Table 1 | Lysosomal storage disorders* Disease Defective protein Main storage materials Sphingolipidoses Fabry α-Galactosidase A Globotriasylceramide and blood-group-B substances Farber lipogranulomatosis Ceramidase Ceramide Gaucher

The extensive diversity of membrane lipids is rarely appreciated by cell and molecular biologists. Although most researchers are familiar with the three main classes of lipids in animal cell membranes, few realize the enormous combinatorial structural diversity that exists within each lipid class, a diversity that enables functional specialization of lipids. In this brief review, we focus on one class of membrane lipids, the sphingolipids, which until not long ago were thought by many to be little more than structural components of biological membranes. Recent studies have placed sphingolipidsincluding ceramide, sphingosine and sphingosine-1-phosphate-at the centre of a number of important biological processes, specifically in signal transduction pathways, in which their levels change in a highly regulated temporal and spatial manner. We outline exciting progress in the biochemistry and cell biology of sphingolipids and focus on their functional diversity. This should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingolipids in regulating defined aspects of cell physiology. Keywords: lipids; sphingolipids; second messenger; ceramide; sphingosine-1-phosphate; raft EMBO reports (2004EMBO reports ( ) 5, 777-782. doi:10.1038 Why biologists can no longer afford to ignore lipids ''Cell membranes are crucial to the life of the cell''. Thus begins a description of the plasma membrane in one of our favourite textbooks (Alberts et al, 2002). Reading on, we learn that "lipids constitute about 50% of the mass of most animal cell membranes," and that there are "about 10 9 lipid molecules in the plasma membrane of a small animal cell." But sadly, the space devoted to further discussion of lipids in this book, and it must be said (lest the reader think that we have an axe to grind against this excellent book), in most other cell biology and biochemistry textbooks, is far less than that expected on the basis of their high abundance. Why is this? Is so much known about lipid cell biology that there is little new to discuss? Are lipids too boring to be worthy of significant attention? The answer is probably related to the perceived difficulties of working with membrane lipids and to a lack of appreciation of the essential and diverse roles that membrane lipids exert in regulating a wide spectrum of physiological events.Cell membranes and lipids are indeed crucial to the life of the cell, but as many famous and wise people have said before us, "the devil is in the details". And the details are complicated. Not only are there three main classes of lipids in cell membranes (glycerolipids, sphingolipids and sterols), but also a huge structural variety exists within each class so that hundreds of individual lipid species (and tens of thousands of subspecies) exist within each cell. Does each individual lipid species have defined functions? How are their levels maintained and regulated? How is their complex intracellular distribution ...

Summary In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their stucture and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases.

Cellular life depends on continuous transport of lipids and small molecules between mitochondria and the endomembrane system. Recently, endoplasmic reticulum-mitochondrial encounter structure (ERMES) was identified as an important yet nonessential contact for such transport. Using a high-content screen in yeast, we found a contact site, marked by Vam6/Vps39, between vacuoles (the yeast lysosomal compartment) and mitochondria, named vCLAMP (vacuole and mitochondria patch). vCLAMP is enriched with ion and amino-acid transporters and has a role in lipid relay between the endomembrane system and mitochondria. Critically, we show that mitochondria are dependent on having one of two contact sites, ERMES or vCLAMP. The absence of one causes expansion of the other, and elimination of both is lethal. Identification of vCLAMP adds to our ability to understand the complexity of interorganellar crosstalk.

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Inhibition of ceramide synthesis prevents diabetes, steatosis, and cardiovascular disease in rodents. Six different ceramide synthases (CerS) that differ in tissue distribution and substrate specificity account for the diversity in acyl-chain composition of distinct ceramide species. Haploinsufficiency for ceramide synthase 2 (CerS2), the dominant isoform in the liver that preferentially makes very-long-chain (C22/C24/C24:1) ceramides, led to compensatory increases in long-chain C16-ceramides and conferred susceptibility to diet-induced steatohepatitis and insulin resistance. Mechanistic studies revealed that these metabolic effects were likely due to impaired β-oxidation resulting from inactivation of electron transport chain components. Inhibiting global ceramide synthesis negated the effects of CerS2 haploinsufficiency in vivo, and increasing C16-ceramides by overexpressing CerS6 recapitulated the phenotype in isolated, primary hepatocytes. Collectively, these studies reveal that altering sphingolipid acylation patterns impacts hepatic steatosis and insulin sensitivity and identify CerS6 as a possible therapeutic target for treating metabolic diseases associated with obesity.

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ~200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with nonneuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α) 8 TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.

Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells. TRH1 or TRH4-overexpression elevated [ 3 H](dihydro)ceramide synthesis from L-[3-3 H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B 1 (FB 1 ). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB 1 . A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acylCoA substrates, although in a FB 1 -sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells. These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3-4) that Lag1p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids.Ceramide is an important intracellular second messenger involved in a number of regulatory processes (1). Intracellular ceramide levels can be altered by a variety of mechanisms, including de novo synthesis, which can impact upon cell growth, regulation, differentiation, and death (2-7). A key enzyme in the de novo pathway is sphinganine:N-acyl transferase (dihydroceramide synthase). Significant steps toward determining the modes of regulation of this enzyme were achieved by the demonstration that the yeast proteins, Lag1p and Lac1p, are required for the synthesis of very long chain (C26) ceramides in yeast (8), and that Alternaria stem canker locus-1 (asc1), a member of the longevity assurance gene (lag) 1 family, mediates resistance to fumonisin B 1 (FB 1 ), a dihydroceramide synthase inhibitor (9 -11), in tomato (12, 13). We subsequently demonstrated that overexpression of a mammalian homolog of these proteins, upstream of growth and differentiation factor 1 (UOG1), conferred FB 1 -resistance in mammalian cells, and unexpectedly, specifically regulated the synthesis of stearoylcontaining sphingolipids (SLs) (14). Data base analyses then revealed a family of Lag1p motif-containing proteins (15-17). UOG1 was classified with fungal and plant Lag1p homologs, and a new branch of the subfamily containing a homeobox-like domain at the N terminus was revealed in animals. Five mammalian proteins, originally characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH), were ...