H,K-ATPase alpha subunit C-terminal membrane topology: epitope tags in theinsect cell expression system

The H,K-ATPase responsible for gastric acidification is a heterodimeric (al pha and beta subunit) P-type ATPase, an integral protein of parietal cell a pical membranes, which promotes the electroneutral exchange of K+ for proto ns, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imida zo[1,2-alpha]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of th e catalytic alpha subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP bi nding and phosphorylation sites, and a C-terminal region with four or six p utative transmembrane domains. Several lines of evidence implicate the C-te rminal region of P-type ATPases in cation-binding and occlusion, conformati onal changes, and interactions with the beta subunit (HK beta), making the definition of topology a prerequisite for understanding the structural basi s of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag ) were inserted in predicted hydrophilic segments of the alpha subunit (HK alpha) to establish the membrane orientation of two amino acids with differ ent predicted topologies in the C-terminal four- and six-transmembrane mode ls. Wild-type and mutated HK alpha and HK beta cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HK alpha- and HK beta-sp ecific and flu-tag-specific antibodies. Functional assays showed K+-stimula ted, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags w as determined by microsomal protease protection assays in Sf9 cells and imm unolabelling of HK alpha and HK beta in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasm ically oriented HK alpha residues. The results indicated cytoplasmic exposu re of Leu(844) and Phe(996), and luminal exposure of Pro(898), leading to a revised secondary structure model of the C-terminal third of HK alpha.