Salvage of glucosylceramide by recycling after ... - Europe PMC

Proc. Nati. Acad. Sci. USA

Vol. 86, pp. 9896-9900, December 1989 Cell Biology

Salvage of glucosylceramide by recycling after internalization along the pathway of receptor-mediated endocytosis (glycosphingolipids/transferrin/lipid recycling)

JAN WILLEM KOK, SINIKKA ESKELINEN*, KARIN HOEKSTRA, AND DICK HOEKSTRAt Laboratory of Physiological Chemistry, State University Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands

Communicated by John D. Baldeschwieler, September 5, 1989

ABSTRACT To examine the (intra)cellular fate of a glycolipid, normally residing at the cell surface, a fluorescent analog of glucosylceramide, 6-[N-(7-nitro-2,1,3-benzoxadiazol4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-glucosylceramide), was inserted into the plasma membrane of baby hamster kidney cells at low temperature. Upon warming the cells to 370C, part of the glycolipid analog was internalized. A comparison with receptor-mediated uptake of transferrin revealed that after 2 min of warming, both C6-NBD-glucosylceramide and the transferrin-transferrin receptor complex are localized in the same intracellular compartment (early endosomes). We conclude that C6-NBD-glucosylceramide is internalized along the pathway of receptor-mediated endocytosis. When, after internalization of part of the membrane-inserted glycolipid analog, the residual pool of plasma membrane C6-NBD-lipid was removed by "back exchange" with a lipid acceptor, C6-NBD-glucosylceramide molecules can be shown to return intact to the plasma membrane. This demonstrates that glycolipids, analogous to a variety of protein receptors, are able to recycle to the plasma membrane after internalization.

compartment (12, 13). Sorting of a glycolipid, after its synthesis from a fluorescently tagged precursor {6-[N-(7-nitro2,1 ,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-ceramide)}, has also been proposed to occur in polarized cells (14, 15). An extensive flow of membranes takes place during the process of endocytosis (1). By receptor-mediated endocytosis, ligand-receptor complexes are internalized by way of coated pits. The coated vesicles formed quickly reach the early endosomal compartment (16, 17) and in this compartment sorting is triggered. Some receptors are transported further to the lysosomes for degradation whereas others recycle back to the plasma membrane, either directly or through the Golgi area (2, 18). Most ligands, irrespective of their dissociation from the receptors, end up in lysosomes. However, the iron transporter transferrin (TfO remains bound to its receptor in the endosome and after iron-discharge the (apo)Tf-Tf receptor complex recycles back to the plasma membrane. This property of Tf, in conjunction with its well-characterized intracellular cycle, can be exploited to characterize internalization pathways, including mechanisms of sorting and recycling of lipids and proteins during intracellular membrane traffic. The present study shows that a fluorescent analog of the glycolipid glucosylceramide is taken into cells together with Tf, by following the pathway of receptor-mediated endocytosis. After its internalization the original glycolipid returns to the plasma membrane, providing direct evidence for a recycling mechanism involved in the trafficking of glycolipids.

Intracellular trafficking and targeting of proteins, as occurs during biosynthesis and endocytosis (1-3), are subject to careful regulation. However, much remains to be resolved with respect to the intracellular flow of lipids. Since organelles differ in lipid composition and maintain their specific composition in spite of a continuous flow of membrane vesicles between those organelles, it seems reasonable to assume that mechanisms also exist to regulate lipid trafficking. Hence, processes like sorting and recycling, well-known phenomena in protein traffic, may also be important in lipid flow (4, 5). Glycolipids may be of special importance in lipid trafficking. Glycolipids have been implicated in a number of cellular functions, including recognition phenomena (6-8). Most glycolipids are believed to reside mainly in the outer leaflet of the plasma membrane, which means that this class of lipids in particular would be subject to sorting (6). Some circumstantial evidence supports this view. For example, the regulation of cell growth by the ganglioside GM3 (9) has been suggested to involve a complex pathway of degradation to lactosylceramide and reutilization of the latter for GM3 synthesis in the Golgi complex (10). The GM3 formed then returns to the cell surface. In liver, gangliosides can be resynthesized from recycled glucosylceramide (11). This implies that the neutral glycolipids lactosylceramide and glucosylceramide are sorted during inbound cellular traffic to avoid degradation in the lysosomal compartment. In this respect, exogenous insertion of various radiolabeled gangliosides in the plasma membrane of cultured human fibroblasts has been shown to result predominantly in their degradation in the lysosomal

