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

The 300-kD man6P receptor was used as a marker for the action of galactosyl-and sialyltransferases. This receptor provides a sensitive means to detect glycosyltransferase action, since it contains 19 potential N-linked oligosaccharide addition sites (30) and is highly glycosylated. The experiment was carried out as follows. CHO cells were labeled with [35S]methionlne and cysteine for 60 rain to label newly synthesized rnan6P receptors. At this time, labeled man6P receptors reside in the ER, since these receptors require up to 3 h to fold and be completely exported from this compartment (19, 40) . Cells were then chased for various times in the presence of BFA, and the potential acquisition of galactose residues was determined by affinity chromatography of isolated man6P receptors on columns of the galactosespecific lectin, RCA-I (1). To further increase the sensitivity of the assay, we used CHO clone 1021 cells that lack apparent sialyltransferase activity (6, 8) . In these cells, any added galactose residues will be present at the termini of N-linked oligosaccharides and fully accessible for optimal lectin binding (1). Fig . I (top) shows the results obtained from such an experiment. Man6P receptors isolated from cells immediately after the labeling period should not have contained galacrose. As expected, none of the man6P receptors bound to RCA-I agarose; all were recovered in the flowthrough fraction. In contrast, with increasing times of BFA treatment, man6P receptors acquired the ability to be retained on an RCA-I column, and could be eluted from such columns with galactose. Quantitative analysis of this experiment (Fig. I, bottom) showed that half of the man6P receptors gained the Triangles, +BFA; squares, -BFA. capacity to bind RCA-I agarose within '~2 h. This was somewhat slower than the rate observed in the absence of BFA (hr~ = 80 rain). A control experiment showed that galactose addition was not due to the action of newly synthesized galactosyltransferase, also accumulated in the ER, since cyclobeximide had essentially no effect on the extent of galactose addition (not shown).