Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.     

Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis

Sphingolipids are important signaling molecules involved in various cellular activities. De novo sphingolipid synthesis is initiated by a rate-limiting enzyme, serine palmitoyltransferase (SPT), a heterodimer consisting of LONG-CHAIN BASE1 (LCB1) and LCB2 subunits. A mutation in the Arabidopsis thaliana LCB1 gene, lcb1-1, was found to cause embryo lethality. However, the underpinning molecular and cellular mechanisms remain largely unclear. Here, we report the identification of the fumonisin B(1) resistant11-2 (fbr11-2) mutant, an allele of lcb1-1. The fbr11-2 mutation, most likely an allele stronger than lcb1-1, was transmitted only through female gametophytes and caused the formation of abortive microspores. During the second pollen mitosis, fbr11-2 initiated apoptotic cell death in binucleated microspores characteristic of nuclear DNA fragmentation, followed by cytoplasm shrinkage and organelle degeneration at the trinucleated stage. In addition, a double mutant with T-DNA insertions in two homologous LCB2 genes showed a phenotype similar to fbr11-2. Consistent with these observations, the FBR11/LCB1 expression was confined in microspores during microgametogenesis. These results suggest that SPT-modulated programmed cell death plays an important role in the regulation of male gametophyte development.     

The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.     

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.     

Death Resulting from Fatty Acid Starvation in Yeast

Mutants of Saccharomyces cerevisiae having the genotypes fas1 (fatty acid synthetase minus) and fas1, ole1 (fatty acid synthetase and fatty acid desaturase minus) were found to undergo logarithmic death when deprived of required fatty acids, whereas ole1 strains did not. During the first 2 to 3 h of fatty acid starvation, macromolecular synthesis occurred at apparently normal rates, although cell division stopped by the end of the 1st h. Cell death commenced at approximately the 2nd to the 3rd h, and within 24 h, depending upon conditions, 2 to 4 log orders of death had occurred. The loss of viability was accelerated by the addition of detergent, but could be largely prevented by the interruption of protein synthesis, either by amino acid starvation or by the use of cycloheximide. The possible significance of this phenomenon in terms of membrane biosynthesis is discussed.     

Analysis of Phosphorylated Sphingolipid Long-Chain Bases Reveals Potential Roles in Heat Stress and Growth Control in Saccharomyces

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in S. cerevisiae cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25°C, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37°C causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44°C heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3 single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.     

Characterization of an Arabidopsis cDNA encoding a subunit of serine palmitoyltransferase, the initial enzyme in sphingolipid biosynthesis.

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis. In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT. The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids. The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding. Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT. Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis. Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum. AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants.     

Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids.

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.     

Sterol-dependent regulation of sphingolipid metabolism in Saccharomyces cerevisiae

We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G. S., Edlind, T., Kurtz, M., and Nickels, J. T., Jr. (2001) J. Biol. Chem. 276, 12702-12711). We have now determined the defects in sphingolipid metabolism in erg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism in Saccharomyces cerevisiae. Using [(3)H]inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells. [(3)H]Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells. Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism. The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation. In addition, [(3)H]inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state. Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1 cells.     

Accumulation of phosphorylated sphingoid long chain bases results in cell growth inhibition in Saccharomyces cerevisiae

Sphingolipid metabolites in mammals can function as signaling molecules with cell-specific functions. In Saccharomyces cerevisiae, phosphorylated long chain bases, such as dihydrosphingosine 1-phosphate and phytosphingosine 1-phosphate, have also been implicated in stress responses. To further explore the biological roles of these molecules, we created disruption mutants for LCB4, LCB5, DPL1, YSR2, YSR3, and SUR2. LCB4 and LCB5 encode kinases that phosphorylate long chain bases. DPL1 and YSR2/YSR3 are involved in degradation of the phosphorylated long chain bases. SUR2 catalyzes conversion of dihydrosphingosine to phytosphingosine. We adapted an HPLC method to measure intracellular concentrations of the phosphorylated long chain bases. Double mutants of dpl1 and ysr2 were inviable, whereas dpl1 ysr2 lcb4 triple mutants were viable. Further, growth inhibition associated with accumulated phosphorylated long chain bases was observed in the triple mutant dpl1 ysr2 lcb4 overexpressing LCB4 or LCB5. These results indicate that phosphorylated long chain bases can inhibit cell growth. Mutants defective in both YSR2 and SUR2, which accumulated dihydrosphingosine 1-phosphate only, grew poorly. The phenotypes of the ysr2 sur2 mutants were suppressed by overexpression of DPL1. Our results clearly show that elevated levels of phosphorylated long chain bases have an antiproliferative effect in yeast.     

