Article: article from journal or magazin.
Polarity of endogenous and exogenous glycosyl-phosphatidylinositol-anchored membrane proteins in Madin-Darby canine kidney cells.
Journal of Cell Science
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.
Animals, Antigens, Surface/metabolism, Antigens, Thy-1, Cell Line, Dogs, Epithelial Cells, Epithelium/metabolism, Glycolipids/physiology, Glycosylphosphatidylinositols, Iodine Radioisotopes/diagnostic use, Kidney/cytology, Membrane Proteins/metabolism, Phosphatidylinositol Diacylglycerol-Lyase, Phosphatidylinositols/physiology, Phosphoinositide Phospholipase C, Phosphoric Diester Hydrolases, Protein Processing, Post-Translational/physiology, Recombinant Proteins/metabolism, Transfection
Web of science
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