SORTING OF P-TYPE ATPASES IN POLARIZED EPITHELIAL-CELLS

The Na,K-ATPase and the H,K-ATPase are highly homologous members of th e P-type family of ion transporting ATPases. Despite their structural similarity, these two pumps are sorted to different destinations in po larized epithelial cells. While the Na,K-ATPase is restricted to the b asolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular s torage compartment in the parietal cells of the stomach. We have gener ated molecular chimeras composed of complementary portions of these tw o pumps' alpha-subunits. By expressing these pump constructs in polari zed epithelial cells in culture, we have been able to identify sequenc e domains which participate in the targetting of the holoenzyme. We fi nd that information embedded within the sequence of the fourth transme mbrane domain of the H,K-ATPase is sufficient to account for this prot ein's apical localization. Stimulation of gastric acid secretion resul ts in insertion of the intracellular H,K-ATPase pool into the apical p lasma membrane and inactivation of acid secretion is accompanied by th e re-internalization of these pumps. We have identified a tyrosine-bas ed signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the crit ical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cel l surface in parietal cells from these animals, and they constitutivel y hypersecrete gastric acid. These results demonstrate that the beta-s ubunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least t wo sorting signals are required to ensure the proper targetting and re gulation of the gastric H,K pump.