The human multidrug resistance protein MRP1 translocates sphingolipid analogs across the plasma membrane

Recently, we have provided evidence that the ABC-transporter MDR1 P-glycoprotein translocates analogs of various lipid classes across the apical plasma membrane of polarized LLC-PK1 cells transfected with MDR1 cDNA. Here, we show that expression of the basolateral ABC-transporter MRP1 (the multidrug resistance protein) induced lipid transport to the exoplasmic leaflet of the basolateral plasma membrane of LLC-PK1 cells at 15°C. C6-NBD-glucosylceramide synthesized on the cytosolic side of the Golgi complex, but not C6-NBD-sphingomyelin synthesized in the Golgi lumen, became accessible to depletion by BSA in the basal culture medium. This suggests the absence of vesicular traffic and direct translocation of C6-NBD-glucosylceramide by MRP1 across the basolateral membrane. In line with this, transport of the lipid to the exoplasmic leaflet depended on the intracellular glutathione concentration and was inhibited by the MRP1-inhibitors sulfinpyrazone and indomethacin, but not by the MDR1 P-glycoprotein inhibitor PSC 833. In contrast to the broad substrate specificity of the MDR1 P-glycoprotein, MRP1 selectively transported C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, the latter only when it was released from the Golgi lumen by brefeldin A. This shows the specific nature of the lipid translocation. We conclude that the transport activity of MDR1 P-glycoprotein and MRP1 must be taken into account in studies on the transport of lipids to the cell surface

Assay for the transbilayer distribution of glycolipids: Selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine-egg phosphatidylethanolamine-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A2 confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containin

Assay for the transbilayer distribution of glycolipids: Selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine-egg phosphatidylethanolamine-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A2 confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containin

Lipid traffic: the ABC of transbilayer movement

Membrane lipids do not spontaneously exchange between the two leaflets of lipid bilayers because the polar headgroups cannot cross the hydrophobic membrane interior. Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other. In addition, cellular membranes contain proteins that facilitate a passive equilibration of lipids between the two membrane halves. In recent years, a growing number of proteins have been put forward as lipid translocators or facilitators. Unexpectedly, some of these appear to be required for efficient translocation of lipids lacking bulky headgroups, like cholesterol and fatty acids. The candidate lipid translocators identified so far belong to large protein families whose other members include pumps for amphiphilic molecules like bile salts and drugs