Transport of newly synthesized glucosylceramide to the plasma membrane by a non-Golgi pathway.

Abstract

High-gradient magnetic affinity chromatography (HDMAC) has been used to obtain highly enriched plasma membranes, free of intracellular membrane contaminmts, from cultured Chinese hamster ovary (CHO) cells in yields of .80%. Using this procedure we have characterized the transport of glucosYceramlde (GlcCer) and the gangoide GM3 to the plasma membrane. Newly synthesized GlcCer reaches the plasma membrane in 7.2 min, whereas GM3 requires 21.5 min to reach the plasma membrane. Brefeldin A prevents transport of newly synthesized GM3 and sWhinmyelmn to the plasma membrane but has no effect on the transport of GIcCer. Similarly, incubation of CHO cells at 15°C blocks transport of GM3 and sphingomyelin to the plasma membrane but has no effect on GicCer movement. We propose that carrier-mediated transport accounts for a major fraction of the plasma membrane GlcCer. Puls-chase studies with either [3Hglcose or [3pH] (2) and ganglioside GM3 (3) as well as short-chain derivatives of sphingomyelin and glucosylceramide (GlcCer) (4-6) to the plasma membrane. (ii) Soluble carriers such as lipid-transfer proteins account for a major portion of the movement of the phospholipids phosphatidylcholine (7) and phosphatidylethanolamine (8, 9) to the plasma membrane. (iii) A vesicular transport pathway which is distinct from the secretory pathway accounts for the transport of newly synthesized cholesterol (10) to the plasma membrane. Transfer ofglucose to ceramide by UDPglucoseceramide glucosyltransferase has been localized to the cytosolic surface of the Golgi apparatus (11)(12)(13)(14), indicating that GlcCer must be translocated to the luminal leaflet for higher glycosylation to occur. We have determined that 45% of GlcCer in Chinese hamster ovary (CHO) cells is located in the plasma membrane (15). Since GlcCer is synthesized on the cytosolic surface of the Golgi membrane and translocated to the luminal surface, GlcCer could be transported to the plasma membrane through the vesicular protein secretory pathway and/or by an alternative pathway through the cytosol, perhaps by lipid-transfer proteins as proposed by Sasaki (16 (vol/vol) heat-inactivated fetal bovine serum. CHO K1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 iron-supplemented heat-inactivated fetal bovine serum, 2 mM glutamine, 1x nonessential amino acids, 1 mM proline, and lx penicillin/streptomycin. Cells were released by trypsin/EDTA treatment and plated on fresh culture dishes 48 hr prior to experimental manipulation.Metabolic Labeling. For pulse-chase experiments, cells were rinsed three times in Dulbecco's phosphate-buffered saline (PBS) and incubated for 1 hr at 370C in methionine-and cysteine-free medium. The medium was replaced with fresh methionine-and cysteine-free medium containing Tran35S-label at 150 pCi/ml. After 20 min of-incubation, incorporation was terminated by washing twice with PBS and the chase was initiated by addition of complete medium. For metabolic labeling of lipids [3H]palmitate [10 puCi/ml ...

Trans-Golgi Network and Subapical Compartment of HepG2 Cells Display Different Properties in Sorting and Exiting of Sphingolipids

“…3, A and B). These different transport kinetics to the basolateral membrane of HepG2 cells are reminiscent of the fast transport of natural GlcCer compared with other sphingolipids in Chinese hamster ovary cells (24). With increasing incubation times the difference between SM and GlcCer with regard to the fraction of the total lipid pools that reached the basolateral membrane diminished, amounting to 75-80% of the pools of both sphingolipids after 60 min.…”

“…has been described (24). In contrast to its natural counterpart, the short chain fluorescent GlcCer analog can be translocated to the exoplasmic leaflet by the basolaterally localized multidrug resistance protein MRP1 to become accessible for BSA (23,37).…”

Combinatorial Ganglioside Biosynthesis

“…This implicates a membrane translocation of glucosylceramide, which might be mediated by an as yet uncharacterized "flippase." Translocation is energy-independent (26) and presumably incomplete, because a part of the GlcCer pool can reach the cytosolic leaflet of the plasma membrane from the cytosolic face of the Golgi apparatus (35). The biosynthesis of higher gangliosides then occurs on the luminal face of the Golgi apparatus.…”

Glycolipid transfer protein: Clear structure and activity, but enigmatic function

“…Due to the limited solubility of the protein and protein denaturation no detailed information about the residues involved in binding of the glycolipid could be obtained, however. (Warnock et al, 1994) and GLTP treated with vesicles containing 30 mol% GlcCer (red). Protein concentrations are 0.3 mM.…”