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

That a true retention mechanism, rather than a mechanism involving retrieval from the Golgi apparatus, is responsible for the segregation of many proteins in the ER, including the ribophorins, was first suggested by the finding that N-linked oligosaccharide chains in ER proteins do not undergo conversion to complex forms, which are resistant to endo H and are capable of binding the lectins WGA and RCA, a process that requires the action of enzymes located in the Golgi apparatus (Rodriguez-Boulan et al., 1987a,b; Rosenfeld et al., 1984; Brands et al., 1985; Yamamoto et al., 1985) . The validity of this argument for any particular protein, however, rests on the premise that the protein could serve as a substrate for the Golgi enzymes, were it to become accessible to them. In the current work this is demonstrated for N-glycosylated ribophorin II molecules, which normally remain sensitive to endo H but in BFA-treated cells become resistant to digestion with this enzyme. A similar behavior was recently observed for ERp99, another ER resident protein Figure 8 . Ribophorin I when overexpressed in HeLa ceils becomes unstable. HeLa ceils (A) and HeLa cells transiently overexpressing ribophorin I (B) were incubated in methionine-free medium for 30 min, pulse labeled with [3SS]methionine for 1 h, and chased for up to 24 h. At the chase times indicated, the cells were lysed with an SDS-containing buffer and processed for immunoprecipitation and SDS-PAGE, followed by fluorography. (Lippincott-Schwartz et al., 1989) . In this regard, our finding that in BFA-treated cells the N-linked oligosaccharide chain in ribophorin I remained endo H sensitive clearly demonstrates that access to the enzymes that could effect its conversion to the endo H resistant form is not sufficient to ensure that the modification takes place. Indeed, the conformation of glycoproteins may very well determine the susceptibility of their sugar chains to the action of the relocated modifying enzymes. In addition, it can not be excluded that the single N-linked oligosaccharide chain of ribophorin I would acquire resistance to digestion with endo H, if the molecule would pass through the Golgi stacks in an ordered fashion, instead of being exposed to the Golgi glycosyltransferases in the BFA-induced ER-Golgi hybrid compartment. In fact, we have observed (unpublished observations) that the five N-linked oligosaccharide chains of the hemagghtinin of influenza, which normally are converted into endo H resistant forms during passage of the protein through the Golgi apparatus, are not modified by relocated Golgi enzymes when the protein remains in the ER in BFA-treated cells.