Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells.
We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domain-selective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation (Le Bivic et al. 1989. Proc. Natl. Acad. Sci USA. 86:9313-9317), we followed the appearance at the cell surface of a major apical sialoglycoprotein, gp114, a basolateral protein, uvomorulin, and a transcytosing protein, the polyimmunoglobulin receptor (pIg-R). We determined that both gp114 and uvomorulin appeared to be delivered directly to their respective surface, with mistargeting levels of 8 and 2%, respectively. Using the same technique, the pIg-R was first detected on the basolateral domain and then on the apical domain, to be finally released into the apical medium, as described (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). To directly determine whether the gp114 pool present on the basolateral surface was a precursor of the apical gp114, we compared it with the equivalent pIg-R pool, by labeling with sulfo-NHS-SS-biotin, a cleavable, tight junction-impermeable probe, and by following the fraction of this probe that became resistant to basal glutathione and accessible to apical glutathione during incubation at 37 degrees C. We found that, contrary to pIg-R, basolateral gp114 was poorly endocytosed and was not transcytosed to the apical side. These results demonstrate that an endogenous apical integral membrane glycoprotein of Madin-Darby canine kidney cells is sorted intracellularly and is vectorially targeted to the apical surface.
Le Bivic
A
A
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
Sambuy
Y
Y
Mostov
K
K
Rodriguez-Boulan
E
E
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D001710:Biotin; D015820:Cadherins / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D004705:Endocytosis / Q000502:physiology; D007553:Isotope Labeling; D007668:Kidney / Q000166:cytology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011971:Receptors, Immunologic / Q000378:metabolism; D012795:Sialoglycoproteins / Q000378:metabolism*
D000954:Antigens, Surface; D015820:Cadherins; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; D012795:Sialoglycoproteins; D001710:Biotin