Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells

-Adaptin and clathrin heavy chain were identified ontubulovesicles of gastric oxyntic cells with the anti--adaptinmonoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb(MAb 23), respectively. In Western blots, crude gastric microsomes fromrabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for-adaptin and clathrin. In immunofluorescent labeling of isolatedrabbit gastric glands, anti--adaptin and anti-clathrin heavy chainimmunoreactivity appeared to be concentrated in oxyntic cells. Inprimary cultures of rabbit oxyntic cells, the immunocytochemicaldistribution of -adaptin immunoreactivity was similar to that of thetubulovesicular membrane marker in oxyntic cells, the H-K-ATPase.Further biochemical characterization of the tubulovesicular-adaptin-containing complex suggested that it has a subunitcomposition that is typical of that for a clathrin adaptor: in additionto the -adaptin subunit, it contains a -adaptin subunit and othersubunits of apparent molecular masses of 50 kDa and 19 kDa. Fromsolubilized gastric microsomes from rabbit, -adaptin could becopurified with the major cargo protein of tubulovesicles, theH-K-ATPase. Thus this tubulovesicular coat may bind directly to theH-K-ATPase and may thereby mediate the regulated trafficking of theH-K-ATPase at the apical membrane of the oxyntic cell during thegastric acid secretory cycle. Given the similarities of the regulatedtrafficking of the H-K-ATPase with recycling of cargo through theapical recycling endosome of many epithelial cells, we propose thattubulovesicular clathrin and adaptors may regulate some part of anapical recycling pathway in other epithelial cells.