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

Clathrin fromH-K-ATPase-rich membranes derived from the tubulovesicular compartmentof rabbit and hog gastric acid secretory (parietal) cells wascharacterized biochemically, and the subcellular localization ofmembrane-associated clathrin in parietal cells was characterizedby immunofluorescence, electron microscopy, and immunoelectronmicroscopy. Clathrin from H-K- ATPase-rich membranes was determinedto be comprised of conventional clathrin heavy chain and a predominanceof clathrin light chain A. Clathrin and adaptors could be induced topolymerize quantitatively in vitro, forming 120-nm-diameter basketlikestructures. In digitonin-permeabilized resting parietal cells, theintracellular distribution of immunofluorescently labeled clathrin wassuggestive of labeling of the tubulovesicular compartment. Clathrin wasalso unexpectedly localized to canalicular (apical) membranes, as were-adaptin and dynamin, suggesting that this membrane domain ofresting parietal cells is endocytotically active. At theultrastructural level, clathrin was immunolocalized to canalicularand tubulovesicular membranes. H-K-ATPase was immunolocalized tothe same membrane domains as clathrin but did not appear to be enrichedat the specific subdomains that were enriched in clathrin. Finally, inimmunofluorescently labeled primary cultures of parietal cells, incontrast to the H-K-ATPase, intracellular clathrin was found not totranslocate to the apical membrane on secretagogue stimulation. Takentogether, these biochemical and morphological data provide a frameworkfor characterizing the role of clathrin in the regulation of membranetrafficking from tubulovesicles and at the canalicular membrane inparietal cells.