SUBUNIT INTERACTIONS IN THE NA,K-ATPASE EXPLORED WITH THE YEAST 2-HYBRID SYSTEM

Subunit interactions of the alpha 1- and beta 1-subunits of the chicke n Na,K-ATPase were explored with the yeast two-hybrid system, Gal4-fus ion proteins containing domains of the alpha 1- and beta 1-subunits we re designed for examining both intersubunit and intrasubunit protein-p rotein interactions, Regions of the alpha- and beta-subunits known to be involved in alpha-beta-subunit assembly were positive in two-hybrid assay, supporting the validity of the assays. A library of beta-subun it ectodomains with C-terminal truncations was screened to find the ma ximal truncation retaining an interaction with the alpha-subunit extra cellular H7H8 loop (where H7 refers to the seventh membrane span, and so on), The maximal truncation removed all the cysteines involved in d isulfide bridges, leaving only 63 amino acids of the beta-subunit ecto domain. Scanning alanine mutagenesis led to identification of an evolu tionarily conserved sequence of four amino acids (SYGQ) in the extrace llular H7H8 loop of the alpha-subunit that is crucial to alpha-beta-in tersubunit interactions. Oligomerization studies with single domains f ailed to detect self-association of either of the two large cytosolic loops (H2H3 and H4H5) within the alpha-subunit. However, evidence was found for an interaction between these two cytoplasmic loops.