beta-subunit assembly is essential for the correct packing and the stable membrane insertion of the H,K-ATPase alpha-subunit

The alpha-subunits of H,K-ATPase (HKA alpha) and Na,K-ATPase require a beta -subunit for maturation. We investigated the role of the beta-subunit in th e membrane insertion and stability of the HKA alpha expressed in Xenopus oo cytes, Individual membrane segments M1, M2, M3, M4, and M9 linked to a glyc osylation reporter act as signal anchor (SA) motifs, and M10 acts as a part ial stop transfer motif. In combined HKA alpha constructs, M2 acts as an ef ficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 a nd M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined a constructs , M1-3 alpha-proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha-proteins are not resistant to cellular degradation. However, c o-expression with beta-subunits increases the membrane insertion of M9 in a M1-9 alpha-protein and completely protects M1-10 alpha-proteins against ce llular degradation. Our results indicate that HKA alpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular ch aracteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha-subunit depend on intramolecular and intermolecular beta-subunit interactions that are similar but not identica l to data obtained for the Na,K-ATPase alpha-subunit.