Uptake of exogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae

AbstractSphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.Author SummarySphingolipids (SPs) are membrane lipids globally required for eukaryotic life. In contrast to other lipid classes, SPs cannot be stored in the cell and therefore their levels have to be tightly regulated. Failure to maintain sphingolipid homeostasis can result in pathologies including neurodegeneration, childhood asthma and cancer. However, we are only starting to understand how SP biosynthesis is adjusted according to need. In this study, we use genetic and biochemical methods to show that the uptake of exogenous serine is necessary to maintain SP homeostasis in Saccharomyces cerevisiae. Serine is one of the precursors of long chain bases in cells, the first intermediate of SP metabolism. Our results suggest that the uptake of serine is directly coupled to SP biosynthesis at ER-plasma membrane contact sites. Overall, our study identifies serine uptake as a novel regulatory factor of SP homeostasis. While we use yeast as a discovery tool, these results also provide valuable insights into mammalian SP biology especially under pathological conditions.

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Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption

Biochemical Society Transactions ◽

10.1042/bst0290831 ◽

2001 ◽

Vol 29 (6) ◽

pp. 831-835 ◽

Cited By ~ 62

Author(s):

S. C. Linn ◽

H. S. Kim ◽

E. M. Keane ◽

L. M. Andras ◽

E. Wang ◽

...

Keyword(s):

De Novo ◽

Uv Light ◽

Wide Spectrum ◽

Sphingolipid Metabolism ◽

Intrinsic Activity ◽

Type I ◽

Serine Palmitoyltransferase ◽

Post Translational Modification ◽

Sphingolipid Biosynthesis ◽

Complex Sphingolipids

Complex sphingolipids are ‘built’ on highly bio-active backbones (sphingoid bases and ceramides) that can cause cell death when the amounts are elevated by turnover of complex sphingolipids, disruption of normal sphingolipid metabolism, or over-induction of sphingolipid biosynthesis de novo. Under normal conditions, it appears that the bioactive intermediates of this pathway (3-keto-sphinganine, sphinganine and ceramides) are kept at relatively low levels. Both the intrinsic activity of serine palmitoyltransferase (SPT) and the availability of its substrates (especially palmitoyl-CoA) can have toxic consequences for cells by increasing the production of cytotoxic intermediates. Recent work has also revealed that diverse agonists and stresses (cytokines, UV light, glucocorticoids, heat shock and toxic compounds) modulate SPT activity by induction of SPTLC2 gene transcription and/or post-translational modification. Mutation of the SPTLC1 component of SPT has also been shown to cause hereditary sensory neuropathy type I, possibly via aberrant oversynthesis of sphingolipids. Another key step of the pathway is the acylation of sphinganine (and sphingosine in the recycling pathway) by ceramide synthase, and up-regulation of this enzyme (or its inhibition to cause accumulation of sphinganine) can also be toxic for cells. Since it appears that most, if not all, tissues synthesize sphingolipids de novo, it may not be surprising that disruption of this pathway has been implicated in a wide spectrum of disease.

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Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5679-5687.1990 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5679-5687

Author(s):

C K Barlowe ◽

D R Appling

Keyword(s):

Saccharomyces Cerevisiae ◽

De Novo ◽

Point Mutations ◽

Gene Replacement ◽

Molecular Genetic Analysis ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

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Feeding Stimulates Sphingosine-1-Phosphate Mobilization in Mouse Hypothalamus

International Journal of Molecular Sciences ◽

10.3390/ijms20164008 ◽

2019 ◽

Vol 20 (16) ◽

pp. 4008

Author(s):

Valentina Vozella ◽

Natalia Realini ◽

Alessandra Misto ◽

Daniele Piomelli

Keyword(s):

De Novo ◽

Sphingolipid Metabolism ◽

Rt Pcr ◽

Liquid Chromatography Tandem Mass ◽

Sphingosine 1 Phosphate ◽

Phosphate Mobilization ◽

S1p Receptor ◽

Free Feeding ◽

G Protein Coupled ◽

Sphingolipid Biosynthesis

Previous studies have shown that the sphingolipid-derived mediator sphingosine-1-phosphate (S1P) reduces food intake by activating G protein-coupled S1P receptor-1 (S1PR1) in the hypothalamus. Here, we examined whether feeding regulates hypothalamic mobilization of S1P and other sphingolipid-derived messengers. We prepared lipid extracts from the hypothalamus of C57Bl6/J male mice subjected to one of four conditions: free feeding, 12 h fasting, and 1 h or 6 h refeeding. Liquid chromatography/tandem mass spectrometry was used to quantify various sphingolipid species, including sphinganine (SA), sphingosine (SO), and their bioactive derivatives SA-1-phosphate (SA1P) and S1P. In parallel experiments, transcription of S1PR1 (encoded in mice by the S1pr1 gene) and of key genes of sphingolipid metabolism (Sptlc2, Lass1, Sphk1, Sphk2) was measured by RT-PCR. Feeding increased levels of S1P (in pmol-mg−1 of wet tissue) and SA1P. This response was accompanied by parallel changes in SA and dihydroceramide (d18:0/18:0), and was partially (SA1P) or completely (S1P) reversed by fasting. No such effects were observed with other sphingolipid species targeted by our analysis. Feeding also increased transcription of Sptlc2, Lass1, Sphk2, and S1pr1. Feeding stimulates mobilization of endogenous S1PR1 agonists S1P and SA1P in mouse hypothalamus, via a mechanism that involves transcriptional up-regulation of de novo sphingolipid biosynthesis. The results support a role for sphingolipid-mediated signaling in the central control of energy balance.

