Regions of association between the alpha and the beta subunit of the gastric H,K-ATPase

Abstract

A binding and a yeast two-hybrid analysis were carried out on the gastric H,K-ATPase to determine interactive regions of the extracytoplasmic domains of the alpha and beta subunits of this P type ATPase. Wheat germ agglutinin fractionation of fluorescein 5-maleimide-labeled tryptic fragments of detergent-solubilized H, K-ATPase showed that a fragment Leu855 to Arg922 of the alpha subunit was bound to the beta subunit. The yeast two-hybrid system showed that the region containing only a part of the seventh transmembrane segment, the loop, and part of the eighth transmembrane segment was capable of giving positive interaction signals with the ectodomain of the beta subunit. The sequence in the extracytoplasmic loop close to the eighth transmembrane segment, namely Arg898 to Thr928, was identified as being the site of interaction using this method. We deduced that the sequence Arg898 to Arg922 in the alpha subunit has strong interaction with the extracytoplasmic domain of the beta subunit. Again, using yeast two-hybrid analysis, two different sequences in the beta subunit Gln64 to Asn130 and Ala156 to Arg188 were identified as association domains in the extracytoplasmic sequence of the beta subunit. These data enable identification of major associative regions of the alpha-beta subunits of the H,K-ATPase.