MATERIALS AND METHODS Materials. N-Palmitoyl-DL-dihydroglucocerebroside, Npalmitoyl-DL-dihydrolactocerebroside, D-sphingosine, and iron-free human Tf were purchased from Sigma. 6-[N-(7Nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoic acid (C6NBD) and lissamine rhodamine B sulfonyl chloride (LR), 10% (wt/wt) on Celite, were from Molecular Probes. Synthesis of C6-NBD-Labeled Glycosphingolipids. C6NBD-glucosylceramide, C6-NBD-lactosylceramide, and C6NBD-ceramide were synthesized from C6-NBD and 1-

,B-DL-glucosyldihydrosphingosine, 1-/3-DL-lactosyldihydrosphingosine, and D-sphingosine, respectively, as described (19). 1-,B-DL-Glucosyldihydrosphingosine and 1-f3-DL-lac-

tosyldihydrosphingosine were prepared (20) from N-palmitoyl-DL-dihydroglucocerebroside and N-palmitoyl-DLdihydrolactocerebroside, respectively. Some experiments, as described below, were also carried out with a C6-NBD Abbreviations: Tf, transferrin; LR, lissamine rhodamine B sulfonyl chloride; BSA, bovine serum albumin; C6-NBD, 6-[N-(7-nitro2,1,3-benzoxadiazol-4-yl)amino]hexanoic acid or -hexanoyl. *Present address: Department of Pathology, University of Oulu, SF-90220 Oulu, Finland. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9896

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derivative of 1-,B-D-glucosylsphingosine, prepared from glucocerebrosides isolated from human (Gaucher disease) spleen (Sigma). The results were identical to those obtained for the derivative of 1-f3-DL-glucosyldihydrosphingosine. The C6-NBD-lipids were quantitated spectrophotometrically in a fluorometer (Perkin-Elmer MPF-43) with an excitation wavelength of 465 nm and an emission wavelength of 530 nm, by reference to a known amount of C6-NBD-phosphatidylcholine. Tf Labeling. Saturation of Tf with iron was carried out by the procedure of Van Renswoude et al. (21). Diferric Tf was conjugated with LR as follows: 10 mg of Tf was incubated with 10 mg of LR-Celite in 1.5 ml of 0.25 M Tris Cl (pH 9.5-9.6) for 30 min at room temperature. The reaction was stopped by removing LR-Celite from the mixture by centrifugation. To ensure the complete removal ofany free dye, the sample was subsequently chromatographed on Sephadex G-50. Cell Culture and Membrane Insertion of Fluorescent Lipids. Monolayer cultures of baby hamster kidney cells (BHK-21) were grown in Glasgow minimum essential medium, supplemented with 5% (vol/vol) fetal calf serum and 10%o (vol/vol) tryptose phosphate broth in a water-saturated atmosphere of 5% C02/95% air. Experiments were carried out 48-72 hr after passage. C6-NBD-lipid insertion was carried out at 2TC. Prior to labeling, the cells were cooled to 2TC (30 min). Two different methods were employed to insert the fluorescent lipid analog into the plasma membrane of the cells. (i) The cells were incubated with C6-NBD-lipid-containing liposomes. To this end, small unilamellar vesicles (190 nmol of dioleoylphosphatidylcholine and 10 nmol of C6-NBD-lipid in

Proc. Natl. Acad. Sci. USA 86 (1989)

