INTERNALIZATION AND SORTING OF A FLUORESCENT ANALOG OF GLUCOSYLCERAMIDE TO THE GOLGI-APPARATUS OF HUMAN SKIN FIBROBLASTS - UTILIZATION OF ENDOCYTIC AND NONENDOCYTIC TRANSPORT MECHANISMS

We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPY(TM))-1-pentanoyl]-glucosyl sphingosine (C-5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C-5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C-5-DMB-galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C-5-DMB-GlcCer. Internalization of C-5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C-5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with; a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C-5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C-5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C-5-DMB-sphingomyelin and C-5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C-5-DMB-sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C-5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane.-