Robert L. Lester

SPHINGOLIPIDS ARE POTENTIAL HEAT STRESS SIGNALS IN SACCHAROMYCES

The ability of organisms to quickly respond to stresses requires the activation of many intracellular signal transduction pathways. The sphingolipid intermediate ceramide is thought to be particularly important for activating and coordinating signaling pathways during mammalian stress responses. Here we present the first evidence that ceramide and other sphingolipid intermediates are signaling molecules in the Saccharomyces cerevisiaeheat stress response. Our data show a 2–3-fold transient increase in the concentration of C18-dihydrosphingosine and C18-phytosphingosine, more than a 100-fold transient increase in C20-dihydrosphingosine and C20-phytosphingosine, and a more stable 2-fold increase in ceramide containing C18-phytosphingosine and a 5-fold increase in ceramide containing C20-phytosphingosine following heat stress. Treatment of cells with dihydrosphingosine activates transcription of the TPS2 gene encoding a subunit of trehalose synthase and causes trehalose, a known thermoprotectant, to accumulate. Dihydrosphingosine induces expression of aSTRE-LacZ reporter gene, showing that the global stress response element, STRE, found in many yeast promoter sequences can be activated by sphingolipid signals. TheTPS2 promoter contains four STREs that may mediate dihydrosphingosine responsiveness. Using genetic and other approaches it should be possible to identify sphingolipid signaling pathways in S. cerevisiae and quantify the importance of each during heat stress.

Sphingolipid functions in Saccharomyces cerevisiae

Recent advances in understanding sphingolipid metabolism and function in Saccharomyces cerevisiae have moved the field from an embryonic, descriptive phase to one more focused on molecular mechanisms. One advance that has aided many experiments has been the uncovering of genes for the biosynthesis and breakdown of sphingolipids. S. cerevisiae seems on the verge of becoming the first organism in which all sphingolipid metabolic genes are identified. Other advances include the demonstration that S. cerevisiae cells have lipid rafts composed of sphingolipids and ergosterol and that specific proteins associate with rafts. Roles for phytosphingosine (PHS) and dihydrosphingosine (DHS) in heat stress continue to be uncovered including regulation of the transient cell cycle arrest, control of putative signaling pathways that govern cell integrity, endocytosis, movement of the cortical actin cytoskeleton and regulation of protein breakdown in the plasma membrane. Other studies suggest roles for sphingolipids in exocytosis, growth regulation and longevity. Finally, some progress has been made in understanding how sphingolipid synthesis is regulated and how sphingolipid levels are maintained.

The LCB4 (YOR171c) and LCB5(YLR260w) Genes of Saccharomyces Encode Sphingoid Long Chain Base Kinases

Sphingolipid long chain bases (LCBs) and phosphorylated derivatives, particularly sphingosine 1-phosphate, are putative signaling molecules. To help elucidate the physiological roles of LCB phosphates, we identified two Saccharomyces cerevisiae genes, LCB4 (YOR171c) andLCB5 (YLR260w), which encode LCB kinase activity. This conclusion is based upon the synthesis of LCB kinase activity in Escherichia coli expressing eitherLCB gene. LCB4 encodes most (97%)Saccharomyces LCB kinase activity, with the remainder requiring LCB5. Log phase lcb4-deleted yeast cells make no LCB phosphates, showing that the Lcb4 kinase synthesizes all detectable LCB phosphates under these growth conditions. The Lcb4 and Lcb5 proteins are paralogs with 53% amino acid identity but are not related to any known protein, thus revealing a new class of lipid kinase. Two-thirds of the Lcb4 and one-third of the Lcb5 kinase activity are in the membrane fraction of yeast cells, a puzzling finding in that neither protein contains a membrane-localization signal. Both enzymes can use phytosphingosine, dihydrosphingosine, or sphingosine as substrate. LCB4 and LCB5 should be useful for probing the functions of LCB phosphates in S. cerevisiae. Potential mammalian cDNA homologs of the LCB kinase genes may prove useful in helping to understand the function of sphingosine 1-phosphate in mammals.

Synthesis of Mannose-(inositol-P)2-ceramide, the Major Sphingolipid in Saccharomyces cerevisiae, Requires the IPT1 (YDR072c) Gene

Knowledge of the Saccharomyces cerevisiaegenes and proteins necessary for sphingolipid biosynthesis is far from complete. Such information should expedite studies of pathway regulation and sphingolipid functions. Using the Aur1 protein sequence, recently identified as necessary for synthesis of the sphingolipid inositol-P-ceramide (IPC), we show that a homolog (open reading frameYDR072c), termed Ipt1 (inositolphosphotransferase1) is necessary for synthesis of mannose-(inositol-P)2-ceramide (M(IP)2C), the most abundant and complex sphingolipid in S. cerevisiae. This conclusion is based upon analysis of an ipt1-deletion strain, which fails to accumulate M(IP)2C and instead accumulates increased amounts of the precursor mannose-inositol-P-ceramide. The mutant also fails to incorporate radioactive precursors into M(IP)2C, and membranes prepared from it do not incorporate [3H-inositol]phosphatidylinositol into M(IP)2C, indicating a lack of M(IP)2C synthase activity (putatively phosphatidylinositol:mannose-inositol-P-ceramide phosphoinositol transferase). M(IP)2C synthase activity is inhibited in the micromolar range by aureobasidin A, but drug sensitivity is over 1000-fold lower than reported for IPC synthase activity. An ipt1-deletion mutant has no severe phenotypic effects but is slightly more resistant to growth inhibition by calcium ions. Identification of the IPT1 gene should be helpful in determining the function of the M(IP)2C sphingolipid and in determining the catalytic mechanism of IPC and M(IP)2C synthases.

METABOLISM AND SELECTED FUNCTIONS OF SPHINGOLIPIDS IN THE YEAST SACCHAROMYCES CEREVISIAE

Our knowledge of sphingolipid metabolism and function in Saccharomyces cerevisiae is growing rapidly. Here we discuss the current status of sphingolipid metabolism including recent evidence suggesting that exogenous sphingoid long-chain bases must first be phosphorylated and then dephosphorylated before incorporation into ceramide. Phenotypes of strains defective in sphingolipid metabolism are discussed because they provide hints about the undiscovered functions of sphingolipids and are one of the major reasons for studying this model eukaryote. The long-chain base phosphates, dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate, have been hypothesized to play roles in heat stress resistance, perhaps acting as signaling molecules. We evaluate the data supporting this hypothesis and suggest future experiments needed to verify it. Finally, we discuss recent clues that may help to reveal how sphingolipid synthesis and total cellular sphingolipid content are regulated.

In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae.

1. 1. Evidence is given for reactions leading to the formation of all the major glycerol containing phospholipids of Saccharomyces cerevisiae. A cell free particulate fraction prepared from S. cerevisiae is capable of incorporating sn-[14C]glycero-3-phosphoric acid into phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerophosphate, phosphatidylglycerol, cardiolipin, CDP-diglyceride and diglyceride. These products were separated by a two-dimensional chromatography system. 2. 2. The incorporation of sn-[14C]glycero-3-phosphoric acid into phosphatidic acid requires CoA and ATP and is greatly stimulated by the addition of divalent cations. The incorporation of this label into CDP-diglyceride, phosphatidylserine, phosphatidylinositol, phosphatidylglycerophosphate, phosphatidylglycerol and cardiolipin has an additional requirement for CTP. Inositol and serine enhance the incorporation of sn-[14C]glycero-3-phosphoric acid into phosphatidylinositol and phosphatidylserine, respectively. Evidence is given for two pathways for phosphatidylethanolamine synthesis, from the reaction of CDP-ethanolamine and diglyceride and by the decarboxylation of phosphatidylserine. There are also two pathways for the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine and the reaction of CDP-choline and diglyceride. CDP-diglyceride may therefore be a precursor, directly or indirectly, of all the phospholipids under study. The direct conversion of labeled CDP-diglyceride to some of the phospholipids is demonstrated. 3. 3. Formation of CDP-diglyceride from endogenous precursors appears to be a major reaction.

SYRINGOMYCIN ACTION GENE SYR2 IS ESSENTIAL FOR SPHINGOLIPID 4-HYDROXYLATION IN SACCHAROMYCES CEREVISIAE

The Saccharomyces cerevisiae geneSYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of asyr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.