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1 ml of Hanks' solution) were freshly prepared for each experiment by probe sonication for 10-15 min on ice and under nitrogen. (ii) Alternatively, appropriate amounts of C6-NBD-lipid, stored in chloroform/methanol [2:1 (vol/ vol)], were dried under nitrogen and subsequently solubilized in absolute ethanol. An aliquot of the ethanolic solution (0.5%, final concentration) was injected into Hanks' solution (pH 7.4) under vigorous vortex mixing. This solution was then added to the cells. Lipid and Tf Internalization. Labeling of the cells with LR-Tf (0.2 mg/ml) was carried out for 30 min at 2°C whereafter the unbound Tf was removed by washing the cells with ice-cold Hanks' solution. To initiate the internalization of the membrane-inserted lipid and the membrane-bound Tf, the cells were warmed to 37°C by adding warm Hanks' solution. At various times, the buffer was removed and replaced by ice-cold (2°C) Hanks' solution. Back Exchange of Membrane-Inserted Analogs. Back exchange was carried out by incubating the cells for 30 min at 2°C with either dioleoylphosphatidylcholine small unilamellar vesicles (500 nmol/ml) or 5% (wt/vol) bovine serum albumin (BSA), both in Hanks' solution. Fluorescence was then measured after lipid extraction of the BSA solution and of cells to determine the back-exchanged fraction. Lipid Extraction and Analysis. Lipids were extracted by the procedure of Bligh and Dyer (22) and analyzed by TLC on silica gel 60 HPTLC plates (Merck), using CHC13/CH30H/ 20% (wt/vol) NH40H [70:30:5 (vol/vol)] as the running solvent system, allowing separation of all three C6-NBDlipids and the C6-NBD-fatty acid. (The Rf values are 0.76, 0.44, 0.25, and 0.16, respectively, for C6-NBD-ceramide,

FIG. 1. Plasma membrane insertion of glucosylceramide. Cells were labeled at 2°C with C6-NBD-glucosylceramide-containing liposomes for 30 min (a and b). Subsequently a back exchange was performed with 5% BSA/Hanks' solution for 30 min at 2°C to remove plasmamembrane-inserted C6-NBD-lipid (c and d). (b and d) Phase-contrast images corresponding to a and c, respectively. Similar results were obtained when the cells were labeled with the ethanol-injection method or when the back exchange was done with dioleoylphosphatidylcholine liposomes. (Bars = 10 ,Lm.)

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associated C6-NBD-lipid pool, and on the cells scraped from the plate with a rubber policeman after the back exchange. Microscopy. Cells were grown on glass coverslips in 35-mm Petri dishes. Prior to experiments, the cells were cooled (30 min) and washed several times with ice-cold Hanks' solution. For colabeling studies, the cells were first labeled with the C6-NBD-lipid for 30 min at 2TC followed by washing with Hanks' solution and subsequent labeling with LR-Tf (0.2 mg/ml). Internalization was initiated by adding warm Hanks' solution, and after various times the traffic processes were stopped by replacing warm buffer with ice-cold buffer. After a back exchange at 20C, the cells were fixed for 30 min at room temperature with 2% (vol/vol) formaldehyde in buffer containing 20 mM sodium phosphate (pH 7.4), 100 mM

C6-NBD-glucosylceramide, C6-NBD, and C6-NBD-lactosylceramide.) Recycling. Microscopic visualization of recycling lipids and chemical analysis of internalized and recycled lipids were done as follows. The cells were labeled at 20C with C6NBD-glycolipid. Internalization was then triggered by warming the cells to 370C. At various times, the cells were cooled and a back exchange was carried out at 20C, as described above. After the back exchange, the cells were again warmed to 370C for 30 min. Subsequently a second back exchange was carried out, if appropriate. For analysis of the fluorescent molecules responsible for the visible labeling pattern, a lipid extraction was performed on the BSA solution used for back exchange, representing the exchangeable plasma membranen-. r

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FIG. 2. Internalization and recycling of glucosylceramide. Cells were labeled at 20C with C6-NBD-glucosylceramide-containing liposomes for 30 min and, after washing, incubated at 370C for 30 min. Thereafter a back exchange was performed with 5% BSA/Hanks' solution at 20C for 30 min (a and b). Subsequently, the cells were again incubated at 370C for 30 min to monitor recycling of the glycolipid from intracellular sites to the plasma membrane (c and d). When a second back exchange was performed, the recycled glycolipid was removed from the plasma membrane (e and f). (b, d, and f) Phase-contrast images corresponding to a, c, and f, respectively. Similar results were obtained with the ethanol-injection labeling method and back exchange with dioleoylphosphatidylcholine liposomes. (Bars = 10,am.)

