Koster J C, Blanco G, Mercer R W
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1995 Jun 16;270(24):14332-9. doi: 10.1074/jbc.270.24.14332.
While most structural studies of the Na,K-ATPase support a subunit stoichiometry of one alpha-subunit to one beta-subunit, the exact quaternary structure of the Na,K-ATPase and its relevance to enzyme function is the subject of much debate. Formation of a higher order enzyme complex is supported by our previous study demonstrating specific alpha/alpha interactions among the rat Na,K-ATPase isoforms (alpha 1, alpha 2, alpha 3), expressed in virally infected Sf-9 insect cells and among native alpha isoforms in rat brain (1). This detergent-resistant association was not observed in insect cells coexpressing the homologous gastric H,K-ATPase alpha-subunit, nor was it dependent on the coexpression of the beta-subunit. To delineate domains necessary for alpha/alpha assembly, a series of H,K-ATPase-Na, K-ATPase chimerase were constructed by combining the N-terminal, cytoplasmic midregion and C-terminal segments derived from the Na,K-ATPase (N) and the H,K-ATPase (H) alpha-polypeptides (HNN, HNH, NHH, NHN, and HHN). The alpha-subunit chimeras were coexpressed with the Na,K-ATPase alpha 1-subunit in Sf-9 cells using the baculovirus expression system. Specific and detergent-stable association is observed between the Na,K-ATPase alpha-subunit and the HNN and HNH chimeras, but not with the NHH, NHN, or HHN chimeras. Consistent with the Na,K-ATPase cytoplasmic domain as being necessary for alpha/alpha interactions, the full-length alpha-subunit stably associates with an alpha N-terminal deletion mutant (delta Gly2-Leu273), but not with an alpha cytoplasmic deletion mutant (delta Arg350-Pro785). In addition, the naturally occurring C-terminal truncated alpha 1 isoform, alpha 1T (delta Gly554 to C terminus), does not associated with the alpha 1-subunit in Sf-9 cells coexpressing both polypeptides. thus, a cytoplasmic region in the alpha-subunit (Gly554-Pro785) is necessary for specific alpha/alpha association. The same cytoplasmic region contains a strongly hydrophobic segment that, by analogy with oligomerization of water-soluble proteins, may form the interface of the extramembranous alpha/alpha contact site.
虽然大多数关于钠钾-ATP酶的结构研究支持一个α亚基与一个β亚基的亚基化学计量比，但钠钾-ATP酶的确切四级结构及其与酶功能的相关性仍是众多争论的主题。我们之前的研究支持形成更高阶的酶复合物，该研究表明在病毒感染的Sf-9昆虫细胞中表达的大鼠钠钾-ATP酶同工型（α1、α2、α3）之间以及大鼠脑中的天然α同工型之间存在特定的α/α相互作用（1）。在共表达同源胃H⁺，K⁺-ATP酶α亚基的昆虫细胞中未观察到这种抗去污剂的缔合，它也不依赖于β亚基的共表达。为了确定α/α组装所需的结构域，通过组合来自钠钾-ATP酶（N）和H⁺，K⁺-ATP酶（H）α多肽的N端、胞质中区和C端片段构建了一系列H⁺，K⁺-ATP酶-钠钾-ATP酶嵌合体（HNN、HNH、NHH、NHN和HHN）。使用杆状病毒表达系统在Sf-9细胞中将α亚基嵌合体与钠钾-ATP酶α1亚基共表达。在钠钾-ATP酶α亚基与HNN和HNH嵌合体之间观察到特异性且抗去污剂稳定的缔合，但与NHH、NHN或HHN嵌合体未观察到这种缔合。与钠钾-ATP酶胞质结构域是α/α相互作用所必需的一致，全长α亚基与α N端缺失突变体（ΔGly2-Leu273）稳定缔合，但与α胞质缺失突变体（ΔArg350-Pro785）未稳定缔合。此外，天然存在的C端截短的α1同工型α1T（ΔGly554至C端）在共表达这两种多肽的Sf-9细胞中不与α1亚基缔合。因此，α亚基中的一个胞质区域（Gly554-Pro785）是特异性α/α缔合所必需的。同一胞质区域包含一个强疏水片段，类似于水溶性蛋白质的寡聚化，它可能形成膜外α/α接触位点的界面。
