Regions of Association between the α and the β Subunit of the Gastric H,K-ATPase

• Published in: The Journal of biological chemistry Vol. 273; no. 18; p. 11075
• Main Authors: Dominique Melle-Milovanovic, Marko Milovanovic, Sunil Nagpal, George Sachs, Jai Moo Shin
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 01.05.1998
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.18.11075

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 α and β 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 Leu 855 to Arg 922 of the α subunit was bound to the β 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 β subunit. The sequence in the extracytoplasmic loop close to the eighth transmembrane segment, namely Arg 898 to Thr 928 , was identified as being the site of interaction using this method. We deduced that the sequence Arg 898 to Arg 922 in the α subunit has strong interaction with the extracytoplasmic domain of the β subunit. Again, using yeast two-hybrid analysis, two different sequences in the β subunit Gln 64 to Asn 130 and Ala 156 to Arg 188 were identified as association domains in the extracytoplasmic sequence of the β subunit. These data enable identification of major associative regions of the α-β subunits of the H,K-ATPase.