title: A cytoplasmic region of the Na,K-ATPase alpha-subunit is necessary for specific alpha/alpha association.
abstract: 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.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Koster
authors.forename: G
authors.initials: G
authors.lastname: Blanco
authors.forename: R W
authors.initials: RW
authors.lastname: Mercer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology*; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
chemical_list: D000254:Sodium-Potassium-Exchanging ATPase