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

Both ribophorin I and its truncated variant (RI332) contain a single N-linked high-mannose oligosaccharide chain attached to Asn27s, which can be cleaved off by endo H digestion (Rosenfeld et al., 1984; Harnik-Ort et al., 1987; Tsao et al., 1992) . To determine whether the apparent increase in the molecular masses of the truncated ribophorin I molecules that appear in BFA-treated cells reflects a processing of the N-linked oligosaccharide chain, the effect of endo H treatment on this protein was examined. As is shown in Fig. 1 A, endo H digestion increased the electrophoretic mobility of all the labeled truncated ribophorin I molecules produced in BFA-treated cells. The same behavior was found for intact ribophorin I (not shown). This demonstrates that the N-linked oligosaccharide chains in these molecules had not undergone the trimming and addition of sugars that have been observed for other proteins as a consequence of the BFA-induced backflow of Golgi enzymes to the ER (Lippincott-Schwartz, 1989; Doms et al., 1989) . However, the deglycosylated truncated products were still of higher molecular weight than those produced by endo H digestion of the labeled molecules present immediately after the pulse, which had not yet undergone the BFA-induced posttranslational modification ( Fig. 1 A, compare R/%2.m and R/%2). This demonstrates that the latter modification affects parts of the ribophorin I molecule other than the N-linked oligosaccharide chain.