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 appearance of trans-Golgi components in the ER as a result of BFA treatment could also be demonstrated by EM using lectin labeling techniques and immunogold detection procedures on ultrathin frozen sections (Gdfliths et al., 1982) . In agreement with previous reports (Fujiwara et al., Figure 5 . The posttranslational modification of RI332 observed in BFA-treated cells is etfeeted by pre-existing enzyme.s. Cultures of HeLa-RI332 cells were pulse labeled with [~S]methionine for 10 min and, after this time, two cultures were placed on ice (lanes a and n). The other cultures were incubated for 30 rain in complete chase medium with (h-m) or without (b-g) cyclobeximide (10 #g/ ml). After the chase, BFA (5/~g/ml) was added and the incubation continued for up to 120 min. At the chase times indicated, the cells were lysed with an SDS-containing buffer and processed for immunoprecipiation and SDS-PAGE, followed by fluorography. 1988; Lippincott-Schwartz et al., 198% 1990; Ulmer and Palade, 1989) , BFA led to profound changes in the structure of the Golgi apparatus which, after treatment with the drug, was apparently reduced to clusters of vesicular and tubular elements located near partly rough and partly smooth ER cistemae with the typical appearance of transitional elements (Jamieson and Palade, 1967) . As expected from previous studies (Tartakoff and Vassalli, 1983) , in control HeLa cells, Figure 6 . BFA treatment of HeLa cells leads to the appearance of WGA binding sites in the ER and nuclear envelope. Ultrathin frozen sections from control HeLa-RI332 cells (A), or cells treated with BFA (5 t~g/rnl) for 5 min (B), or 2 h (C and D) were incubated with WGA. The sites of lectin binding were detectexl using anti-WGA antibodies and protein A-gold. In control cells (A), the nuclear envelope (NE) and ER are not labeled, but dense labeling is observed in trans-Golgi cisternae (GA) and the plasma membrane (PM). Within 5 mitt after BFA treatment (B), some WGA binding is observed in the nuclear envelope and ER, as well as in profiles corresponding to remnants of the Golgi apparatus (/arge arrows). After 2 h of BFA treatment, the ER and NE are much more intensely labeled. WGA, which recognizes sialic acid and exposed N-acetyl glucosamine residues (Bhavanandan and Katlic, 1979) , labeled intensely the trans-cisternae of the Golgi apparatus, but not the membranes or luminal content of the ER (Fig. 6 A) . On the other hand, in cells treated with BFA (5/~g/ml) for times as short as 5 min (Fig. 6 B) and up to 2 h (Fig. 6 C) , WGA binding situs were present throughout the ER and in the nuclear envelope, as well as in the apparent remnants of the Golgi apparatus (Fig. 6 D) . Similar results were obtained with the lectin RCA, which recognizes terminal galactose residues (Baenziger and Fiete, 1979) , or when the cells were treated with cycloheximide for 1 h before the addition of BFA to deplete the Eli of newly synthesized Golgi proteins (results not shown). These findings are in accord with the preceding biochemical demonstration that BFA induces a redistribution of O-glycosylating enzymes of the trans-region of the Golgi apparatus, such as galactosyl-and sialyltransferases, into the ER.