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 w e compared its behavior to that of the corresponding fluorescent analo gues of sphingomyelin, galactosylceramide, and lactosylceramide. All f our fluorescent analogues were readily transferred from defatted BSA t o the plasma membrane during incubation at 4 degrees C. When cells tre ated with C-5-DMB-GlcCer were washed, warmed to 37 degrees C, and subs equently incubated with defatted BSA to remove fluorescent lipid at th e cell surface, strong fluorescence was observed at the Golgi apparatu s, as well as weaker labeling at the nuclear envelope and other intrac ellular membranes. Similar results were obtained with C-5-DMB-galactos ylceramide, except that labeling of the Golgi apparatus was weaker tha n with C-5-DMB-GlcCer. Internalization of C-5-DMB-GlcCer was not inhib ited by various treatments, including ATP depletion or warming to 19 d egrees 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 a ccumulation of C-5-DMB-GlcCer at the Golgi apparatus was a saturable p rocess. In contrast, cells treated with C-5-DMB-analogues of sphingomy elin or lactosylceramide internalized the fluorescent lipid into a pun ctate 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 wer e documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429 -442). Incubation of perforated cells with C-5-DMB-sphingomyelin resul ted in prominent labeling of the nuclear envelope and other intracellu lar 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 galactosylce ramide at the plasma membrane and early endosomes of human skin fibrob lasts, and suggest that both endocytic and nonendocytic pathways are u sed in the internalization of these lipids from the plasma membrane.-