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

To estimate the time interval during which the newly synthesized ribophorin I molecules are accessible to the O-glycosylation system, HeLa cells were labeled for 1 h in the presence or absence of BFA, and the extent to which the posttranslational modification occurred in a subsequent chase period in the presence of the drug was assessed. After this relatively long labeling period, which facilitates the detection of the labeled ribophorin I molecules, modified full length polypeptides were detected only in cells labeled in the presence of the drug (Fig. 7 A) . The failure of BFA to cause the modification of previously labeled ribophorin molecules, however, could result in part from the time required for the drug to exert its effect and the Golgi enzymes to reach the ER. The latter has recently been estimated to be considerably less than 15 min (Donaldson et al., 1991) , which is in accordance with our observations of the appearance of lectin binding sites in the ER within 5 rain of BFA treatment (Fig. 6 B) . Since ribophorin I is a stable protein that can only be weakly labeled during a short pulse, it is difficult to carry out this type of experiment with a shorter labeling period, to obtain accurate kinetics for the modification of the intact Figure 7 . In BFA-treated cells, intact ribophorin I and ribophorin II molecules can be posttranslationally glycosylated only during a limited period after their synthesis. HeLa cells (A and C) and HeLa cells transiently overexpressing ribophorin I (B) were preincubated in methionine-free medium for 30 min with (lanes a-c), or without (d-g) BFA (5 t~g/ml) and then labeled with [~SS]methionine for 1 h in the presence (a-c) or absence (d-g) of the drug. Some cultures (a, d, and g) were placed on ice immediately after the labeling period, whereas the others were incubated in chase medium containing BFA for 2 h (b and e) or 4 h (c and f). Immunoprecipitates obtained from cell lysates with antibodies against ribophorin I (A and B) or ribophorin II (C) were analyzed by SDS-PAGE, followed by fluorography. The labeled bands are marked using the abbreviations introduced in the legends to Figs. 1 and 4. molecule. If we assume that, when available to the modifying enzymes, the intact and truncated molecules are modified with the same kinetics, it can be concluded that within 15 rain or less after their synthesis is completed, ribophorin I molecules are no longer susceptible to modification by the relocated Golgi enzymes.