Subunit assembly of the sodium,potassium-ATPase

The Na,K-ATPase is an integral membrane protein that maintains concentration gradients of Na+ and K+ ions across the plasma membrane. These ionic gradients are important for cellular homeostasis, excitability and solute transport. The minimum functional Na,K-ATPase consists of one α- and one β-subunit that are assembled in the endoplasmic reticulum during or shortly after biosynthesis. This assembly is critical for the functional expression of the enzyme on the cell surface.; Since subunit assembly is important to the Na,K-ATPase, we are interested in the nature of its subunit interaction. Previous studies using chimeras between the Na,K-ATPase and the endoplasmic/sarcoplasmic reticulum Ca-ATPase identified the extracellular loop between transmembrane helices 7 and 8 (H7H8) of the α-subunit as being sufficient for subunit assembly. Studies using chimeras between the β-subunit and dipeptidyl peptidase IV further indicated that the subunit interaction sites are in the ectodomain of the β-subunit In the yeast two-hybrid system, C-terminal truncations of the β-ectodomain narrowed the region that makes physical contacts with the H7H8 loop of the α-subunit to 63 amino acids proximal to the transmembrane domain.; In this thesis, we examined the assembly of the Na,K-ATPase subunits in greater details. We searched for amino acids that are involved in inter subunit interaction. We constructed a library of randomly mutagenized H7H8 loop of the α-subunit and screened this library in the yeast reverse two-hybrid system for clones that failed to interact with the β-subunit ectodomain. Several identified mutants carried mutations at residues that are either conserved or semi-conserved among the vertebrate NaK-ATPases. We also searched for a minimal domain of the β-subunit that interacts with the α-subunit. We constructed a library of the β-subunit ectodomain randomly truncated from the N-terminus. The result from screening this library in the reverse two-hybrid and the result from the previous study using C-terminal truncations of the β-subunit suggested that the minimal domain required for subunit assembly consists of 36 amino acids. Lastly, we explored subunit assembly in vitro. We constructed and purified the α-subunit H7H8 loop as a glutathione S-transferase fusion protein, and obtained the β-subunit as a secretory protein produced by cultured mammalian cells. We explored and characterized the α-β subunit interaction in solution.