Abstract
We have established a ricin-resistant glycosylation-defective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells. The strategy employed in this study is a modification of that used previously (Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628) to demonstrate transport of the cation-independent and -dependent mannose 6-phosphate receptors from the cell surface to the trans-Golgi network in nonsecretory cell types. In ricin-resistant PC12 cells, radiolabeled galactose was incorporated enzymatically into surface glycoconjugates, primarily glycoproteins. Resistance to beta-galactosidase was acquired upon reculture at 37 degrees C due to further terminal glycosylation of the galactose residues. Treatment of N-linked oligosaccharides isolated from recultured cells with a variety of glycosidases in conjunction with beta-galactosidase demonstrated the addition of sialic acid N-acetylglucosamine and fucose residues to the galactose residues in recultured cells. Resistance to beta-galactosidase was not acquired in cells recultured at 19 degrees C, indicating that subsequent glycosylation of galactose residues did not occur at the cell surface or in endosomes. While glycosylation of galactose incorporated into asparagine oligosaccharides in Chinese hamster ovary clone 13 cells was not significant (less than 1%) after 6 h of reculture, approximately 10% of the galactose incorporated into surface oligosaccharides was further glycosylated in PC12 cells in this time. Analysis of total labeled versus beta-galactosidase-resistant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that endocytic traffic to the site of glycosylation activity in mutant PC12 cells was highly selective, but included a much greater number of proteins than were detected in Chinese hamster ovary clone 13 fibroblasts.
Highlights
From the Department of Biochemistry and Biophysics, University of California, San Francisco, We have established a ricin-resistant glycosylationdefective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells
Stategy-One approach that has been used to study endocytic traffic to the Golgi apparatus entails selecting cell lines that are deficient in addition of terminal galactose residues to N-acetylglucosamine residues on nascent glycoproteins
If glycoproteins radiolabeled in this way are recycled to the Golgi apparatus, they will be sustrates for glycosyltransferases that normally act on terminal galactose residues, which were not incorporated when the proteins were synthesized
Summary
From the Department of Biochemistry and Biophysics, University of California, San Francisco, We have established a ricin-resistant glycosylationdefective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells. Resistance to B-galactosidase was acquired upon reculture at 37 *C due to further terminal glycosylation of the galactose residues. Resistance to /3-galactosidase was not acquired in cells recultured at 19 “C, indicating that subsequent glycosylation of galactose residues did not occur at the cell surface or in endosomes. While glycosylation of galactose incorporated into asparagine oligosaccharides in Chinese hamster ovary clone 13 cells was not significant (~1%) after 6 h of reculture, approximately of the galactose incorporated into surface oligosaccharides was further glycosylated in PC12 cells in this time. PC12 cells was highly selective, but included a much greater number of proteins than were detected in Chinese hamster ovary clone 13 fibroblasts
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