Molecular machinery for non-vesicular trafficking of ceramide.
Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.
Hanada
Kentaro
K
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. hanak@nih.go.jp
Kumagai
Keigo
K
Yasuda
Satoshi
S
Miura
Yukiko
Y
Kawano
Miyuki
M
Fukasawa
Masayoshi
M
Nishijima
Masahiro
M
D017398:Alternative Splicing / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005816:Genetic Complementation Test; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013109:Sphingomyelins / Q000096:biosynthesis
D002518:Ceramides; D018129:Phosphatidylinositol Phosphates; D013109:Sphingomyelins; C037178:phosphatidylinositol 4-phosphate; C119090:CERT1 protein, human; D017346:Protein Serine-Threonine Kinases