Sphingolipids and phosphoinositides both play signaling roles in Saccharomyces cerevisiae. Although previous data indicate independent functions for these two classes of lipids, recent genetic studies have suggested interactions between phosphatidylinositol (PtdIns) phosphate effectors and sphingolipid biosynthetic enzymes. The present study was undertaken to further define the effects of phosphatidylinositol 4-phosphate (PtdIns(4)P) metabolism on cell sphingolipid metabolism. The data presented indicate that deletion of SAC1, a gene encoding a PtdIns(4)P phosphatase, increased levels of most sphingolipid species, including sphingoid bases, sphingoid base phosphates, and phytoceramide. In contrast, sac1⌬ dramatically reduced inositol phosphosphingolipids, which result from the addition of a PtdIns-derived phosphoinositol head group to ceramides through Aur1p. Deletion of SAC1 decreased PtdIns dramatically in both steady-state and pulse labeling studies, suggesting that the observed effects on sphingolipids may result from modulation of the availability of PtdIns as a substrate for Aur1p. Supporting this hypothesis, acute attenuation of PtdIns(4)P production through Stt4p immediately increased PtdIns and subsequently reduced sphingoid bases. This reduction was overcome by the inhibition of Aur1p. Moreover, modulation of sphingoid bases through perturbation of PtdIns(4)P metabolism initiated sphingolipid-dependent biological effects, supporting the biological relevance for this route of regulating sphingolipids. These findings suggest that, in addition to potential signaling effects of PtdInsP effectors on sphingolipid metabolism, PtdIns kinases may exert substantial effects on cell sphingolipid profiles at a metabolic level through modulation of PtdIns available as a substrate for complex sphingolipid synthesis.Sphingolipids play vital roles in Saccharomyces cerevisiae, including regulation of translation, cell cycle, sporulation, ubiquitin-dependent proteolysis, actin cytoskeleton rearrangements, endocytosis, stress responses, and numerous other processes (see Refs. 1-3 for review). Phosphoinositides comprise another group of key lipid mediators that regulate numerous cellular functions, including organization of the actin cytoskeleton, endocytosis, cytokinesis, vacuolar morphology, and translation initiation (4 -7). The apparent overlap in biological roles of these distinct classes of lipids implies interaction between them. Indeed, recent studies support regulation of sphingolipid metabolism by phosphoinositides (8 -11).Sphingolipid metabolism has been reviewed extensively, but, in brief, sphingolipid synthesis begins with the condensation of palmitoyl-coenzyme A and serine by serine-palmitoyl transferase to form 3-ketodihydrosphingosine (12) (Fig. 1). This shortlived intermediate is quickly converted to the sphingoid bases dihydrosphingosine (DHS) 2 and phytosphingosine (PHS), which undergo phosphorylation to form sphingoid base phosphates or N-acylation to form ceramides (13, 14). Phytoceramide and phosphati...

Much data implicate saturated fatty acids in deleterious processes associated with obesity, diabetes, and the metabolic syndrome. Many of these changes may be due to aberrant generation of bioactive lipids when saturated fatty acid availability to tissues is increased. On the other hand, studies are emerging that implicate the monounsaturated fatty acid oleate in protection from saturated fat mediated toxicity; however, the mechanisms are not well understood. Our data demonstrate a novel role for palmitate in increasing mRNA encoding DES1, which is the enzyme responsible for generating ceramide from its precursor dihydroceramide and thus controls synthesis of the bioactive lipid ceramide. Moreover, co-treatment with oleate prevented the increase in ceramide, and this occurred through attenuation of the increase in message and activity of DES1. Knockdown of DES1 also protected from palmitate-induced insulin resistance, and overexpression of this enzyme ameliorated the protective effect of oleate. Together, these findings provide insight into the mechanisms of oleate-mediated protection against metabolic disease and provide novel evidence for fatty acid-mediated regulation of a key enzyme of ceramide biosynthesis.The rise in obesity in recent years has initiated a pandemic of metabolic disease including heart disease and diabetes (1, 2). Perturbations in lipid metabolism and/or endocrine function that occur in obesity likely mediate the development of a disorders, including insulin resistance, nonalcoholic fatty liver disease, pancreatic cell death, hypertension, and the "metabolic syndrome" (3, 4). Although the mechanistic links between obesity and these pathophysiological processes remain incompletely understood, one proposed mechanism is that the elevation in plasma lipids associated with obesity overloads tissues with precursors for synthesis of bioactive lipids, including diacylglycerols (DAG) 2 and ceramides (5, 6).Epidemiological data link diets high in saturated fatty acids with increased incidence of metabolic disease (6, 7). Supporting a mechanistic connection between saturated fatty acids and metabolic syndrome, many studies demonstrate that the saturated fatty acid palmitate promotes insulin resistance in heart, adipose, and skeletal muscle (8 -10). On the other hand, data are emerging which support that unsaturated fatty acids such as oleate have protective effects against palmitate toxicity, though the mechanisms are not fully understood (11,12).Several studies demonstrate that ceramide plays a key role in insulin resistance in human skeletal muscle cells (13-15). Biosynthesis of sphingolipids including ceramide begins with condensation of serine with acyl-CoA such as palmitoyl-CoA. Exposing muscle cells to palmitate increased ceramide synthesis and inhibited insulin stimulation of Akt/protein kinase B, a serine/threonine kinase that is a central mediator of insulinstimulated anabolic metabolism (13); moreover, inhibiting ceramide synthesis negated the antagonistic effect of saturated free fatty acid...

Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein.More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1⌬ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5⌬ yor1 strains and reduced in rsb1⌬ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.Sphingolipids represent one of the major components of the lipid fraction of the eukaryotic plasma membrane. Biosynthesis of these lipids proceeds through production of ceramide that is formed from the linkage of a long chain base (LCB) 4 with a very long chain fatty acid. In the yeast Saccharomyces cerevisiae, one of the two LCBs produced in vivo is referred to as phytosphingosine (PHS) (for reviews see Refs.1, 2). PHS is required for sphingolipid production but also has regulatory properties in terms of subcellular localization of proteins. Elevated levels of PHS cause mislocalization of nutrient permeases from the plasma membrane to the vacuole where these proteins are degraded (3, 4). Regulation of PHS levels in the cell is tightly controlled and important to ensure normal metabolism.One of the best described routes of PHS degradation is provided by the LCB-phosphate lyase Dpl1 (5). This enzyme breaks LCB phosphate into an aldehyde and ethanolamine phosphate limiting accumulation of LCBs. Strains lacking Dpl1 are hypersensitive to PHS. This phenotype was exploited to identify an integral membrane protein designated Rsb1 that, when overproduced, suppressed the PHS hypersensitivity of a dpl1⌬ strain (6). Evidence was presented that elevated levels of Rsb1 led to increased LCB efflux. More recent work demonstrated that loss of the multidrug transporters Pdr5 and Yor1 from cells led to a strong increase in PHS tolerance...

Sphingolipids are an important class of structural and signaling molecules within the cell. As sphingolipids have been implicated in the development and pathogenesis of insulin resistance and the metabolic syndrome, it is important to understand their regulation and metabolism. Although these lipids are initially produced through a common pathway, there is no "generic" sphingolipid. Indeed, the biophysical and signaling properties of lipids may be manipulated by the subunit composition or isoform of their synthetic enzymes, via regulation of substrate integration. Functionally distinct pools of chemically-equivalent lipids may also be generated by de novo synthesis and recycling of existing complex sphingolipids. The highly integrated metabolism of the many bioactive sphingolipids means that manipulation of one enzyme or metabolite can result in a ripple effect, causing unforeseen changes in metabolite levels, enzyme activities, and cellular programmes. Fortunately, a suite of techniques, ranging from thin-layer chromatography to liquid chromatography-mass spectrometry approaches, allows investigators to undertake a functional characterization of all or part of the sphingolipidome in their systems of interest.

Background:The serine deaminase CHA1 responds to heat stress in a sphingolipid-dependent manner. Results: CHA1 requires de novo sphingoid base production for induction by serine, limiting growth-suppressing accumulation of sphingoid bases. Conclusion: Sphingoid bases are feedback sensors of serine availability, forming a feedforward/feedback loop through CHA1. Significance: This study defines a fundamental connection between sphingolipid and amino acid metabolic pathways with implications for disease.Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1⌬ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1⌬ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.Sphingolipids constitute a unique class of lipids that have been implicated in a variety of functions in yeast, including nutrient uptake and cell cycle regulation. In particular, several responses to heat stress have been shown to require de novo synthesis of sphingolipids, and these include cell cycle arrest, proteolysis, and nutrient import (1-6).Microarray analysis analyzing the effects of heat stress on gene expression in the lcb1-100 strain, defective in de novo synthesis, was previously used to define genes whose regulation depended on de novo synthesis of sphingolipids (7). An especially intriguing gene among these is CHA1, which encodes a serine deamidase/dehydratase, known to be up-regulated by exogenous serine (8,9). This was of great interest because the enzyme serves to attenuate serine levels and because serine also serves as a limiting substrate in the ...