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

In the absence of BFA, high mannose oligosaccharidecontaining man6P receptors within and beyond the TGN would not be expected to gain access to the mannosidases and glycosyltransferases that lie proximal to the TGN (10). This was verified by isolating man6P receptors after various chase intervals and subjecting them to RCA-I chromatography. Fig. 5 (bottom) presents the results of such an experiment. In the absence of BFA, ,o7% of the mantP receptors bound to RCA-I at time zero, showing that the dMM was >90 % effective in inhibiting o~-mannosidase I. After a chase period of up to 6 h, there was only a very slight increase ('o5 %) in the fraction of man6P receptors that bound to an RCA-I column. These results confirm that the majority of CHO cell man6P receptors do not appear to return to the site of Golgi o~-mannosidase I (cis/medial Golgi) after transport to the TGN (10) . Fig, 5 (top) shows the results obtained if man6P receptors were pulse-labeled either in the ER (squares) or chased to compartments beyond the TGN (triangles) in the presence of dMM, and then further chased for various times without DMM, but in the presence of BFA. If the receptors were present in the ER, they were efficiently modified by galactosyltransferase. The rate of galactose addition was slightly slower than that observed in the absence of dMM pretreatmerit ( Fig. 1) , probably due to the time required to reverse the effect of dMM (4, 10, 33) . On the other hand, if the receptors were permitted to pass through the TGN prior to the addition of BFA, only a very small increase was detected in the amount of man6P receptors containing galactose, even after 6 h of incubation (Fig. 5, triangles) . The small increase observed ('~8%) was very close to that observed in the absence of BFA (Fig. 5, bottom) . Duncan and Kornfeld have shown that only '~10% of man6P receptors labeled at the surface of CHO cells can be detected to acquire sialic acid upon their return to the TGN after 6 h of reculture (10) . In our hands, only ,x,4-6% of the surface-labeled receptor acquired sialic acid after 6 hours (Fig. 4) . If this value represents the total amount of man6P receptor transitting through the TGN in 6 h, it would be difficult to detect the potential return of such a small quantity of man6P receptors from the TGN to the ER, especially with the ,'~2-h lag for galactose addition in that compartment (Fig. 5) . However, results identical to those shown in Fig. 5 were obtained using BW5147 ceils, in which 40% of surfacelabeled man6P receptors return to the TGN in 3 h (10). This strongly supports the conclusion that the majority of man6P receptors within and beyond the TGN do not return to the ER in BFA.