EPITHELIAL SPHINGOLIPID SORTING IS INSENSITIVE TO REORGANIZATION OF THE GOLGI BY NOCODAZOLE, BUT IS ABOLISHED BY MONENSIN IN MDCK CELLS ANDBY BREFELDIN-A IN CACO-2 CELLS

In epithelial MDCK and Caco-2 cells, short-chain analogs of glucosylce ramide and sphingomyelin are delivered from the Golgi to the cell surf ace with different apical/basolateral polarities, which results in an apical enrichment of the glycolipid glucosylceramide over the phosphol ipid sphingomyelin. Here, we have interfered with the integrity of the Golgi complex in various ways and tested the effects on lipid transpo rt and sorting. Nocodazole, which depolymerizes microtubules, disperse d the Golgi over the cytoplasm of MDCK cells and reduced transport of newly synthesized -benzoxadiazol-4-yl]aminocaproyl)-glucosylceramide a nd C6-NBD-sphingomyelin to the apical surface by 40%. The lipids were not mistargeted to the basolateral surface and upon removal of nocodaz ole, apical transport recovered. Nocodazole did not affect the apical enrichment of glucosylceramide over sphingomyelin. The ionophore monen sin led to swelling of the Golgi of MDCK cells and -inhibited lipid tr ansport to the cell surface by 30-50%. Whereas sphingomyelin transport to both surface domains was equally affected, monensin mainly inhibit ed apical transport of glucosylceramide. At 10-20 muM of monensin, the two lipids displayed the same polarity of delivery: sorting between t he two lipids was abolished. Brefeldin A at 1 mug/ml, which resulted i n disruption of the Golgi in HepG2 cells and completely inhibited prot ein secretion, had no inhibitory effect on transport of the C6-NBD-lip ids to the surface. The same was observed in Caco-2 cells. However, br efeldin A selectively shifted transport of sphingomyelin towards the a pical direction which abolished the apical enrichment of glucosylceram ide over sphingomyelin. Caco-2 cells were used because in MDCK cells b refeldin A did not change Golgi structure nor lipid transport and sort ing. In summary, modification of the Golgi by monensin and brefeldin A , but not nocodazole, interfered with the sorting event by which gluco sylceramide is enriched over sphingomyelin in the transport pathway fr om the Golgi to the apical surface.