Yeast Phospholipid Research Articles

Combinatorial regulation of phospholipid biosynthetic gene expression by the UME6, SIN3 and RPD3 genes.

The Ume6p-Sin3p-Rpd3p complex negatively regulates expression of genes containing a Ume6p binding site. However, these regulatory proteins also function independently to regulate gene expression both negatively and positively. The model system for this combinatorial regulation is the yeast phospholipid biosynthetic pathway. Sin3p negatively regulates the INO1, CHO1, CHO2 and OPI3 genes while Ume6p negatively regulates the INO1 gene and positively regulates the other genes. We have suggested that the positive regulation results from indirect effects on expression of the INO2 transcriptional activator gene. Here, we demonstrate that the effect of Ume6p on INO2 gene expression is also indirect. We also show that Rpd3p is a negative regulator of phospholipid biosynthetic gene expression. The ability of Ume6p, Sin3p and Rpd3p to differentially regulate expression of the phospholipid biosynthetic genes affects phospholipid composition. A sin3 mutant strain lacks detectable levels of phosphatidylethanolamine and elevated levels of phosphatidylcholine (PC) and a rpd3 mutant strain has reduced levels of PC. These alterations in membrane composition suggest that there may exist additional differences in regulation of phospholipid biosynthetic gene expression and that membrane compositions may be coordinated with other biological processes regulated by Ume6p, Sin3p and Rpd3p.

Regulation of yeast phospholipid biosynthetic genes in phosphatidylserine decarboxylase mutants.

In the yeast Saccharomyces cerevisiae, the products of two genes (PSD1 and PSD2) are able to catalyze the decarboxylation of phosphatidylserine (PS) to produce phosphatidylethanolamine (PE) (C. J. Clancey, S. Chang, and W. Dowhan, J. Biol. Chem. 268:24580-24590, 1993; P. J. Trotter, J. Pedretti, and D. R. Voelker, J. Biol. Chem. 268:21416-21424, 1993; P.J. Trotter, and D. R. Voelker, J. Biol. Chem. 270:6062-6070, 1995). I report that the major mitochondrial PS decarboxylase gene (PSD1) is transcriptionally regulated by inositol in a manner similar to that reported for other coregulated phospholipid biosynthetic genes. The second PS decarboxylase gene (PSD2) is not regulated on a transcriptional level by inositol and/or ethanolamine. In yeast, phosphatidylcholine (PC) biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol. I show that the presence of a functional major mitochondrial PS decarboxylase encoded by the PSD1 gene is necessary for proper regulation of INO1 in response to inositol in the absence of ethanolamine. Disruption of the second PS decarboxylase gene (PSD2) does not affect the INO1 regulation. Analysis of phospholipid content of PS decarboxylase mutants suggests that the proportion of PC on total cellular phospholipids is not correlated to the cell's ability to repress INO1 in response to inositol. Rather, yeast cells are apparently able to monitor the flux through the phospholipid biosynthetic pathway and modify the transcription of phospholipid biosynthetic genes accordingly.

Regulation of yeast phospholipid biosynthetic gene expression in response to inositol involves two superimposed mechanisms.

Transcription of phospholipid biosynthetic genes in the yeast Saccharomyces cerevisiae is maximally derepressed when cells are grown in the absence of inositol and repressed when the cells are grown in its presence. We have previously suggested that this response to inositol may be dictated by regulating transcription of the cognate activator gene, INO2. However, it was also known that cells which harbor a mutant opi1 allele express constitutively derepressed levels of target genes (INO1 and CHO1), implicating the OPI1 negative regulatory gene in the response to inositol. These observations suggested that the response to inositol may involve both regulation of INO2 transcription as well as OPI1-mediated repression. We investigated these possibilities by examining the effect of inositol on target gene expression in a strain containing the INO2 gene under control of the GAL1 promoter. In this strain, transcription of the INO2 gene was regulated in response to galactose but was insensitive to inositol. The expression of the INO1 and CHO1 target genes was still responsive to inositol even though expression of the INO2 gene was unresponsive. However, the level of expression of the INO1 and CHO1 target genes correlated with the level of INO2 transcription. Furthermore, the effect of inositol on target gene expression was eliminated by deleting the OPI1 gene in the GAL1-INO2-containing strain. These data suggest that the OPI1 gene product is the primary target (sensor) of the inositol response and that derepression of INO2 transcription determines the degree of expression of the target genes.