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FIG. 3. TLC analysis of internalized and recycled glucosylceramide. Cells were labeled at 20C with C6-NBD-glucosylceramide with the ethanol-injection method, incubated at 37TC for 30 min, and back-exchanged at 20C with 5% BSA. Subsequently, the cells were again incubated at 370C for 30 min to allow recycling to occur. Thereafter, a second back exchange was performed and the BSA solution and the cells were extracted for lipid analysis. In the BSA fraction, which represents the plasma membrane fraction (lane 3), only C6-NBD-glucosylceramide is present (the faint second spot with higher Rf represents BSA contamination). In the cell fraction [i.e., the internal fluorescent lipid pool (lane 4)], a small amount of C6-NBD-ceramide is found. Lane 5 shows the total NBD-lipid pool when the second back exchange was omitted. Lanes 6, 7, and 8 show the plasma membrane (i.e., the lipid fraction obtained upon back exchange), the internal lipid pool (as obtained after back exchange of plasma membrane C6-NBD-lipid), and the total C6-NBD-lipid pool, respectively, after an incubation of the cells at 370C for 60 min. Bands are (from top to bottom) C6-NBD-ceramide (lanes 1 and 9), C6NBD-glucosylceramide (lanes 2-9), C6-NBD (lanes 2 and 9), and C6-NBD-lactosylceramide (lanes 2 and 9).

lysine, 60 mM sucrose, and 100 mM sodium periodate, post-fixed sequentially with 4% formaldehyde and with 6% formaldehyde, both in the same buffer, for 5 and 10 min, respectively. Fluorescence microscopy was performed with a Leitz Orthoplan microscope equipped with a Leitz Vario Orthomat 2 photography system. Photomicrographs were taken at 30-sec exposition times using Kodak T-max P3200 film that was processed at 12,800 ASA.

RESULTS AND DISCUSSION C6-NBD-Glycolipid Incorporation into the Plasma Membrane. C6-NBD-glucosylceramide and C6-NBD-lactosylceramide can be readily inserted into the plasma membrane of BHK cells, using either the ethanol-injection method or a

procedure involving incubation with C6-NBD-lipid containing dioleoylphosphatidylcholine liposomes (Fig. 1). In contrast to C6-NBD-ceramide, which rapidly internalizes at 20C due to flip-flop, and then labels membranes of cellular organelles (data not shown, cf. refs. 14 and 15), the C6NBD-glycolipids label only the plasma membrane at 20C, which suggests that they remain in the outer leaflet of the membrane. That this is indeed the case can be further shown by a back-exchange procedure that removes all of the C6NBD-glycolipid after insertion into the plasma membrane at 2°C (Fig. 1). This localization of the C6-NBD-glycolipids enables us to study the fate of these lipids during the process of endocytosis, initiated upon warming the cells to 37°C. Furthermore, the exchangeability of these fluorescent lipid analogs (i.e., their ability to be removed completely from the plasma membrane by a back-exchange procedure) makes it possible to determine whether and to what extent these lipids can be

FIG. 4. Cointernalization of C6-NBD-glucosylceramide and LRTf. Cells were labeled at 2°C with C6-NBD-glucosylceramide containing liposomes for 30 min, washed, and labeled with LR-Tf (30 min). Subsequently, the cells were incubated at 37°C for 2 min, immediately cooled to 2°C, and subjected to a back exchange with 5% BSA/Hanks' solution for 30 min. The cells were fixed at room temperature and examined by fluorescence microscopy, using appropriate filters for visualizing rhodamine fluorescence. (a) Tf. (b) NBD (lipid). (c) Corresponding phase-contrast image. (Bars = 10

m-.) recycled to the plasma membrane after they have been internalized by endocytosis. Internalization and Recycling. When the cells with C6NBD-glucosylceramide inserted into the plasma membrane are warmed to 37°C, part of the C6-NBD-lipid is internalized. Indeed, back exchange revealed that =20% becomes internalized during a 30-min incubation at 37°C. In the presence of metabolic inhibitors (5 mM sodium azide/50 mM 2-deoxyglucose) lipid internalization was inhibited and >97% of the total cell-associated C6-NBD-lipid fraction was recovered by

