CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake

dThe carboxy terminus (CT) of the colonic H+-K+-ATPase is required for stable assembly with the beta-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK alpha(2), we selected 84 amino acids in the CT of the alpha-subunit of mouse colonic H+-K+-ATPase (CT-HK alpha(2)) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK alpha(2) with CD63, recombinant CT-HK alpha(2) and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK alpha(2) protein (but not CT-HK alpha(1)) coprecipitated with CD63, confirming stable assembly of HK alpha(2) with CD63. In HEK-293 transfected with HK alpha(2) plus beta(1)-Na+-K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK alpha(2)/NK beta(1) and Rb-86(+) uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK alpha(2)-NK beta(1) complex in the cell membrane.