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 also found that both ribophorins, which normally do not acquire O-linked oligosaccharides, do so after BFA treatment. This demonstrates that they, indeed, can serve as substrates for the enzymes that carry out this modification, and therefore, in the absence of the drug must not be exposed to them. O-glycosylation involves the addition of N-acetylgalactosamine to serine and threonine residues in the polypeptide backbone, followed by linkage to galactose and sialic acid (Sadier, 1984) . The exact subcellular location of the N-acetyl galactosaminyl transferase that initiates growth of the O-linked oligosaccharide chain has not been established, although the relatively rapid kinetics with which this modification takes place (Tooze et al., 1988) , as well as the fact that certain mutant low density lipoprotein (LDL) receptors that fail to transverse the Golgi apparatus nevertheless acquire O-linked sugars (Cummings et al., 1983) , has sug-gested that this enzyme could be located in a pre-Golgi compartment. In fact, a recent study of the E1 glycoprotein of the mouse hepatitis coronavirus virus MHV-A59 (Tooze et al., 1988 ) concluded that addition of N-acetylgalactosamine to this protein occurs in a pre-Golgi compartment, which is also the site of budding of the virion into the lumen of the endomembrane system and morphologically resembles transitional elements and vesicles found between the ER and the Golgi apparatus. Since ribophorins I and II, as well as the truncated ribophorin I variant, RI332, undergo O-glycosylation only in BFA-treated cells, we can conclude that under normal circumstances these molecules do not even reach this intermediate compartment, which may be the same as the "salvage ~ compartment from where KDEL-containing luminal proteins that escape from the ER are retrieved to this organelle (Pelham, 1989) . This reinforces the conclusion that the segregation of ribophorins I and II does not involve a retrieval mechanism. It should be noted that if, indeed, O-glycosylation takes place in the "salvage " compartment, one would expect that some luminal ER proteins that recycle through this compartment would contain O-linked sugars, a prediction that can be tested experimentally.