Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization.
Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin 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, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.
Okamoto
C T
CT
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles 90089-9121, USA. cokamoto@hsc.,usc.edu
Duman
J G
JG
Tyagarajan
K
K
McDonald
K L
KL
Jeng
Y Y
YY
McKinney
J
J
Forte
T M
TM
Forte
J G
JG
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D005591:Chemical Fractionation; D002845:Chromatography; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D017886:Durapatite; D005615:Freezing; D005744:Gastric Acid / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D007158:Immunologic Techniques; D013058:Mass Spectrometry; D008861:Microsomes / Q000378:metabolism; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D002966:Clathrin; D011108:Polymers; D017886:Durapatite; D017506:H(+)-K(+)-Exchanging ATPase