Compartmentation of the Golgi complex: brefeldin-A distinguishes trans- Golgi cisternae from the trans-Golgi network

Sialic acid addition was monitored in BFA-treated, CHO wild-type cells, using the sialic acid-specific lectin from the slug, L/max flavus. Fig. 3 shows the kinetics with which man6P receptors acquired the capacity to bind to slug lectin columns and be eluted by excess sialic acid. In the absence of BFA, man6P receptors acquired sialic acid during their transit through the Golgi complex. Sialic acid addition was extensive after 2 h of chase, and by 4 h, 66 % of newly synthesized man6P receptors had acquired the ability to bind to slug lectin-Afligel, as we have previously shown (17) . In the presence of BFA, <10% of total man6P receptor molecules bound to slug lectin-Atfigel after 8 h of incubation (Fig. 3 ). This small amount of sialic acid addition was not due to newly synthesized sialyltransferase because it was also observed in ~e presence of cycloheximide (not shown). The electrophoretic mobility of the sialic acid-containing man6P isolated, digested with pronase to prepare glycopeptides, and then further digested with/3-galactosidase to cleave [3H]galactose from oligosaccharides that had not received sialic acid. Cleavage products were resolved by Sephadex G25 chromatography; fractions eluting at the void volume (which resisted B-galactosidase cleavage and thus contain sialic acid) are shown. The counts per minute in the excluded peak of the control reaction (-BFA) represents 4% of the total glycopeptide radioactivity recovered. In other experiments, as much as 6% resialylation was observed.