Coupaye-Gerard B, Zuckerman J B, Duncan P, Bortnik A, Avery D I, Ernst S A, Kleyman T R
Department of Medicine, University of Pennsylvania, Philadelphia, USA.
Am J Physiol. 1997 Jun;272(6 Pt 1):C1781-9. doi: 10.1152/ajpcell.1997.272.6.C1781.
Na(+)-K(+)-ATPase is localized to the basolateral cell surface of most epithelial cells. Conflicting results regarding the intracellular trafficking of Na(+)-K(+)-ATPase in Madin-Darby canine kidney cells have been reported, with delivery to both apical and basolateral membranes or exclusively to the basolateral cell surface. We examined the delivery and steady-state distribution of Na(+)-K(+)-ATPase in the amphibian epithelial cell line A6 using an antibody raised against Na(+)-K(+)-ATPase alpha-subunit and sulfo-N-hydroxysuccinimidobiotin to tag cell surface proteins. The steady-state distribution of the Na(+)-K(+)-ATPase was basolateral, as confirmed by immunocytochemistry. Delivery of newly synthesized Na(+)-K(+)-ATPase to the cell surface was examined using [35S]methionine and [35S]cysteine in a pulse-chase protocol. After a 20-min pulse, the alpha-subunit and core glycosylated beta-subunit were present at both apical and basolateral cell surfaces. The alpha-subunit and core glycosylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na(+)-K(+)-ATPase alpha-subunit and core glycosylated beta-subunits are not retained in the endoplasmic reticulum of A6 cells and apparently lack sorting signals. Mature Na(+)-K(+)-ATPase is targeted to the basolateral surface, suggesting that basolateral targeting of the protein is conformation dependent.
钠钾ATP酶定位于大多数上皮细胞的基底外侧细胞表面。关于钠钾ATP酶在麦迪逊-达比犬肾细胞内的运输，已有相互矛盾的报道，即它会运输到顶端和基底外侧膜，或者仅运输到基底外侧细胞表面。我们使用针对钠钾ATP酶α亚基的抗体和磺基-N-羟基琥珀酰亚胺生物素标记细胞表面蛋白，来研究两栖类上皮细胞系A6中钠钾ATP酶的运输和稳态分布。免疫细胞化学证实，钠钾ATP酶的稳态分布是在基底外侧。采用脉冲追踪实验，使用[35S]甲硫氨酸和[35S]半胱氨酸来检测新合成的钠钾ATP酶向细胞表面的运输。经过20分钟的脉冲后，α亚基和核心糖基化的β亚基出现在顶端和基底外侧细胞表面。运输到顶端细胞表面的α亚基和核心糖基化的β亚基在2小时内被降解。在1至24小时的追踪后，成熟的α/β异二聚体几乎只在基底外侧表面被发现。这些数据表明，未成熟的钠钾ATP酶α亚基和核心糖基化的β亚基不会保留在A6细胞的内质网中，而且显然缺乏分选信号。成熟的钠钾ATP酶靶向基底外侧表面，这表明该蛋白的基底外侧靶向是依赖于构象的。
