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

We have previously shown (Tsao et al., 1992) that in permanently transfected HeLa cells expressing RI332, a truncated form of ribophorin I that lacks the membrane and cytoplasmic segments and consists of only the amino-terminal 332 amino acids of the luminal domain the truncated molecules are degraded with biphasic kinetics. Furthermore, in cells treated with BFA, the degradation of RI332 proceeded with monophasic kinetics and a rate constant intermediate between the two rates observed in control cells (Tsao et al., 1992) . Upon careful analysis it was apparent that in contrast to control cells, in the presence of BFA the apparent molecu- Figure 1 . The BFA-induced modification of RI332 and ribophorin I does not affect its N-linked oligosaccharide, which remains endo H sensitive, and also occurs in cells treated with tunicamycin. (A) HeLa-RI332 cells were incubated in methionine-free medium for 30 min, pulse labeled with [3SS]methionine for 5 min, and chased for up to 2 h. BFA (5/zg/ml) was present during all these incubations. Immunoprecipitates from samples taken at different times of chase were incubated with endo H or in the absence of the enzyme, as indicated, and analyzed by SDS-PAGE followed by fluorography. R/%z indicates the position of the polypeptide generated from RI332 after removal of the N-linked oligosaccharide by endo H. The asterisks alongside lane a and on the band in lane f mark the position of RI'332.~, the polypeptide generated by endo H digestion of RI332.m. The position of the latter is marked with dots in lane e and alongside lane a. (B) HeLa-RI332 cells were preincubated with tunicamycin (5 tzg/ml) for 1.5 h and in methionine-free medium containing tunicamycin and BFA (5 ~g/ml) for 30 min. They were then pulse labeled with [35S]methionine for 5 min, and chased for up to 90 min in the presence of both drugs (c'-g'). Control cultures, not treated with the drugs, were harvested immediately after the pulse (a'), or after 90 min of chase (b'). Immunoprecipitates were obtained and analyzed by SDS-PAGE, followed by fluorography. (m), A modified form of the ribophorin I and RI332 polypeptides observed in the presence of BFA; (*) a form lacking the N-linked oligosaccharide. lar weight of the labeled RI332 molecules underwent a progressive increase throughout the chase ( Fig. 1 A, lanes a, c, e, g, i, k, and m) . After 10 min of chase •50% of the RI332 molecules are modified (lane c), and after 30 min, the posttranslational modification is essentially completed (lane g). After prolonged chase periods, the decaying pool contained a heterogenous population of RI332 molecules, represented by several closely spaced electrophoretic bands ( Fig. 1 A, lanes k and m).