Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells.
Madin-Darby canine kidney cells (MDCK) were transfected with a cDNA encoding the glycosyl-phosphatidylinositol (GPI)-anchored protein mouse Thy-1 in order to study the steady-state surface distribution of exogenous and endogenous GPI-linked proteins. Immunofluorescence of transfected cells grown on collagen-coated coverslips showed that expression of Thy-1 was variable throughout the epithelium, with some cells expressing large amounts of Thy-1 adjacent to very faintly staining cells. Selective surface iodination of cells grown on collagen-coated or uncoated transwell filters followed by immunoprecipitation of Thy-1 demonstrated that all the Thy-1 was present exclusively in the apical plasma membrane. Although cells grown on uncoated filters had much smaller amounts of Thy-1, it was consistently localized on the apical surfaces. Immunofluorescent localization of Thy-1 on 1 micron frozen sections of filter-grown cells demonstrated that all the Thy-1 was on the apical surface and there was no detectable intracellular pool. Phosphatidylinositol-specific phospholipase C digestion of intact iodinated monolayers released Thy-1 only into the apical medium, indicating that Thy-1 was processed normally in transfected cells and was anchored by a GPI-tail. In agreement with previous findings, endogenous GPI-linked proteins were found only on the apical plasma membrane. These results suggest that there is a common mechanism for sorting and targeting of GPI-linked proteins in polarized epithelial cells.
Wilson
J M
JM
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, MA 02115.
Fasel
N
N
Kraehenbuhl
J P
JP
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D002460:Cell Line; D004285:Dogs; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006017:Glycolipids / Q000502:physiology*; D017261:Glycosylphosphatidylinositols; D007457:Iodine Radioisotopes; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000502:physiology*; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D018800:Thy-1 Antigens; D014162:Transfection
D000954:Antigens, Surface; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D018800:Thy-1 Antigens; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase