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J. Biol. Chem., Vol. 279, Issue 9, 7867-7876, February 27, 2004

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Role of Multiple Drug Resistance Protein 1 in Neutral but Not Acidic Glycosphingolipid Biosynthesis^*

María Fabiana De Rosa, Daniel Sillence¶, Cameron Ackerley, and Clifford Lingwood||**

From the Research Institute and Department of Pediatric Laboratory Medicine, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, the Departments of Laboratory Medicine & Pathobiology and ^||Biochemistry, University of Toronto, Toronto M5G 1L5, Canada, and the ^¶Department of Biochemistry, Glycobiology Institute, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom

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.

Received for publication, May 29, 2003 , and in revised form, December 5, 2003.

^* This work was supported by the Canadian Cancer Society via Grant 011090 from the National Cancer Institute of Canada and Canadian Institutes for Health Research Grant MT13073. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 416-813-5998; Fax: 416-813-5993; E-mail: cling{at}sickkids.on.ca.

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