Differences in the intracellular lipids of sake yeast in main mash seeded respectively with two kinds of seed mash: Kimoto and sokujo-moto

In sake brewing, yeast cells grown in the traditional seed mash ( kimoto) are enriched in palmitic acid, and those in the popular seed mash ( sokujo-moto) are enriched in linoleic acid. Lipid composition of yeast in the main mash seeded with respective seed mash was determined. The amount of intracellular phospholipids (PLs) of yeast showed little change throughout the fermentation period, while intracellular triglyceride (TG) content decreased dramatically during the growth phase in the main mash. In the main mash seeded with sokujo-moto, the linoleic acid content of intracellular phosphatidyl ethanolamine (PE) was reduced from 30.6% of the original seed mash yeast to 6–9%, whereas the content of intracellular phosphatidyl choline (PC) was merely reduced from the original 48.4% to 26–30% during fermentation. Moreover, even after 4–5 generations of growth in the main mash, the percent of linoleic acid in PC did not notably decrease. The intracellular TG of seed mash yeast is therefore supposed to be a major source of linoleic acid in the early stage of growth in main mash. On the other hand, linoleic acid content in PLs of yeast were detectable only in low quantities (5–10%) during the fermentation of main mash seeded with kimoto as well as that seeded with kimoto (5.5%). Since there was no increase in linoleic acid content, the main mash during rapid growth phase seemed to be deficient in linoleic acid and, therefore, the digestion of raw materials, which contains large amounts of linoleic acid, failed to actively proceed. The ratio of PC PE of the cells was 0.67 in kimoto, and 1.62 in sokujo-moto. The same sort of difference in the PC PE ratio was also seen between the corresponding main mash. The presence of linoleic acid increased the PC PE ratio at the stationary phase of growth, and was decreased in the presense of 16% ethanol. Thus, kimoto could be differentiated from sokujo-moto on the basis of fatty acid composition in PC and by the PC PE ratio of the yeast in the main mash.

Effect of lactic acid bacteria on fatty acid composition of phospholipids of yeast grown in Kimoto

Effect of lactic acid bacteria on fatty acid composition of phospholipids of yeast grown in Kimoto

Cloning and Characterization of the SCS1 Gene Required for the Expression of Genes in Yeast Phospholipid Synthesis1

A dominant mutation of Saccharomyces cerevisiae, CSE1, caused a decrease in the expression of the INO1 gene product, inositol-1-phosphate synthase. The residual activity was completely repressed by the addition of choline to the medium. A mutant carrying this mutation could not grow in the presence of choline unless inositol was added to the medium. Here we report a suppressor gene of the CSE1 mutation, SCS1 (suppressor of CSE1), which was cloned by complementation of CSE1 with a wild-type multicopy yeast genomic library. The cloned SCS1 gene contained an open reading frame which encoded 304 amino acid residues with a calculated molecular mass of 34,234 Da, and the sequence coincided with residues with a calculated molecular mass of 34,234 Da, and the sequence coincided with that of the INO2 gene. An scs1/ino2 null mutant constructed by gene replacement was viable, but auxotrophic for inositol and choline, and used for determination of the mRNA levels of various phospholipid-synthesizing enzymes. In agreement with the reported data for ino2 mutants the disruptant showed decreased expression of the INO1 and PSS genes, which are known to be regulated by inositol and choline. In addition, we newly found that the disruption of SCS1/INO2 also caused a decrease in the expression of the CKI, PEM1, and PEM2 genes, which we previously showed to belong to the inositol-choline-regulated gene family. These results confirm and strengthen the conclusion that the SCS1/INO2 gene is required for expression of inositol-choline-regulated genes in phospholipid synthesis.

Regulatory gene INO4 of yeast phospholipid biosynthesis is positively autoregulated and functions as a transactivator of fatty acid synthase genes FAS1 and FAS2 from Saccharomyces cerevisiae.

