Reconstitution Assay System for Ceramide Transport With Semi-Intact Cells
ABSTRACT The intracellular transport of lipids from the sites of their synthesis to their appropriate destination is a critical step for lipid metabolism. One well-defined inter-organelle lipid movement is the transport of ceramide by ceramide transport protein (CERT). Ceramide, a key intermediate for both sphingomyelin and glycosphingolipids, is synthesized at the endoplasmic reticulum and delivered to the Golgi apparatus to be converted to sphingomyelin. CERT delivers ceramide from the ER to the Golgi apparatus in a non-vesicular and ATP-dependent manner. This chapter describes a reconstitution assay system for ceramide transport with semi-intact cells, which is useful for the study of the CERT-mediated inter-organelle transport of ceramide.
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ABSTRACT: The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We here showed that the phosphorylation of CERT at serine-315 (S315), which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at S315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine rich motif; SRM) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the SRM and the phosphorylation of S315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.Journal of Biological Chemistry 02/2014; 289(15). DOI:10.1074/jbc.M113.528380 · 4.57 Impact Factor