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back exchange (data not shown). This indicates that the internalization is energy dependent and accomplished by endocytosis (see below). Due to intensive membrane-staining, internalized lipid can best be visualized microscopically when a back exchange is performed. The intracellular labeling pattern is described as follows. Internalization starts rapidly after warming and within 2 min irregularly shaped vesicles at the periphery of the cells are labeled. After 5 min at 370C, large spherical vesicles become labeled that are localized more toward the cell center and 15 min after warming fluorescence also becomes apparent in the Golgi area, which is a distinct juxtanuclear area in BHK cells. After 30 min at 370C, a steady state is reached (Fig. 2a)-i.e., the labeling pattern as such does not change any more after prolonged incubation. To investigate whether C6-NBD-glucosylceramide molecules are able to recycle to the plasma membrane from these intracellular structures, cells were subjected to a back exchange at 2TC to remove all plasma membrane-associated fluorescence, after internalization had been allowed to proceed for 30 min. A subsequent incubation at 37TC showed that fluorescence reappeared in the plasma membrane (Fig. 2c). That this fluorescence is indeed localized in the plasma membrane can be shown by performing another back exchange at 20C, which removes all membrane-associated fluorescence (Fig. 2e) and indicates that the fluorescent molecules were located in the outer leaflet. To establish whether intact C6-NBD-glucosylceramide reappeared at the cell surface (i.e., that genuine recycling had taken place), the intracellular and plasma membrane C6NBD-lipid fractions were analyzed separately on TLC after lipid extraction of the BSA solution, used for back exchange (plasma membrane fraction), and the cells (intracellular fraction; Fig. 3). This analysis showed that after a 60-min incubation at 370C a small (3 hr). This implies that recycling of intact C6-NBD-glucosylceramide molecules has occurred after internalization. Furthermore, from Fig. 2, it is clear that when the plasma membrane pool is removed by a back exchange, the subsequent incubation at 37TC results in a shift in intracellular labeling pattern-i.e., fluorescence disappears from the large spherical vesicles and becomes localized exclusively in the Golgi area. From these results it can be inferred that recycling of the C6-NBD-glucosylceramide takes place by way of the Golgi area. However, it remains to be determined whether recycling also takes place from early endosomes (see below) and from the large spherical vesicles. Pathway of Glycolipid Internalization. To investigate in more detail the pathway along which the C6-NBD-glycolipid is internalized, cointernalization experiments were performed with LR-Tf. Tf is taken up into cells (including BHK cells) (S.E., J.W.K., and D.H., unpublished results) by receptor-mediated endocytosis. After a short incubation at 370C (2-5 min) Tf resides in early endosomes from which part of the molecules can recycle back to the plasma membrane. Another fraction proceeds toward the Golgi area, from which recycling to the plasma membrane can also occur (18). These trafficking routes can also be monitored in BHK cells with LR-Tf. When double-labeling experiments were carried out using C6-NBD-glucosylceramide and LR-Tf, both probes colocalized perfectly after 2 min at 37°C (Fig. 4). This implies that the C6-NBD-glycolipid is internalized during receptormediated endocytosis and resides in early endosomes after


