Gatto C, McLoud S M, Kaplan J H
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
Am J Physiol Cell Physiol. 2001 Sep;281(3):C982-92. doi: 10.1152/ajpcell.2001.281.3.C982.
The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na(+)-K(+)-ATPase activity. We expressed sheep kidney Na(+)-K(+)-ATPase alpha- and beta-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na(+)-K(+)-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8-2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na(+) pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [(3)H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na(+)-K(+)-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive (86)Rb(+) uptake in whole cells as a means to specifically evaluate Na(+)-K(+)-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the alpha-subunit or beta-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the alpha-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the beta-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the alpha-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the alpha-beta heterodimer within the endoplasmic reticulum apparently does not require a Na(+) pump-specific chaperone.
钠钾ATP酶是一种异源二聚体质膜蛋白，负责几乎所有动物细胞的细胞离子稳态。研究表明，一些昆虫细胞（如High Five细胞）没有（或极低）钠钾ATP酶活性。我们通过杆状病毒表达系统在High Five细胞中分别或共同表达绵羊肾钠钾ATP酶的α亚基和β亚基。我们使用定量斑点印迹分析确定，在这些细胞中，表达的钠钾ATP酶占总膜蛋白的0.5%至2%。使用五步蔗糖梯度（0.8 - 2.0 M）分离内质网、高尔基体和质膜组分，我们在每个膜组分中观察到功能性钠泵分子并对其特性进行了表征。几乎所有表达的蛋白功能正常，类似于在纯化的犬肾酶制剂中发现的情况。因此，这里描述的测量没有因大量无功能的异源表达酶而变得复杂。具体而言，对每个组分测量了哇巴因敏感的ATP酶活性、[³H]哇巴因结合以及阳离子依赖性。钠钾ATP酶在内质网组装后其功能特性基本未改变。此外，我们测量了全细胞中哇巴因敏感的⁸⁶Rb⁺摄取，以此作为特异性评估正确折叠并转运到质膜的钠钾ATP酶分子的一种方法。当单独表达α亚基或β亚基时，我们无法测量到任何哇巴因敏感活性。对单独的膜组分进行免疫染色表明，单独表达时，α亚基在蛋白质成熟途径（即内质网）早期被降解，但β亚基正常加工并转运到质膜。因此，似乎只有α亚基在成熟和转运到质膜方面有寡聚体需求。此外，内质网内α - β异源二聚体的组装显然不需要钠泵特异性伴侣蛋白。