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TORC1-regulated protein kinase Npr1 phosphorylates Orm to stimulate complex sphingolipid synthesis

Molecular Biology of the Cell ◽

10.1091/mbc.e12-10-0753 ◽

2013 ◽

Vol 24 (6) ◽

pp. 870-881 ◽

Cited By ~ 72

Author(s):

Mitsugu Shimobayashi ◽

Wolfgang Oppliger ◽

Suzette Moes ◽

Paul Jenö ◽

Michael N. Hall

Keyword(s):

Saccharomyces Cerevisiae ◽

Physiological Response ◽

De Novo ◽

Serine Palmitoyltransferase ◽

De Novo Synthesis ◽

Amino Acid Permease ◽

Evolutionarily Conserved ◽

General Amino Acid Permease ◽

And Function ◽

Complex Sphingolipids

The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.

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Effects of Furfural on the Respiratory Metabolism of Saccharomyces cerevisiae in Glucose-Limited Chemostats

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.4076-4086.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 4076-4086 ◽

Cited By ~ 98

Author(s):

Ilona Sárvári Horváth ◽

Carl Johan Franzén ◽

Mohammad J. Taherzadeh ◽

Claes Niklasson ◽

Gunnar Lidén

Keyword(s):

Saccharomyces Cerevisiae ◽

Furfuryl Alcohol ◽

Inhibitory Effect ◽

Anaerobic Growth ◽

Yeast Cells ◽

Flux Analysis ◽

Complete Replacement ◽

Furoic Acid ◽

Conversion Rates ◽

Glycerol Formation

ABSTRACT Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.

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Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5166 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5166-5178 ◽

Cited By ~ 13

Author(s):

H Jakubowski ◽

E Goldman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Density ◽

De Novo ◽

High Cell Density ◽

Degradation Products ◽

Yeast Cells ◽

High Cell ◽

Yeast Saccharomyces Cerevisiae ◽

Type Locus ◽

Low Cell Density

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.

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Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.02522-08 ◽

2009 ◽

Vol 75 (8) ◽

pp. 2304-2311 ◽

Cited By ~ 174

Author(s):

Dawid Brat ◽

Eckhard Boles ◽

Beate Wiedemann

Keyword(s):

Saccharomyces Cerevisiae ◽

Heterologous Expression ◽

Xylose Fermentation ◽

Thermus Thermophilus ◽

Xylose Isomerase ◽

Functional Expression ◽

Yeast Cells ◽

Highly Active ◽

Excellent Starting Point ◽

Starting Point

ABSTRACT In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

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Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2176-2181.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2176-2181

Author(s):

R C Dickson ◽

G B Wells ◽

A Schmidt ◽

R L Lester

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Lipids ◽

Molecular Genetic ◽

Suppressor Gene ◽

Molecular Genetic Analysis ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Chain Base ◽

Long Chain Base ◽

Long Chain

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.

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Lipid nutrition of Saccharomyces cerevisiae in winemaking

Canadian Journal of Microbiology ◽

10.1139/w04-051 ◽

2004 ◽

Vol 50 (9) ◽

pp. 669-674 ◽

Cited By ~ 23

Author(s):

Simona Belviso ◽

Laura Bardi ◽

Alessandra Biondi Bartolini ◽

Mario Marzona

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid ◽

Cell Growth ◽

Acid Production ◽

Unsaturated Fatty Acids ◽

Membrane Lipids ◽

Synthetic Medium ◽

Wine Industry ◽

Yeast Cells ◽

Acetic Acid Production

Biosynthesis of cell membrane lipids is a crucial metabolic pathway for the growth and viability of eucaryotic microorganisms. In Saccharomyces cerevisiae, unsaturated fatty acids and ergosterol synthesis needs molecular oxygen. Stuck and sluggish fermentations are related to this aspect of metabolism and constitute a major problem in the wine industry. Anaerobiosis, when lipids are not available in the growth medium, highly stresses cells. They release lipid biosynthesis metabolites and soon cease to multiply. This paper describes an investigation of the nutritional role of exogenous lipids from inactivated yeast cells (IYCs). Fermentations were carried out in a nitrogen-rich synthetic medium similar to grape juice with glucose and fructose as carbon sources, without lipid sources, and in anaerobiosis. The effect of the addition of IYC was assessed. Cell growth, cell lipid composition, glucose and fructose consumption, and acetic acid production were measured during fermentation. Addition of IYC boosted cell growth and sugar consumption, whereas acetic acid production decreased. Biomass yield was influenced by ergosterol availability and increased when IYCs were added. Fatty acid composition of yeast cells was changed by IYC addition.Key words: fermentation, lipids, nutrition, Saccharomyces cerevisiae, wine.

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