Abstract
Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.
Highlights
Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex
We show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested
Metabolic labeling in wild type and knockout (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity
Summary
MDCK cells transfected with the human MDR1 cDNA were a gift from Dr M. HEp-2, SF-539 astrocytoma, ovarian carcinoma SK VLB (MDR variant of the parental SKOV3 cell line [15]), MDR1-MDCK cells were maintained in ␣-minimal essential medium supplemented with 5% or 10% FBS and 40 g/ml gentamicin. Daudi Burkitt’s lymphoma and Jurkat E6.1 cells were maintained in RPMI 1640 medium supplemented with 10% FBS. For MDR1 inhibition, cells were grown in medium containing inhibitor at the highest concentration (Ϯ4 or 8 M CsA for 4 days, or 5, 10, or 15 M ketoconazole for 5 days), which our prior studies had shown to have no effect on cell growth. For MDR1 inhibition studies, CsA or ketoconazole was added to cells 4 or 5 days, respectively, prior to VT1 and maintained during the cytotoxicity assay
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