The sequence motif 5' TYTTCACATGY 3' functions as an upstream activation site common to both yeast fatty acid synthase genes, FAS1 and FAS2. In addition, this UASFAS element is shared by all so far characterized genes of yeast phospholipid biosynthesis. We have investigated the influence of a functional INO4 gene previously described as a regulator of inositol biosynthesis on the expression of FAS1 and FAS2. In a delta ino4 null allele strain, both genes are expressed at only 50% of wild type level. Using individual UASFAS sequence motifs inserted into a heterologous test system, a drastic decrease of reporter gene expression to 2-10% of the wild type reference was observed in the delta ino4 mutant. In gel retardation assays, the protein-DNA complex involving the previously described FAS binding factor 1, Fbf1, was absent when using a protein extract from the delta ino4 mutant. On the other hand, this signal was enhanced with an extract from cells grown under conditions of inositol/choline derepression. Subsequent experiments demonstrated that INO4 expression is itself affected by phospholipid precursors, mediated by an UASFAS element in the INO4 upstream region. Thus, in addition of being an activator of phospholipid biosynthetic genes, INO4 is also subject to a positive autoregulatory loop in its own biosynthesis.

60] Phospholipid transfer proteins from yeast

This chapter discusses the phospholipid transfer proteins from yeast. Baker's yeast, Saccharomyces cerevisiae , contains at least two cytosolic proteins, which catalyze transport of phospholipids between natural membranes and phospholipid vesicles in vitro. One protein is specific for phosphatidylinositol (Ptdlns) and phosphatidylcholine (PtdCho) and closely resembles Ptdlns-transfer protein from mammalian tissues with respect to size, isoelectric point, substrate specificity, and inhibition by negatively charged phospholipids. The second yeast phospholipid transfer protein has a broad substrate specificity in that it catalyzes preferentially the transfer ofphosphatidylserine (PtdSer) as well as phosphatidylethanolamine (PtdEtn), cardiolipin, phosphatidic acid, and ergosterol. In contrast to the mammalian nonspecific lipid transfer protein, the yeast protein does not mediate intermembrane movement of PtdIns or PtdCho. Yeast is considered a useful biological model to study the role of proteins in vivo by a combination of biochemical and molecular genetics techniques. Recent experiments have revealed that the PtdIns transfer protein is essential for cellular growth, and it is identical to the SEC 14 gene product which is part of the protein secretory machinery and acts in the Golgi complex.

Heparin-binding growth factors exhibit differential affinities for B/A4 amyloid-containing structures in Alzhiemer's disease: ∗E.G. Stopa, A. Baird, A.M. Gonzalez, V. Kuo-Leblanc, R.C. Corona, I. Kovesdi, and P. Bohlen. Departments of Pathology, SUNY-HSC, Syracuse and Albany Medical College, Albany, NY, Dept. of Molecular and Cellular Growth Biology, The Whittier Institute, La Jolla, CA, Brain Tissue Resource Ctr., McLean Hospital, Belmont, MA

This chapter discusses the phospholipid transfer proteins from yeast. Baker's yeast, Saccharomyces cerevisiae, contains at least two cytosolic proteins, which catalyze transport of phospholipids between natural membranes and phospholipid vesicles in vitro. One protein is specific for phosphatidylinositol (Ptdlns) and phosphatidylcholine (PtdCho) and closely resembles Ptdlns-transfer protein from mammalian tissues with respect to size, isoelectric point, substrate specificity, and inhibition by negatively charged phospholipids. The second yeast phospholipid transfer protein has a broad substrate specificity in that it catalyzes preferentially the transfer ofphosphatidylserine (PtdSer) as well as phosphatidylethanolamine (PtdEtn), cardiolipin, phosphatidic acid, and ergosterol. In contrast to the mammalian nonspecific lipid transfer protein, the yeast protein does not mediate intermembrane movement of PtdIns or PtdCho. Yeast is considered a useful biological model to study the role of proteins in vivo by a combination of biochemical and molecular genetics techniques. Recent experiments have revealed that the PtdIns transfer protein is essential for cellular growth, and it is identical to the SEC 14 gene product which is part of the protein secretory machinery and acts in the Golgi complex.

Genetic analysis of yeast phospholipid biosynthesis.

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . POTENTIAL SIGNAL TRANSDUCTION PATHWAyS: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PHOSPHOLIPID BIOSYNTHESIS IN yEAST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Localization of Enzymatic Activi ties and Movement of Lipids . Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural Genes . Regulatory Genes . ROLE OF PtdC and PtdDME BIOSYNTHESIS IN CROSS PATHWAY REGULATION CONCLUSIONS . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1- 14C]oleate-labeled autoclaved yeast: A membranous substrate for measuring phospholipase A 2 activity in vitro

Radiolabeled, autoclaved yeast were tested as a substrate for mammalian phospholipase A 2 activity because the only other membranous substrate used for this purpose, autoclaved Escherichia coli, totally lacks a major mammalian phospholipid, phosphatidylcholine. Candida albicans were grown in the presence of [1- 14C]oleate and then autoclaved. Sixty three percent of the incorporated label was in yeast phospholipid, and more than 95% of that was in the 2-acyl position. The distribution of label in the yeast phospholipids (phosphatidylcholine and -ethanolamine, -serine + -inositol, and phosphatidic acid corresponded closely to the chemical distribution of phosphorus in those phospholipids. Snake venom ( Naja naja) and human synovial fluid phospholipase A 2 hydrolyzed yeast phospholipid exclusively to release 14C-labeled fatty acid. When 50–60% of the yeast phospholipid was hydrolyzed, the radioactive fatty acids as determined by gas-liquid chromatographic analysis were predominantly oleate (45%) and linoleate (>54%). Hydrolysis of yeast phospholipid by both enzymes was near-linear with protein and time under conditions of optimal pH (neutral-alkaline) and Ca 2+ (1–5 m m) previously reported for optimal hydrolysis of autoclaved E. coli phospholipid. N. naja phospholipase A 2 showed less preference for phosphatidyleth-anolamine than -choline as liposomes or yeast phospholipid as compared to human synovial fluid phospholipase A 2 which clearly preferred phosphatidylethanolamine to -choline as a liposome or yeast phospholipid. These results illustrate that radiolabeled phospholipids of autoclaved yeast, enriched in phosphatidylcholine, are readily hydrolyzed by snake venom and human nonpancreatic phospholipases A 2 and may, therefore, be useful in the measurement of in vitro enzymatic activity.

Primary structure of the yeast choline transport gene and regulation of its expression.

The yeast Saccharomyces cerevisiae possesses a choline transport system encoded by the CTR gene. Incorporated choline is exclusively utilized for the synthesis of phosphatidylcholine. Determination of the complete nucleotide sequence of the CTR gene showed that the deduced primary sequence of the choline transporter comprised 563 amino acid residues with a calculated molecular weight of 62,055. Both the amino- and carboxyl-terminal regions were hydrophilic whereas the rest of the sequence was highly hydrophobic and contained several membrane-spanning regions. There were four potential glycosylation sites in the hydrophilic parts of the sequence. The transporter showed significant sequence similarities to the yeast arginine and histidine transporters. A 2.0-kilobase RNA species was identified as the CTR transcript. Disruption of the CTR locus completely abolished the choline transport activity, indicating that the CTR product is the sole choline transporter in yeast. The regulation of choline transport mRNA was investigated by Northern blot analysis using a CTR probe. The abundance of CTR mRNA significantly decreased on incubation of cells with a combination of choline and myo-inositol. The CTR mRNA level was high in the exponential growth phase but decreased dramatically when cells entered the stationary phase. Similar control had been observed for some enzymes in yeast phospholipid synthesis.

Catalytic properties of the yeast phospholipid transfer protein

The properties of the phosphatidylcholine (PC) transfer reaction catalyzed by the yeast phospholipid transfer protein (TP-I) were examined in vitro. Donor and acceptor membranes consisted of unilamellar (ULV) and multilamellar (MLV) vesicles, respectively. The phospholipid composition of the membranes participating in the transfer reaction, and in particular that of the MLV acceptors, have a tremendous effect upon the rate of PC-catalyzed transfer. Phosphatidylethanolamine (PE) is an essential component of the acceptor membrane, but it alone is not sufficient to sustain appreciable transfer rates. If combined in an equimolar ratio with PC, there is only a modest increase in transfer rates. On the other hand, when combined with alternate substrates such as phosphatidylinositol (PI) or phosphatidylserine (PS), very high rates of PC transfer occur. The measurement of transfer rates is not affected by the molecular species of PC used as the radioactive tracer. Evidence is also presented to indicate that the two forms of the transfer protein (TP-I and TP-II) are not identical in terms of their interactions with a membrane surface: differences occur in the levels of transfer of PC, PE, PI, and PS at equilibrium. Finally, by kinetic analysis, the mechanism of the protein-catalyzed transfer of PC is shown to conform to a ping-pong bibi model with excess substrate inhibition, analogous to ordinary two-substrate enzyme-catalyzed reactions. Both the rates of desorption and adsorption of the protein from the surface of the ULV are much greater than those describing the similar interactions of the protein with MLV.(ABSTRACT TRUNCATED AT 250 WORDS)

Appearance of cell-bound β-galactosidase activity in Kluyveromyces bulgaricus resting cells

The conditions involved in the appearance of cell-bound β-galactosidase activity in Kluyveromyces bulgaricus resting cells were determined in relation to phospholipid content. The cells displayed an optimum β-galactosidase activity when they were anaerobically growing in whey medium and kept at 25°C for more than 14 days. The activity was stimulated by a preliminary 3–5 hours shaking period and an adequate protein concentration of whey medium. The expression of the cell-bound β-galactosidase was correlated to a 60% decrease in yeast phospholipid content.

Changes in phospholipids of Saccharomyces cerevisiae associated with inositol-less death

Two inositol-requiring strains of Saccharomyces cerevisiae were examined for changes in levels of phospholipids occurring after inositol deprivation. Lack of inositol results in loss of cell viability (inositol-less death) and in very large increases in two phospholipid precursors, phosphatidic acid and CDP-diacylglycerol; the accumulation of other glycerophospholipids continues for a considerable time at normal rates. Phosphatidylinositol accumulation does not occur in the absence of inositol; however, the further metabolism of this lipid continues, with 80 to 90% of this lipid disappearing. This disappearance is matched by increases in the phosphoinositol containing sphingolipids and extracellular glycerophosphoinositol. These changes are not observed when growth is blocked by cycloheximide or by omission of lysine from a lysine auxotroph, most lipids continuing to accumulate long after growth stops. There appears to be no close coordination in the synthesis of the major yeast phospholipids or between protein synthesis and phospholipid synthesis. However, despite very large changes in the composition of yeast phospholipids that can be achieved by altering culture conditions, it appears that the average charge per phospholipid molecule remains fairly constant.

Evidence for a functional change in the plasma membrane of yeasts associated with the mechanism of arsenate resistance

In previous reports experimental evidence has been presented indicating a possible relationship between the formation of arseno-phosphoinositides and the active transport of arsenate-phosphate in yeast cells. There is an increment in the amount of inositides in yeast cells adapted to grow in the presence of toxic concentrations of arsenate. These cells exhibit a highly reduced arsenate uptake but maintain their capacity to transport phosphate. Since, in normal (nonadapted) yeast cells, both arsenate and phosphate anions share the same transport system, a study was conducted to obtain further information about the plausible role played by the phosphoinositides in the active transport system of arsenate and their inhibition that allows the cells to grow in the presence of the toxic. Studies on [ 32P]orthophosphate and [ 74As]arsenate incorporation into phospholipids in normal and arsenate-adapted yeast show that: The 32P incorporation into phospholipids is two times larger in normal yeast as compared to arsenateadapted ones. The 32P labeling was maximum for phosphatidylinositol in normal yeasts while in the arsenate-adapted cells it was maximum for phosphatidylcholine. This incorporation was largely inhibited by arsenate in normal yeasts and minimal in the arsenate-adapted ones. Cell fractionation shows that the maximum incorporation of [ 32P]orthophosphate resides in the microsomal fraction, while the incorporation of [ 74As]arsenate resides mainly in the cell envelope fraction which incorporates 86% of the 74As label. Phosphate is capable of inhibiting the 74As-inositide complex formation and destroying the previously formed one. Yeast cells prelabeled with [2C- 3H]myoinositol showed a reduced turnover rate of phosphoinositides even when transporting nontoxic amounts of arsenate. The involvement of the inositides as a regulatory mechanism in the phosphate-arsenate active transport system in yeast cells is discussed.

