Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity.
Sphingomyelin is a major lipid of the mammalian cell surface. The view that sphingomyelin, after synthesis in the Golgi lumen, reaches the outer leaflet of the plasma membrane on the inside of carrier vesicles has been challenged by inconsistencies in the results of transport studies. To investigate whether an alternative pathway to the cell surface exists for sphingomyelin, brefeldin A and mitotic cells were used to block vesicular traffic between the Golgi complex and the plasma membrane. Exogenous sphingomyelinase was applied in the cold to assay for the presence of sphingomyelin on the surface of CHO cells. Newly synthesized radiolabeled sphingomyelin was found to equilibrate with cell surface sphingomyelin within 1.5 hours at 37 degrees C. Brefeldin A and mitosis inhibited this transport but, surprisingly, not the surface appearance of the short-chain sphingomyelin analog N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl(C6-NBD)-sphingo myelin as assayed by depletion of this lipid in the medium by the scavenger albumin. Transport of C6-NBD-sphingomyelin in the presence of brefeldin A was blocked by cyclosporin A and PSC 833, inhibitors of the multidrug resistance P-glycoprotein. The same was observed in HepG2 and HeLa cells, and for short-chain glucosylceramide, which demonstrates the general nature of the transporter-dependent sphingolipid translocation across the plasma membrane.
van Helvoort
A
A
Department of Cell Biology, Medical School AZU H02.314, University of Utrecht, The Netherlands.
Giudici
M L
ML
Thielemans
M
M
van Meer
G
G
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D016466:CHO Cells; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D005963:Glucosylceramides / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology; D010069:Oxadiazoles / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D017855:Transferases (Other Substituted Phosphate Groups) / Q000302:isolation & purification
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002518:Ceramides; D003517:Cyclopentanes; D005963:Glucosylceramides; C104907:N-6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminohexanoysphingomyelin; D010069:Oxadiazoles; D013109:Sphingomyelins; D020126:Brefeldin A; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase