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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.

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Figure 1: CERT complements the mutant phenotypes of LY-A2 cells.
Figure 2: CERT specifically extracts ceramide from phospholipid bilayers.
Figure 3: CERT catalyses the intermembrane transfer of ceramide.
Figure 4: CERT from the mutant LY-A cell line contains a mutation.
Figure 5: Intracellular distribution of GFP fusions of CERT.
Figure 6: CERT-dependent trafficking of ceramide in semi-intact cells.


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We thank J. Cunningham for the mCAT-1 cDNA; T. Kitamura for the Plat-E cells; O. Kuge for the 5′-RACE- and 3′-RACE-ready CHO cDNAs and the E. coli expression plasmids for CHO Sar1a and Sar1a[T39N]; and Y. Sekizawa for lysenin. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

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Correspondence to Kentaro Hanada.

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Hanada, K., Kumagai, K., Yasuda, S. et al. Molecular machinery for non-vesicular trafficking of ceramide. Nature 426, 803–809 (2003).

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