TRANSPORT OF BIOSYNTHETIC SPHINGOLIPIDS FROM GOLGI TO PLASMA-MEMBRANEIN HT29 CELLS - INVOLVEMENT OF DIFFERENT CARRIER VESICLE POPULATIONS

Intracellular transport of the sphingolipids glucosylceramide (GlcCer) and sphingomyelin (SM), was examined in HT29 human colon adenocarcino ma cells. After synthesis from a fluorescent precursor, ro-2,1,3-benzo xadiazol-4-yl)amino]hexanoylceramide (C-6-NBD-Cer), transfer of SM fro m the Golgi complex to the plasma membrane can occur independently of that of GlcCer, as revealed by temperature-dependent experiments. Thus , at 20 degrees C, SM trafficking to the cell surface is essentially u naffected, whereas GlcCer transport to the plasma membrane is ininhibi ted by approximately 75 %, when compared to the transfer of both lipid s at 37 degrees C. The mechanism by which SM and GlcCer are transporte d to the cell surface involves at least in part a vesicular mechanism. Transport vesicles, containing both lipids at their luminal surface, as revealed by the inaccessibility of the NBD fluorescence to the quen cher sodium dithionite, have been isolated from cells, permeabilized b y filter stripping. As evidenced by electron microscopic and biochemic al criteria, no vesicles or lipids were released when cell permeabiliz ation had been carried out with streptolysin. Density gradient analysi s indicates the potential existence of several vesicle populations, di stinctly enriched in either lipid, involved in transport of sphingolip ids to the plasma membrane in HT29 cells.