(GLYCO)SPHINGOLIPIDS ARE SORTED IN SUB-APICAL COMPARTMENTS IN HEPG2 CELLS - A ROLE FOR NON-GOLGI-RELATED INTRACELLULAR SITES IN THE POLARIZED DISTRIBUTION OF (GLYCO)SPHINGOLIPIDS

In polarized HepG2 cells, the fluorescent sphingolipid analogues of gl ucosylceramide (C-6-NBD-GlcCer) and sphingomyelin (C-6-NBD-SM) display a preferential localization at the apical and basolateral domain, res pectively, which is expressed during apical to basolateral transcytosi s of the lipids (van IJzendoorn, S.C.D., M.M.P. Zegers, J.W. Kok, and D. Hoekstra. 1997. J. Cell Biol. 137:347-457). In the present study we have identified a non-Golgi-related, sub-apical compartment (SAC), in which sorting of the lipids occurs. Thus, in the apical to basolatera l transcytotic pathway both C-6-NBD-GlcCer and C-6-NBD-SM accumulate i n SAC at 18 degrees C. At this temperature, transcytosing IgA also acc umulates, and colocalizes with the lipids. Upon rewarming the cells to 37 degrees C, the lipids are transported from the SAC to their prefer red membrane domain. Kinetic evidence is presented that shows in a dir ect manner that after leaving SAC, sphingomyelin disappears from the a pical region of the cell, whereas GlcCer is transferred to the apical, bile canalicular membrane. The sorting event is very specific, as the GlcCer epimer C-6-NBD-galactosylceramide, like C-6-NBD-SM, is sorted in the SAC and directed to the basolateral surface. It is demonstrated that transport of the lipids to and from SAC is accomplished by a ves icular mechanism, and is in part microtubule dependent. Furthermore, t he SAC in HepG2 bear analogy to the apical recycling compartments, pre viously described in MDCK cells. However, in contrast to the latter, t he structural integrity of SAC does not depend on an intact microtubul e system. Taken together, we have identified a non-Golgi-related compa rtment, acting as a ''traffic center'' in apical to basolateral traffi cking and vice versa, and directing the polarized distribution of sphi ngolipids in hepatic cells.