Abstract
Using monoclonal antibodies directed against the plasma membrane of Madin-Darby canine kidney (MDCK) cells, we demonstrated previously that a glycoprotein with an Mr = 23,000 (gp23) had a non-polarized cell surface distribution and was observed on both the apical and basolateral membranes (Ojakian, G. K., Romain, R. E., and Herz, R. E. (1987) Am. J. Physiol. 253, C433-C443). However, in parallel studies on MDCK clonal lines (D11, D18) with high transepithelial electrical resistances and in kidney cells in vivo it was determined that gp23 had a polarized cell surface distribution, being localized only to the basolateral membrane. The cell surface distribution of other glycoproteins was identical in both MDCK and MDCK clonal lines, indicating that MDCK cells were not deficient in the ability to properly sort membrane glycoproteins. Metabolic labeling with radioactive substrates followed by immunopurification and gel electrophoresis demonstrated that gp23 from both MDCK and MDCK clone D11 had many biochemical similarities including electrophoretic mobility, glycosylation, and palmitate incorporation. However, proteolytic digestion of gp23 from MDCK and clone D11 cells produced unique peptide maps suggesting that these closely related glycoproteins may have different primary sequences. In this report, we present evidence that the differential targeting of gp23 may be due to differences between the primary sequences of the basolateral and non-targeted proteins. The possibility that the observed differences in gp23 targeting are due to the presence of a basolateral recognition signal in gp23 from clone D11 cells is discussed.
Highlights
From the Department of Anatomy and Cell Biology, State University of New York Health Science Center, Brooklyn, New York 11203
Using monoclonal antibodies produced against the MDCK cell surface, we have demonstrated previously that gp23 has a non-polarized distributionon the plasma membrane of MDCK cells but istargeted to the basolateral surface of high resistance MDCK clonal lines [5],including strain I MDCK used by other laboratories [2, 11, 14]
We demonstrate here that the difference in gp23 sorting in MDCK and MDCK clonal cells is not aproperty of the intrinsic sortingpathway, since both apical and basolateral plasma membrane proteins are correctly targeted in MDCK and MDCK clonal lines
Summary
Medical College of Pennsylvania) and MDCK clone D University of California, San Diego) were maintained by serial passage in complete Eagle’s minimal essential medium containing Earle’s salts (MEM) supplemented with gentamycin (50 &g/ml) and 10%. Clonal lines were isolated from clone D MDCK cells [34] biplating cells/lOO-mm culture dish (Falcon) and removing individual colonies from cloning cylinders with 0.2% trypsin, 2 mM EDTA. The MDCK cells used in this study grow into confluent monolayers which have transepithelial electrical resistances of 150-200 ohms. Cm*, while the clone Dll and D18 cells form extremely tight monolayers with electrical resistances of 1000-3000 ohms. Cell culture medium components were purchased from GIBCO and plasticware from Falcon
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