Internalization and recycling of glycoprotein 280 in epithelial cells of yolk sac

The luminal plasma membrane of the epithelial cells lining the visceral layer of the yolk sac and the renal proximal tubule display a well developed brush border defining numerous clathrin-coated intermicrovillar areas which are further characterized by expressing two glycoproteins, gp280 and gp330, the latter also known as the Heymann nephritis antigen. The present study analyzes the distribution, the internalization and intracellular trafficking of gp280 and gp330 in yolk sac epithelium by immunoultrastructural and cell surface labeling techniques. Immunocytochemistry revealed that gp280 and gp330 were distributed very similarly in the endocytic pathway including dense apical tubules, with the exception that gp330 was found in lysosomes to a much greater extent than gp280. To demonstrate internalization of gp280, apical cell membrane proteins of paired yolk sacs were labeled at 4 degrees C with biotin, linked via a disulfide bond cleavable under mild reducing conditions by glutathione, and either kept at 4 degrees C or incubated at 37 degrees C. These experiments showed that biotin could be cleaved from gp280 by glutathione in yolk sacs kept at 4 degrees C, whereas it became inaccessible to glutathione after incubation at 37 degrees C, suggesting internalization of gp280. Furthermore, incubation of yolk sacs in the presence of colloidal gold-labeled antibodies to gp280 demonstrated that gp280, initially expressed on the cell membrane, was translocated into endocytic vacuoles and accumulated in dense apical tubules, whereas only a small fraction reached the lysosomes. Under similar conditions, gold-labeled antibodies to gp330 were also internalized and followed a similar intracellular routing, but lysosomal accumulation was also found. Bovine serum albumin-labeled gold particles accumulated in lysosomes but were virtually absent from dense apical tubules. These observations suggest that gp280 and gp330, visualized by anti-gp280 and anti-gp330 antibodies coupled to gold particles, returned to the cell surface via dense apical tubules, whereas albumin gold particles dissociated from a potential binding protein in the early endocytic compartment and were subsequently accumulated in lysosomes. Since gp280 is internalized and apparently translocated to a recycling compartment as is gp330, our results suggest that gp280 may play a role as a receptor for endocytosis of as yet unknown ligands.