Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?
Article first published online: 30 DEC 2003
Copyright © 1998 American Association for the Study of Liver Diseases
Volume 27, Issue 4, pages 1089–1097, April 1998
How to Cite
Zegers, M. M. P., Zaal, K. J. M. and Hoekstra, D. (1998), Functional involvement of proteins, interacting with sphingolipids, in sphingolipid transport to the canalicular membrane in the human hepatocytic cell line, HepG2?. Hepatology, 27: 1089–1097. doi: 10.1002/hep.510270426
- Issue published online: 30 DEC 2003
- Article first published online: 30 DEC 2003
- Manuscript Accepted: 8 DEC 1997
- Manuscript Received: 8 AUG 1997
- Netherlands Foundation for Chemical Research with financial aid from the Netherlands Foundation for Scientific Research (M.Z.)
A photoreactive sphingolipid precursor was used to investigate the potential involvement of protein-lipid interactions that may convey specificity to sphingolipid transport in the human hepatoma cell line, HepG2. A 125I-labeled, photoreactive ceramide, 125I-N3 -Cer, was incubated with the cells and became incorporated into two sphingolipid products. The major product was photoreactive sphingomyelin (125I-N3 -SM) (25% of total radioactivity), while only minor amounts of photoreactive glucosylceramide (125I-N3 -GlcCer) were formed (<2%). After photoactivation, a restricted number of proteins was labeled. Given the absolute amounts of the newly synthesized, photoreactive lipids and their precursor present in the cells, labeling of the proteins can be assumed to be derived from interaction with either ceramide (Cer) or sphingomyelin (SM), or both. To discriminate between these possibilities, photoactivation and protein analysis was performed in cells treated with D-threo-1-phenyl-2-decanoyl amino-3-morpholino-1-propanol (PDMP), an inhibitor of sphingolipid biosynthesis. In treated cells, the radioactive SM pool was reduced by ≈80% Concomitantly, labeling of a 60-kd protein, seen in control cells, decreased. Furthermore, the 60-kd protein is membrane-associated and insoluble in detergent at low temperature. Moreover, when cells containing photoreactive sphingolipids after a preincubation with the photoreactive Cer were photoactivated and subsequently incubated with fluorescent sphingolipid analogs, transport of the latter to the bile canalicular membrane, as observed in control cells, was inhibited. Taken together, the data suggest that distinct proteins, among them a 60-kd protein, may play a specific and functional role in sphingolipid transport to the bile canalicular membrane.