Polarized insertion of an intracellular glycoprotein pool into the apical membrane of MDCK cells

Abstract

A monoclonal antibody that recognizes a 135-kDa glycoprotein (GP135) on the apical membrane of Madin-Darby canine kidney (MDCK) cells was used to identify and characterize an intracellular pool of GP135. Mild trypsin treatment at 4 degrees C removed approximately 95% of the GP135, and after warming to 37 degrees C, the reappearance of GP135 on the apical membrane was monitored by radioimmunoassay. Incorporation of GP135 into the apical cell surface after trypsin treatment consisted of two components, a rapidly inserted, cycloheximide-insensitive portion (defined as the GP135 pool), which leveled off within 1 h, followed by a slower insertion of newly synthesized GP135. Immunogold electron microscopy demonstrated that the GP135 pool was targeted in a polarized manner and was only detected on the apical membrane. Temperature shift and retrypsinization experiments provided evidence that the GP135 pool consisted of intracellular vesicles that could fuse with the plasma membrane. This was confirmed by immunofluorescence microscopy demonstrating that GP135 was localized within large cytoplasmic vesicles residing at varying distances from the apical cell surface. These data provide evidence for the presence of a regulated pathway in MDCK cells and support the possibility that the GP135 pool functions as an intracellular reserve which can exhibit polarized insertion into the plasma membranes similar to that described for other epithelial cells.