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

core sugars of N-linked oligosaccharides, on the electrophoretic mobility of the modified ribophorins was investigated ( Fig. 2) . Digestions with these enzymes were carded out after different times of chase, since O-linked chains grow by sequential addition of monosaccharide units (Sadler, 1984) , which progressively alters their susceptibility to cleavage by specific enzymes. After 30 min of chase, only endo-/3-galactosidase (Fig. 2 , lane e), but neither O-glycosidase, which removes O-linked oligosaccharides with terminal galactose residues (Umemoto et al., 1977) , nor the exoenzyme/3-galactosidase (Fig. 2 , lane d) substantially increased the electrophoretic mobility of the modified RI3a2 molecules. The apparent molecular mass of the endo-/3galactosidase-treated molecules was, however, still higher than that of the protein synthesized in control cells (Fig. 2 , compare lane e with lanes a and h). It appears, therefore, that the modified molecules contain O-linked oligosaccharides with internal galactose residues. Neuraminldase, an enzyme that cleaves off terminal sialic acid residues, already had a detectable effect on molecules obtained after 30 min of chase (Fig. 2, lane f ) and, when applied to samples chased for 90 min, it completely eliminated the slower migrating components of the modified ribophorin population, corresponding to the diffuse upper portion of the RI332,m band in Fig. 2 (compare lanes i and k). It is noteworthy that, even after removal of the terminal sialic acid residues by neuraminldase, the oligosaccharide chains remained resistant to O-glycosidase (Fig. 2 , compare lanes k and l). These data indicate that the galactose residues in the chains are still covered by other distal sugars.