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes ( AtLCB2a and AtLCB2b ) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.     

Control of vacuole permeability and protein degradation by the cell cycle arrest signal in Saccharomyces cerevisiae.

Saccharomyces cerevisiae responds to deperivation of nutrients by arresting cell division at the unbudded G1 stage. Cells situated outside of G1 at the time of deperivation complete the cell cycle before arresting. This prompted an investigation of the source of nutrients used by these cells to complete division and the mechanisms controlling their availability. We found a close correlation between accumulation of unbudded cells and loss of previously formed allophanate hydrolase activity after nutrient starvation. These losses were not specific to the allantoin, system since they have been observed for a number of other enzymes and also when cellular protein levels were monitored with [3H]leucine. Loss of hydrolase activity was also observed when protein synthesis was inhibited either by addition of inhibitors or loss of the prtl gene product. We found that onset of nutrient starvation brought about release of large quantities of arginine and allantoin normally sequestered in the cell vacuole. Treatment of a cells with alpha-factor resulted in both the release of allantoin and arginine from the cell vacuole and the onset of intracellular protein degradation. These effects were not observed when either alpha cells or a/alpha diploid strains were treated with alpha-factor. These data suggest that release of vacuolar constitutents and protein turnover may be regulated by the G1 arrest signal.     

Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states

Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from reserve carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.     

Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity

Serine palmitoyltransferase catalyzes the first step of sphingolipid synthesis, condensation of serine and palmitoyl CoA to form the long chain base 3-ketosphinganine. The LCB1/TSC2 and LCB2/TSC1 genes encode homologous proteins of the alpha-oxoamine synthase family required for serine palmitoyltransferase activity. The other alpha-oxoamine synthases are soluble homodimers, but serine palmitoyltransferase is a membrane-associated enzyme composed of at least two subunits, Lcb1p and Lcb2p. Here, we report the characterization of a third gene, TSC3, required for optimal 3-ketosphinganine synthesis in Saccharomyces cerevisiae. S. cerevisiae cells lacking the TSC3 gene have a temperature-sensitive lethal phenotype that is reversed by supplying 3-ketosphinganine, dihydrosphingosine, or phytosphingosine in the growth medium. The tsc3 mutant cells have severely reduced serine palmitoyltransferase activity. The TSC3 gene encodes a novel 80-amino acid protein with a predominantly hydrophilic amino-terminal half and a hydrophobic carboxyl terminus that is membrane-associated. Tsc3p coimmunoprecipitates with Lcb1p and/or Lcb2p but does not bind as tightly as Lcb1p and Lcb2p bind to each other. Lcb1p and Lcb2p remain tightly associated with each other and localize to the membrane in cells lacking Tsc3p. However, Lcb2p is unstable in cells lacking Lcb1p and vice versa.     

Deoxyribonucleic Acid Synthesis and Cell Division in a lon− Mutant of Escherichia coli

The lon(-) mutants of Escherichia coli form long filamentous cells after temporary inhibition of deoxyribonucleic acid (DNA) synthesis by ultraviolet irradiation, treatment with nalidixic acid, or thymine starvation. The kinetics of DNA synthesis and cell division after a period of thymine starvation have been compared in lon(+) and lon(-) cells. After this treatment, both kinds of cells recover their normal DNA to mass ratio with the same kinetics. In contrast to previous reports, cell division is found to recommence in both lon(+) and in lon(-) cells after such a temporary period of inhibition of DNA synthesis. However, the delay separating the recommencement of DNA synthesis and of cell division is approximately three times as long in lon(-) as in lon(+) cells. Low concentrations of penicillin inhibit cell division in both lon(+) and lon(-) cells. In this case, cell division recommences with the same kinetics in both strains after the removal of penicillin. This suggests that different steps in the cell division process are blocked by inhibition of DNA synthesis and by penicillin treatment. The lon(-) mutation appears to affect the former of these steps.