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 behavior of ribophorin II, a resident glycoprotein of the ER membrane with the same transmembrane disposition as ribophorin I (Crimaudo et al., 1987; Pirozzi et al., 1991) , was also examined in BFA-treated cells. It must first be noted that in control HeLa cells only "~50% of the newly synthesized ribophorin II molecules acquire an N-linked oligosaccharide chain (Fig. 4, RI] vs RII* lane a) and are therefore sensitive to endo H treatment (lane b). A pulse-chase experiment demonstrates that in BFA-treated cells both types of ribophorin II molecules (that is, those that bear [RII] , as well as those that lack [RH*] N-linked oligosaccharides) are convetted to more slowly migrating forms. Moreover, in contrast to the situation with ribophorin I, after a 2-h chase period, the modified molecules were almost completely resistant to endo H digestion (Fig. 4, compare lanes e and f). This indicates that the high mannose N-linked oligosaccharities in ribophorin II were processed by the relocated Golgi enzymes, and that O-linked sugars were added to the ribophorin II polypeptides, whether or not they contain an N-linked oligosaccharide. The addition of O-linked sugars to ribophorin II in BFA-treated cells was confirmed by the finding that the drug led to the appearance of more slowly migrating forms even in cells that were treated with tunica- addition of BFA (5 tzg/ml) 30 rain before labeling. One BFA-treated sample (e and f) was incubated in chase medium containing BFA for 2 h. After lysis and immunoprecipitation with anti-ribophorin II antibody the samples were divided into two equal aliquots that were either mock treated (a, c, and e) or treated with endo H (b, d, and f) and then analyzed by SDS-PAGE and fluorography. HeLa cell cultures were also pretreated with tunicamycin (5/~g/ml) for 2 h and one culture was treated with BFA (5/~g/ml) for 30 min before labeling for 1 h with [35S]methionine. RII* is the membraneinserted but completely unglycosylated ribophorin II polypeptide that, to some degree, is always found in control cells, but it is the only form present in cells treated with tunicamycin in the absence of BFA. R/I is the ribophorin II molecule containing a high mannose (endo H sensitive) N-linked oligosaccharide chain, which is found normally in the ER membrane. R/Ira and R/Fro are O-glycosylated forms found in BFA-treated cells that are derived from RII and RII'. In the presence of BFA the N-linked oligosaccharide in RII and in its O-glycosylated derivative (R/1,) is converted into an endo H resistant form. mycin to suppress N-glycosylation (Fig. 4, lanes g and h) . In this case, also in contrast to the situation with ribophorin I, two distinct modified forms of ribophorin II were produced, which most likely correspond to molecules containing different numbers of O-linked oligosaccharide chains.