AJP - Cell Physiology, Vol 258, Issue 3 C390-C398, Copyright © 1990 by American Physiological Society
Polarized insertion of an intracellular glycoprotein pool into the apical membrane of MDCK cells
G. K. Ojakian, R. Schwimmer and R. E. Herz
Department of Anatomy and Cell Biology, State University of New York Health Science Center, Brooklyn 11203.
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.
