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 presence of galactose and sialic acid residues in the modified ribophorin I molecules indicates that after BFA treatment the respective glycosyltransferases, which are nor-maUy present only in the trans-cisternae of the Golgi apparatus (Kornfeld and Kornfeld, 1985; Cummings et al., 1983; Elhammer and Kornfeld, 1984) , are found in the ER. This could result either from a relocation of preexisting Golgi enzymes, as has been demonstrated for o~-mannosidase H and galactosyltransferase (Lippincott-Schwartz et al., 1989 , 1990 , or from the accumulation of newly synthesized glycosyltransferases that fail to leave the ER in the presence of the drug. To distinguish between these possibilities, the effect of BFA was assessed in cells that, after pulse labeling with psS]methionine, were incubated with the protein synthesis inhibitor cycloheximide for 30 min, a period sufficient to allow the egress from the ER of newly synthesized proteins destined to the Golgi apparatus. As shown in Fig. 5 , even under these conditions, BFA treatment resulted in the appearance of the more slowly migrating forms of RI332 with the same kinetics as in the absence of cycloheximide. It therefore can be concluded that the modifications induced by BFA are carried out by preexisting glycosyltransferases, relocated from the Golgi apparatus to the ER as a result of treatment with the drug. It may also be noted in Fig. 5 that treatment with cycloheximide appeared to slow down the degradation of RI332 molecules.