Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane.

Abstract

When monolayer cultures of Chinese hamster lung fibroblasts are briefly incubated at 2°C with the fluorescent sphingolipid analogue, C6-NBD-ceramide (N-[7-(4-nitrobenzo-2-oxa-l,3-diazole)] aminocaproyl sphingosine), fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occur. During further incubation at 37°C, the Golgi apparatus, and later the plasma membrane, become intensely fluorescent. Within this period, the C6-NBD-ceramide is converted to equal amounts of fluorescent sphingomyelin and glucocerebroside (Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA., 80:2608-2612. In the present study, the intracellular translocation of these metabolites and their subsequent appearance at the plasma membrane were investigated by fluorescence microscopy, the addition of the ionophore monensin, and the technique of "back exchange," in which the amounts and types of fluorescent lipids present at the cell surface are identified after their transfer from the cell surface into recipient vesicles. In control cells, the amount of fluorescent glucocerebroside and sphingomyelin that could be removed from the cell surface by back exchange increased during incubation at 37°C, correlating with the increased fluorescence of the plasma membrane observed by microscopy. In the presence of 10 #M monensin, visible labeling of the plasma membrane was greatly diminished, whereas the Golgi apparatus became highly fluorescent and distended. The ability to remove fluorescent metabolites from the cell surface by back exchange was significantly but reversibly inhibited by monensin. Monensin also increased the total amount of fluorescent sphingomyelin, but not the glucocerebroside found in cells.Subcellular fractions were assayed for their ability to convert radiolabeled and fluorescent ceramides to the corresponding sphingomyelins and glucocerebrosides. The activities of parallel fractions coincided, suggesting that the presence of the NBD moiety did not affect the cellular metabolism of ceramide. Furthermore, the major peak of sphingomyelin-and glucocerebroside-synthesizing activity appeared to coincide with an enriched Golgi fraction.These results strongly suggest that fluorescent sphingomyelin was not synthesized at the plasma membrane as has recently been suggested for endogenous sphingomyelin. Rather, both the sphingomyelin and glucocerebroside analogues were synthesized intracellularly from C6-NBD-ceramide and translocated through the Golgi apparatus to the cell surface.The sites of lipid biosynthesis in animal cells and the mechanism(s) by which newly synthesized molecules are subsequently translocated to various intracellular membranes pose many fundamental but, as yet, unresolved questions for biochemists and cell biologists. Sphingomyelin, the predominant sphingolipid in many cell types, illustrates this point (1).

Sorting of sphingolipids in the endocytic pathway of HT29 cells

“…Similar observations have been described for other cell types (7,14) . The two main lipid products synthesized are C6-NBD-glucosylceramide and C6-NBD-sphingomyelin .…”

Effects of Brefeldin A on Phosphatidylcholine Phospholipase D and Inositolphospholipid Metabolism in HL‐60 Cells

“…The disappearance of galactosyltransferase activity in the lower density of the sucrose gradient (fractions 9-15) reflects the redistribution of Golgi markers induced by brefeldin A, as has been reported previously [22,231. 80% inhibition of the galactosyltransferase activity was observed in the postnuclear supernatant obtained from HL-60 cells treated with 0.5 pg/ml brefeldin A for 1 h (1 84 cpm .…”

Tales and Mysteries of the Enigmatic Sphingomyelin Synthase Family

“…In this case, no appearance of newly synthesized SM on the plasma membrane could be detected, suggesting that the main site for SM synthesis resided in the cell and that vescicular trafficking was required for SM delivery to the plasma membrane. 16,17 To complicate matters, the idea that, in BHK cells, SM destined to the plasma membrane is synthesized in recycling endosomes was also put forward [21][22][23] but later unequivocally disproved. 24 Also, SMS activity seemed absent from an intermediate ER-Golgi compartment (ERGIC) and, also in the Vero cells employed in this study, it was mostly enriched in the Golgi fraction.…”

“…[11][12][13] SMS activity at the Golgi was later detected also in the same mouse SV40-transformed fibroblasts and in Chinese hamster lung fibroblasts and epithelial Madin-Darby canine kidney cells. [14][15][16][17] In order to clarify the issue of subcellular localization of SM synthesis, a thorough study was performed in 1990 by Futerman and coworkers using the well-characterized protocol for subcellular fractionation of rat liver and by paying particular attention to inhibit the activity of the SM-metabolizing enzyme Figure 1. Schematic of the biochemical structures of SM, CPE and CPI.…”