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 a truncated variant of ribophorin I that contains only the first 332 amino acids of the luminal domain (RI332), when synthesized in permanent transformants of HeLa cells, is rapidly degraded by a nonlysosomal pathway with biphasic kinetics. The first phase of degradation of RI332 begins immediately after synthesis is completed and takes place in the ER itself. The second phase, however, appears to require vesicular transport of the remaining molecules from the ER to a second compartment, where degradation takes place at an accelerated rate. The two degradative compartments appear to fuse in cells treated with brefeldin A (BFA) ~, in which the truncated ribophorin is degraded with monophasic kinetics at a rate intermediate between those of the two normal degradative phases (Tsao et al., 1992) . BFA is an antibiotic (H/irri et al., 1963) that profoundly affects the structure of the Golgi apparatus and causes the backflow of Golgi enzymes to the ER (Fujiwara et al., 1988; Lippincott-Schwartz et al., 1989 , 1990 Doms et al., 1989; Ulmer and Palade, 1989) . In this report we show that in the presence of BFA both the newly synthesized normal ribophorins and the truncated ribophorin I variant undergo posttranslational modifications which do not take place in untreated cells. Both ribophorins undergo O-glycosylation and the N-linked oligosaccharide chain in ribophorin II, but not that in ribophorin I, is converted into an endoglycosidase H (endo H) resistant form. However, whereas the truncated ribophorin I molecules remain accessible to the relocated Golgi enzymes throughout their lifetime, the intact ribophorins I and II are susceptible to the modifying enzymes only during or immediately after their synthesis. It would appear that only during this brief period the intact newly synthesized molecules are in a conformational state or location, which, after BFA treatment, permits access to the relocated Golgi enzymes. Only during a subsequent maturation process do the intact ribophorins become assembled and sequestered in a supramolecular complex from which the truncated variant is excluded.