抄録
The gastric proton pump, H+,K+-ATPase, is responsible for gastric acid secretion and is almost exclusively found in the parietal cells of stomach. Under physiological conditions, this pump generates and maintains a proton gradient in excess of a million-fold across the luminal membrane. This pump consists of the catalytic α- (114 kDa) and non-catalytic glycoprotein β-subunits (33 kDa as a protein core). Correct assembly between them is essential for the functional expression of the pump at the cell surface. To study the trafficking and cellular regulation of membrane proteins, good culture systems are required. However, at present, there are no cell lines retaining the function of parietal cells. Recently, we constructed stable cell lines expressing the α- and/or β-subunits from human renal HEK-293 cells. Here, we studied the quality control mechanism of the proton pump on the ER, and the fate of the α- and β-subunits before and after the cell surface expression by immunofluorescence, pulse chase labeling, and cell surface labeling with Sulfo-NHS-SS-biotin. The α-subunit was expressed at the cell surface only in the presence of the β-subunit, whereas the β-subunit can travel to the cell surface even in the absence of the α-subunit. The α/β complex expressed at the cell surface was active for ion transport. The unassembled α-subunit was retained in the ER, polyubiquitinated and degraded by the proteasomes. These findings suggest that the ubiquitin/proteasome system is important in controlling the cell surface expression of functional α/β holoenzymes. [Jpn J Physiol 54 Suppl:S58 (2004)]