Diol Lipids in the Phospholipid Fraction of Lipomyces starkeyi Grown in the Medium Containing 1, 2-Propanediol

After training for 1, 2-propanediol, a yeast, Lipomyces starkeyi, grew well in the media containing the diol, showing a diauxie-like pattern. The yeast phospholipid was converted into monoacetyl-diglyceride by acetolysis. The presence of monoacetyl-mono fatty acyl esters of the diol in this fraction was found by GC-MS. The data suggested that the diol was present as 2-palmitoyl-ester and 2-oleoyl-ester in the form of phospholipid analogue, in which phosphorus bound in 1-position of the diol. Molecular species of the phospholipid was also deduced from the GC-MS data. Diol diester was not found in the triglyceride fraction of the yeast.

Studies on the diversity of inositol-containing yeast phospholipids: incorporation of 2-deoxyglucose into lipid.

Seven inositol-containing phospholipids in Saccharomyces cerevisiae can be resolved by two-dimensional chromatography on silicic acid-impregnated paper. Four of these lipids are stable to mild alkaline methanolysis; one of these has already been characterized as a mannosyl di(inositolphosphoryl) ceramide. Addition of labeled 2-deoxy-d-glucose to a growing culture results in label appearing in five lipids, some of which are alkali-stable and have the same R(F) values as the alkali-stable inositol-containing phospholipids. These lipids are labeled rapidly. The deoxyglucose is incorporated intact, probably in a glycosidic link, since the deoxyglucose is released by mild acid treatment.

Inadequacy of myelin phospholipids for restoration of succinoxidase activity in lipid-depleted mitochondria

Succinoxidase activity in lipid-depleted mitochondria was not restored efficiently by mixed myelin phospholipids at difference with the natural mitochondrial phospholipids, yeast phospholipids, and Asolectin. Since similar differences in activity were present between pure phosphatidyl-ethanolamine fractions separated from myelin phospholipids and Asolectin, they should be due to the different fatty acid composition of the phospholipids. In contrast with the differentability in restoration of succionoxidase, all the phospholipids studied were bound to the lipid-depleted membranes to similar extents.

Specificity of lipids and coenzyme Q in mitochondrial NADH and succin-oxidase of beef heart and S. cerevisiae

The organic structural specificity of coenzyme Q in NADH- and succin-oxidase has been studied with mitochondria from the yeast, S. cerevisiae, and compared with the specificity for mitochondrial enzyme systems from beef heart. The specificity of lipids in CoQ-enzyme systems has also been studied. The origin and concentration of micellar phospholipids used as carriers for the CoQ homologs profoundly affects the restoration of NADH-oxidase in pentane-extracted mitochondria both from yeast and beef heart. High concentrations of phospholipids induce a decrease of the NADH-oxidase activity which had been restored by the lower homologs of CoQ. The activity with CoQ 1-CoQ 4 was higher than that with the natural CoQ 6 in yeast mitochondria, and was lower than that with the natural CoQ 10 in beef heart mitochondria, irrespective of the concentration of phospholipids. Mitochondrial phospholipids of yeast were more effective than soybean phospholipids in supporting restoration of NADH-oxidase by the higher homologs in pentane-extracted mitochondria of yeast. This difference was not evident for succin-oxidase from yeast and beef heart. Succin-oxidase from both sources was similarly restored by the homologs CoQ 2-Q 10 independently of the phospholipid concentration. According to these data, there are two sites for CoQ 6 in yeast mitochondria as there are two sites for CoQ 10 in beef heart mitochondria. The nature of the site in NADH-oxidase of yeast and of beef heart is different.