In mammalian cells, ceramide is synthesized in the endoplasmic reticulum and transferred to the Golgi apparatus for conversion to sphingomyelin. Ceramide transport occurs in a nonvesicular manner and is mediated by CERT, a cytosolic 68-kDa protein with a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. The CERT START domain efficiently transfers natural D-erythro-C16-ceramide, but not lipids with longer (C20) amide-acyl chains. The molecular mechanisms of ceramide specificity, both stereo-specific recognition and length limit, are not well understood. Here we report the crystal structures of the CERT START domain in its apo-form and in complex with ceramides having different acyl chain lengths. In these complex structures, one ceramide molecule is buried in a long amphiphilic cavity. At the far end of the cavity, the amide and hydroxyl groups of ceramide form a hydrogen bond network with specific amino acid residues that play key roles in stereo-specific ceramide recognition. At the head of the ceramide molecule, there is no extra space to accommodate additional bulky groups. The two aliphatic chains of ceramide are surrounded by the hydrophobic wall of the cavity, whose size and shape dictate the length limit for cognate ceramides. Furthermore, local high-crystallographic B-factors suggest that the ␣-3 and the ⍀1 loop might work as a gate to incorporate the ceramide into the cavity. Thus, the structures demonstrate the structural basis for the mechanism by which CERT can distinguish ceramide from other lipid types yet still recognize multiple species of ceramides. crystal structure ͉ lipid transport ͉ sphingomyelin ͉ diacylglycerol

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 resistant11-1 (fbr11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B 1 (FB 1 ), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base1 (LCB1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB 1 . By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine-1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.

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Protein phosphatase 2C⑀ (PP2C⑀), a mammalian PP2C family member, is expressed in various tissues and is implicated in the negative regulation of stress-activated protein kinase pathways. We show that PP2C⑀ is an endoplasmic reticulum (ER) transmembrane protein with a transmembrane domain at the amino terminus and the catalytic domain facing the cytoplasm. Yeast two-hybrid screening of a human brain library using PP2C⑀ as bait resulted in the isolation of a cDNA that encoded vesicle-associated membrane protein-associated protein A (VAPA). VAPA is an ER resident integral membrane protein involved in recruiting lipidbinding proteins such as the ceramide transport protein CERT to the ER membrane. Expression of PP2C⑀ resulted in dephosphorylation of CERT in a VAPA expression-dependent manner, which was accompanied by redistribution of CERT from the cytoplasm to the Golgi apparatus. The expression of PP2C⑀ also enhanced the association between CERT and VAPA. In addition, knockdown of PP2C⑀ expression by short interference RNA attenuated the interaction between CERT and VAPA and the sphingomyelin synthesis. These results suggest that CERT is a physiological substrate of PP2C⑀ and that dephosphorylation of CERT by PP2C⑀ may play an important role in the regulation of ceramide trafficking from the ER to the Golgi apparatus.Vesicle-associated membrane protein-associated protein A (VAPA) 3 is an endoplasmic reticulum (ER)-resident type II transmembrane protein with homologs widely distributed from yeast to human (1-3). Recently, evidence has accumulated that in mammalian cells VAPA participates in the regulation of inter-organelle transport of membrane lipids by recruiting lipid transfer proteins to the ER membrane. VAPA associates with a short, conserved peptide sequence termed the "two phenylalanines in an acidic tract" (FFAT) motif that is found in several lipid transfer proteins including ceramide transport protein CERT, oxysterol-binding protein, Opi1 protein, and PITP/Nir/ rdgB families (4 -11). VAPA is composed of two conserved domains, an amino-terminal immunoglobulin-like ␤ sheet responsible for FFAT motif binding and a carboxyl-terminal transmembrane domain (8). In addition to its role in recruiting FFAT motif-targeted proteins to ER membranes, VAPA has been proposed to function in vesicle trafficking (1,(12)(13)(14), in the organization of the microtubule network (10, 15), and in the replication of hepatitis C virus RNA (16,17). In mammalian cells ceramide is synthesized in the ER and transported to the Golgi apparatus where it is converted to sphingomyelin (SM). The ceramide transport protein CERT plays a key role in the ER-to-Golgi trafficking of ceramide (18 -20). CERT consists of several distinct domains including a Steroidogenic acute regulator-related lipid transfer (START) domain capable of specifically extracting ceramide from membrane, a pleckstrin homology (PH) domain that serves to target the Golgi apparatus by recognizing phosphatidylinositol 4-monphophatate, and a FFAT motif, which interacts with VA...

SummaryRecent discoveries of two sphingolipid transfer proteins, CERT and FAPP2, have brought the field of sphingolipid metabolism to a more dynamic stage. CERT transfers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, a step crucial for sphingomyelin (SM) synthesis. The pleckstrin homology (PH) domain and the FFAT motif of CERT restrict the direction of transfer and destination of ceramide through binding to phosphatidylinositol 4-monophosphate (PI4P) at the Golgi and the ER resident proteins, VAPs, respectively. CERT is regulated by the phosphorylation and dephosphorylation of serine/threonine, in which protein kinase D, possibly casein kinase I, and PP2Ce are involved. On the other hand, FAPP2 transfers glucosylceramide (GlcCer) to appropriate sites for the synthesis of complex glycosphingolipids. Like CERT, FAPP2 contains a PH domain, the binding of which to PI4P is required for its localization to the Golgi. These observations indicate that lipid transfer proteins, CERT and FAPP2, spatially regulate lipid metabolism on the cytosolic side.2008 IUBMB IUBMB Life, 60(8): 511-518, 2008

Intracellullar trafficking of lipids is fundamental to membrane biogenesis. For the synthesis of sphingomyelin, ceramide is transported from the endoplasmic reticulum to the Golgi apparatus by the ceramide transfer protein CERT. CERT is phosphorylated by protein kinase D at S132 and subsequently multiple times in a serine-repeat motif, resulting in its inactivation. However, the kinase involved in the multiple phosphorylation remains unclear. Here, we identify the ␥2 isoform of casein kinase I (CKI␥2) as a kinase whose overexpression confers sphingomyelin-directed toxin-resistance to Chinese hamster ovary cells. In a transformant stably expressing CKI␥2, CERT was hyperphosphorylated, and the intracellular trafficking of ceramide was retarded, thereby reducing de novo sphingomyelin synthesis. The reduction in the synthesis of sphingomyelin caused by CKI␥2 was reversed by the expression of CERT mutants that are not hyperphosphorylated. Furthermore, CKI␥2 directly phosphorylated CERT in vitro. Among three ␥ isoforms, only knockdown of ␥2 isoform caused drastic changes in the ratio of hypo-to hyperphosphorylated form of CERT in HeLa cells. These results indicate that CKI␥2 hyperphosphorylates the serine-repeat motif of CERT, thereby inactivating CERT and down-regulating the synthesis of sphingomyelin.

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