short incubations at 37TC. It is worth noting that in BHK cells another distinct endocytic pathway exists that can be monitored by plasma membrane-inserted LR-labeled phosphatidylethanolamine and presumably represents fluid-phase uptake (unpublished observation). Thus in spite of the apparent homogenous distribution of the C6-NBD-glucosylceramide in the plasma membrane, this glycolipid is taken up only (i.e., specifically) along the pathway of receptor-mediated endocytosis. At later stages of the endocytic pathway the trafficking routes of the glycolipid and Tf diverge; i.e., the glycolipid is routed toward large spherical vesicles, where the Tf is never found (data not shown). Eventually the Golgi area seems to be the intracellular site where both pathways taken by the glycolipid and Tf merge again and from which both molecules can recycle to the plasma membrane. The nature of the large spherical vesicles (late endosomes?) that are part of the glycolipid-trafficking route remains to be established. In conclusion, the results indicate that fluorescent analogs of glycolipids may be helpful tools in investigating the intracellular trafficking of this biologically highly relevant class of lipids. Taking advantage of the known trafficking pathways of the Tf--Tf receptor complex, we have shown that such lipids can be internalized during receptor-mediated endocytosis and that they are engaged in recycling. A divergence in their pathways between early endosome and Golgi is highly intriguing and merits further investigation. To the best of our knowledge, a direct demonstration of glycolipid recycling has not been shown before. The secretarial assistance of Mrs. Rinske Kuperus and Mrs. Lineke Klap is much appreciated. This investigation was carried out under the auspices of the Netherlands Foundation for Chemical Research (SON) with financial support from the Netherlands Organization for Scientific Research (NWO). S.E. was supported by the Natural Science Research Council of the Academy of Finland, Magnus Ehrnrooth Foundation, and Oulu University Foundation. 1. Steinman, R. M., Mellman, I. S., Muller, W. A. & Cohn, Z. A. (1983) J. Cell Biol. 96, 1-27. 2. Brown, M. S., Anderson, R. G. W. & Goldstein, J. L. (1983) Cell 32, 663-667. 3. Tartakoff, A. M. (1987) in Cell Biology: A Series of Monographs, ed. Bittar, E. E. (Wiley, New York), Vol. 6, pp. 1-230. 4. Simons, K. & Van Meer, G. (1988) Biochemistry 27, 6197-6202. 5. Pagano, R. E. & Sleight, R. G. (1985) Science 229, 1051-1057. 6. Hakomori, S. (1981) Ann. Rev. Biochem. 50, 733-764. 7. Curatolo, W. (1987) Biochim. Biophys. Acta 906, 137-160. 8. Hoekstra, D. & Duzgune§, N. (1989) Subcell. Biochem. 14,229-278. 9. Bremer, E. G., Schlessinger, J. & Hakomori, S. I. (1986) J. Biol. Chem. 261, 2434-2440. 10. Usuki, S., Lyu, S. C. & Sweeley, C. C. (1988) J. Biol. Chem. 263, 6847-6853. 11. Tettamanti, G., Ghidoni, R. & Trinchera, M. (1988) Indian J. Biochem. Biophys. 25, 106-111. 12. Sonderfeld, S., Conzelmann, E., Schwarzman, G., Burg, J., Hinrichs, H. & Sandhoff, K. (1985) Eur. J. Biochem. 149, 247-255. 13. Ghidoni, R., Trinchera, M., Venerando, B., Fiorilli, A., Sonnino, S. & Tettamanti, A. (1986) Biochem. J. 237, 147-155. 14. Lipsky, N. G. & Pagano, R. E. (1985) J. Cell Biol. 100, 27-34. 15. Van Meer, G., Stelzer, E. H. K., Wijnaendts-Van Resandt, R. W. & Simons, K. (1987) J. Cell Biol. 105, 1623-1635. 16. Geuze, H. J., Slot, J. W., Strous, G. J., Lodish, H. F. & Schwartz, A. L. (1983) Cell 32, 277-287. 17. Schmid, S. L., Fuchs, R., Male, P. & Mellman, I. (1988) Cell 52, 73-83. 18. Stoorvogel, W., Geuze, H. J., Griffith, J. M. & Strous, G. J. (1988) J. Cell Biol. 106, 1821-1829. 19. Kishimoto, Y. (1975) Chem. Phys. Lip. 15, 33-36. 20. Goda, S., Kobayashi, T. & Goto, I. (1987) Biochim. Biophys. Acta 920, 259-264. 21. Van Renswoude, J., Bridges, K. R., Harford, J. B. & Klausner, R. D. (1982) Proc. Natl. Acad. Sci. USA 79, 6186-6190. 22. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917.