亚基部分重新分布到基底外侧膜。人β2亚基有8个N-糖基化位点，而β1亚型只有3个。因此，通过定点诱变，在β1亚基中依次引入了多达5个与β2亚基中存在的位点同源的额外N-糖基化位点。突变的β1亚基在顶端膜和基底外侧膜上均被检测到。存在于顶端膜上的突变β1亚基的比例与插入的糖基化位点数量成比例增加，对于具有5个额外位点的β1突变体，该比例达到总表面量的80%。通过共聚焦显微镜检测到内源性β2亚基和具有额外糖基化位点的β1突变体与小窝蛋白-1呈聚集分布并共定位，但野生型β1亚基未出现这种情况。因此，与钠钾ATP酶β2亚基相连的N-聚糖包含顶端分选信息，富含N-聚糖的β2亚基亚型的高丰度以及β1亚基的缺失，导致了HGT-1细胞中钠钾ATP酶异常定位于顶端。
