O-glycosylation of intact and truncated ribophorins in brefeldin A- treated cells: newly synthesized intact ribophorins are only transiently accessible to the relocated glycosyltransferases

The permanent HeLa cell transformant (HeLa-RI332) that expresses a truncated version of ribophorin I, consisting of the 332 NH2-terminal amino acids of the luminal domain of this type I transmembrane glycoprotein, has been described (Tsao et al., 1992) . Procedures for cell culture, radioactive labeling, immunopreeipitation, endo H treatment, and PAGE, in either 8 or 6-11% gradient gels, were also described previously (Tsao et al., 1992) . In one experiment (see legend to Fig. 3 ) in which cells were labeled with tritium-labeled sugars or [~5S]methionine, methionine-and glucose-free MEM was used. Before use, the tritium-labeled sugars were dried in a SVCI00H Savant Speed Vac concentrator (Savant Instruments, Inc., Farmingdale, NY) and dissolved in medium to a concentration of 280 ~Cilml. Unless otherwise indicated, cultures treated with BFA were preincubated in methionine-free medium containing the drug for 30 rain before pulse labeling, and the drug was present in the culture medium throughout the labeling and chase periods at a concentration of 5 ~g/ml.