Pil1p and Lsp1p Negatively Regulate the 3-Phosphoinositide-dependent Protein Kinase-like Kinase Pkh1p and Downstream Signaling Pathways Pkc1p and Ypk1p

The Saccharomyces cerevisiae homologs, Pkh1/2p, of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1) regulate the Pkc1-MAP kinase cascade and the partially parallel Ypk1/2p pathway(s) that control growth and cell integrity. Mammalian PDK1 is regulated by 3-phosphoinositides, whereas Pkh1/2p are regulated by sphingolipid long-chain bases (LCBs). Recently Pkh1/2p were found to complex with two related proteins, Pil1p (Ygr086) and Lsp1p (Ypl004). Because these two proteins are not related to any known protein we sought to characterize their functions. We show that Pkh1p phosphorylates both proteins in vitro in a reaction that is only weakly regulated by LCBs. In contrast, LCBs inhibit phosphorylation of Pil1p by Pkh2p, whereas LCBs stimulate phosphorylation of Lsp1p by Pkh2p. We find that Pil1p and Lsp1p down-regulate resistance to heat stress and, specifically, that they down-regulate the activity of the Pkc1p-MAP and Ypk1p pathways during heat stress. Pil1p and Lsp1p are thus the first proteins identified as regulators of Pkh1/2p. An unexpected finding was that the level of Ypk1p is greatly reduced in pkc1Δ cells, indicating that Pkc1p controls the level of Ypk1p. Homologs of Pil1p and Lsp1p are widespread in nature, and our results suggest that they may be negative regulators of PDK-like protein kinases and their downstream cellular pathways that control cell growth and survival.

The Sphingoid Long Chain Base Phytosphingosine Activates AGC-type Protein Kinases in Saccharomyces cerevisiae Including Ypk1, Ypk2, and Sch9

The Pkh1 protein kinase of Saccharomyces cerevisiae, a homolog of the mammalian 3-phosphoinositide-dependent kinase (PDK1), regulates downstream AGC-type protein kinases including Ypk1/2 and Pkc1, which control cell wall integrity, growth, and other processes. Phytosphingosine (PHS), a sphingoid long chain base, is hypothesized to be a lipid activator of Pkh1 and thereby controls the activity of Ypk1/2. Here we present biochemical evidence supporting this hypothesis, and in addition we demonstrate that PHS also stimulates autophosphorylation and activation of Ypk1/2. Greatest stimulation of Ypk1/2 phosphorylation and activity are achieved by inclusion of both PHS and Pkh1 in an in vitro kinase reaction. We also demonstrate for the first time that Pkh1 phosphorylates the Sch9 protein kinase in vitro and that such phosphorylation is stimulated by PHS. This is the first biochemical demonstration of Sch9 activators, and the results further support roles for long chain bases in heat stress resistance in addition to implying roles in chronological aging and cell size determination, since Sch9 functions in these processes. Thus, our data support a model in which PHS, rather than simply being an upstream activator of Pkh1, also activates kinases that are downstream targets of Pkh1 including Ypk1/2 and Sch9.

Identification of a Saccharomyces Gene,LCB3, Necessary for Incorporation of Exogenous Long Chain Bases into Sphingolipids

To identify genes necessary for sphingolipid synthesis in Saccharomyces cerevisiae we developed a procedure to enrich for mutants unable to incorporate exogenous long chain base into sphingolipids. We show here that a mutant strain, AG84-3, isolated by using the enrichment procedure, makes sphingolipids from endogenously synthesized but not from exogenously supplied long chain base. A gene termed LCB3 (YJL134W, GenBank designation X87371x21), which complements the long chain base utilization defect of strain AG84-3, was isolated from a genomic DNA library. The gene is predicted to encode a protein with multiple membrane-spanning domains and a COOH-terminal glycosylphosphatidylinositiol cleavage/attachment site. Deletion of thelcb3 gene in a wild type genetic background reduces the rate of exogenous long chain base incorporation into sphingolipids and makes the host strain more resistant to growth inhibition by long chain bases. Only one protein in current data bases, the S. cerevisiae open-reading frame YKR053C, whose function is unknown, shows homology to the Lcb3 protein. The two proteins are not, however, functional homologs because deletion of theYKR053C open reading frame does not impair long chain base utilization or enhance resistance of cells to growth inhibition by long chain bases. Based upon these data we hypothesize that the Lcb3 protein is a plasma membrane transporter capable of transporting sphingoid long chain bases into cells. It is the first candidate for such a transporter and the first member of what appears to be a new class of membrane-bound proteins.