Subunit Interactions in the Na,K-ATPase Explored with the Yeast Two-hybrid System

Subunit interactions of the α1- and β1-subunits of the chicken Na,K-ATPase were explored with the yeast two-hybrid system. Gal4-fusion proteins containing domains of the α1- and β1-subunits were designed for examining both intersubunit and intrasubunit protein-protein interactions. Regions of the α- and β-subunits known to be involved in α-β-subunit assembly were positive in two-hybrid assay, supporting the validity of the assays. A library of β-subunit ectodomains with C-terminal truncations was screened to find the maximal truncation retaining an interaction with the α-subunit extracellular H7H8 loop (where H7 refers to the seventh membrane span, and so on). The maximal truncation removed all the cysteines involved in disulfide bridges, leaving only 63 amino acids of the β-subunit ectodomain. Scanning alanine mutagenesis led to identification of an evolutionarily conserved sequence of four amino acids (SYGQ) in the extracellular H7H8 loop of the α-subunit that is crucial to α-β-intersubunit interactions. Oligomerization studies with single domains failed to detect self-association of either of the two large cytosolic loops (H2H3 and H4H5) within the α-subunit. However, evidence was found for an interaction between these two cytoplasmic loops.