Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular co mpartment of rabbit and hog gastric acid secretory (parietal) cells was cha racterized biochemically, and the subcellular localization of membrane-asso ciated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K-ATPas e-rich membranes was determined to be comprised of conventional clathrin he avy chain and a predominance of clathrin light chain A. Clathrin and adapto rs could be induced to polymerize quantitatively in vitro, forming 120-nm-d iameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathr in was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, cla thrin was immunolocalized to canalicular and tubulovesicular membranes. H-K -ATPase was immunolocalized to the same membrane domains as clathrin but di d not appear to be enriched at the specific subdomains that were enriched i n clathrin. Finally, in immunofluorescently labeled primary cultures of par ietal cells, in contrast to the H-K-ATPase, intracellular clathrin was foun d not to translocate to the apical membrane on secretagogue stimulation. Ta ken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane traff icking from tubulovesicles and at the canalicular membrane in parietal cell s.