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Molecular and Cellular Biology, July 2006, p. 5055-5069, Vol. 26, No. 13
0270-7306/06/$08.00+0     doi:10.1128/MCB.02107-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide

Paola Giussani ,^1,, Michael Maceyka,^1, Hervé Le Stunff,^1,|| Aki Mikami,¹ Sandrine Lépine,¹ Elaine Wang,² Samuel Kelly,² Alfred H. Merrill Jr.,² Sheldon Milstien,³ and Sarah Spiegel^1*

Department of Biochemistry, Virginia Commonwealth University School of Medicine and Massey Cancer Center, Richmond, Virginia 23298,¹ School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30322,² National Institute of Mental Health, Bethesda, Maryland 20892³

Received 31 October 2005/ Returned for modification 14 December 2005/ Accepted 28 March 2006

Previous studies demonstrated that sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1), which is located mainly in the endoplasmic reticulum (ER), regulates sphingolipid metabolism and apoptosis (H. Le Stunff et al., J. Cell Biol. 158:1039-1049, 2002). We show here that the treatment of SPP-1-overexpressing cells with S1P, but not with dihydro-S1P, increased all ceramide species, particularly the long-chain ceramides. This was not due to inhibition of ceramide metabolism to sphingomyelin or monohexosylceramides but rather to the inhibition of ER-to-Golgi trafficking, determined with the fluorescent ceramide analog N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (DMB-Cer). Fumonisin B1, an inhibitor of ceramide synthase, prevented S1P-induced elevation of all ceramide species and corrected the defect in ER transport of DMB-Cer, readily allowing its detection in the Golgi. In contrast, ceramide accumulation had no effect on either the trafficking or the metabolism of 6-([N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl)-sphingosine, which rapidly labels the Golgi even at 4°C. Protein trafficking from the ER to the Golgi, determined with vesicular stomatitis virus ts045 G protein fused to green fluorescent protein, was also inhibited in SPP-1-overexpressing cells in the presence of S1P but not in the presence of dihydro-S1P. Our results suggest that SPP-1 regulates ceramide levels in the ER and thus influences the anterograde membrane transport of both ceramide and proteins from the ER to the Golgi apparatus.

Supplemental material for this article may be found at http://mcb.asm.org/.

P.G. and M.M. contributed equally to this study.

Present address: Department of Medical Chemistry, Biochemistry, and Biotechnology, Faculty of Medicine, University of Milan, via Fratelli Cervi 93, Segrate, Milan 20090, Italy.

^|| Present address: Laboratoire de Physiopathologie de la Nutrition, Université Paris 7, CNRS UMR 7059, 75251 Paris Cedex 05, France.

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