Fatty acid synthesis and elongation in yeast

Fatty acids are essential compounds in the cell. Since the yeast Saccharomyces cerevisiae does not feed typically on fatty acids, cellular function and growth relies on endogenous synthesis. Since all cellular organelles are involved in--or dependent on--fatty acid synthesis, multiple levels of control may exist to ensure proper fatty acid composition and homeostasis. In this review, we summarize what is currently known about enzymes involved in cellular fatty acid synthesis and elongation, and discuss potential links between fatty acid metabolism, physiology and cellular regulation.     

Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling

In the present study, acyl-CoA synthetase mutants of Saccharomyces cerevisiae were employed to investigate the impact of this activity on certain pools of fatty acids. We identified a genotype responsible for the secretion of free fatty acids into the culture medium. The combined deletion of Faa1p and Faa4p encoding two out of five acyl-CoA synthetases was necessary and sufficient to establish mutant cells that secreted fatty acids in a growth-phase dependent manner. The mutants accomplished fatty acid export during exponential growth-phase followed by fatty acid re-import into the cells during the stationary phase. The data presented suggest that the secretion is driven by an active component. The fatty acid re-import resulted in a severely altered ultrastructure of the mutant cells. Additional strains deficient of any cellular acyl-CoA synthetase activity revealed an almost identical phenotype, thereby proving transfer of fatty acids across the plasma membrane independent of their activation with CoA. Further experiments identified membrane lipids as the origin of the observed free fatty acids. Therefore, we propose the recycling of endogenous fatty acids generated in the course of lipid remodelling as a major task of both acyl-CoA synthetases Faa1p and Faa4p.     

Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation

CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.     

Effect of Unsaturated Fatty Acids on the Development of Respiration and on Protein Synthesis in an Unsaturated Fatty Acid Mutant of Saccharomyces cerevisiae

The extent of development of respiratory function induced by aeration of an anaerobically grown unsaturated fatty acid auxotroph of Saccharomyces cerevisiae is determined by the availability, endogenous or externally supplied, of unsaturated fatty acid. The synthesis of mitochondrial and cytoplasmic enzymes during aeration appears to have a similar basis of regulation by available unsaturated fatty acid. Levels of unsaturated fatty acid that permit the synthesis of mitochondrial enzymes also result in a substantial stimulation of cellular protein synthesis.     

Biogenesis of mitochondria: The effects of altered steady-state membrane lipid composition on mitochondrial-energy metabolism in Saccharomyces cerevisiae

A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression.The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated.The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%.In cells which contained lipids with 22–28% unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the levels of cytochromes aa₃ and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.     

Expression of Human CTP synthetase in Saccharomyces cerevisiae reveals phosphorylation by protein kinase A

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Delta ura8Delta mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Delta ura8Delta mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from (32)P(i)-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation.     

Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae.

Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants.     

Glucolipotoxicity of the pancreatic beta cell

The concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and fatty acid levels on pancreatic beta-cell function and survival. Significant progress has been made in recent years towards a better understanding of the cellular and molecular basis of glucolipotoxicity in the beta cell. The permissive effect of elevated glucose on the detrimental actions of fatty acids stems from the influence of glucose on intracellular fatty acid metabolism, promoting the synthesis of cellular lipids. The combination of excessive levels of fatty acids and glucose therefore leads to decreased insulin secretion, impaired insulin gene expression, and beta-cell death by apoptosis, all of which probably have distinct underlying mechanisms. Recent studies from our laboratory have identified several pathways implicated in fatty acid inhibition of insulin gene expression, including the extracellular-regulated kinase (ERK1/2) pathway, the metabolic sensor Per-Arnt-Sim kinase (PASK), and the ATF6 branch of the unfolded protein response. We have also confirmed in vivo in rats that the decrease in insulin gene expression is an early defect which precedes any detectable abnormality in insulin secretion. While the role of glucolipotoxicity in humans is still debated, the inhibitory effects of chronically elevated fatty acid levels has been clearly demonstrated in several studies, at least in individuals genetically predisposed to developing type 2 diabetes. It is therefore likely that glucolipotoxicity contributes to beta-cell failure in type 2 diabetes as well as to the decline in beta-cell function observed after the onset of the disease.     

Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.     

Cellular localization and phosphorylation of Hrb1p is independent of Sky1p

Protein phosphorylation plays a major role in regulating cellular functions. We have previously demonstrated that Sky1p, the SR protein kinase of the budding yeast Saccharomyces cerevisiae, is a regulator of polyamine transport and ion homeostasis. Since its kinase activity was demonstrated essential for fulfilling these roles, we assumed that Sky1p function via substrates phosphorylation. Using an in vitro phosphorylation assay, we have identified Hrb1p as a putative Sky1p substrate. However, phosphorylation analysis in WT and sky1Delta cells and localization studies disproved Hrb1p as a true Sky1p substrate, although a segment of the RS domain is required for determining its subcellular localization. Furthermore, we demonstrate that Hrb1p and additional putative Sky1p substrates, identified by computational approach, are not involved in mediating the spermine tolerant phenotype of sky1Delta cells.     

Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. Implications for the conservation of oxygen sensing in eukaryotes

An organism's ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we have studied the Delta9 fatty acid desaturase gene OLE1 in Saccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals. OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3' to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in a lacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar to OLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.     

Identification of genes affecting lipid content using transposon mutagenesis in Saccharomyces cerevisiae

Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions. Lipid analysis of disruptants of these ORFs indicated that the Deltasnf2, and Deltaira2 disruptants had significantly higher lipid content. Cultivation in a nitrogen-limited medium increased the lipid content in all disruptants, among which the Deltapre9 disruptant was the most sensitive to nitrogen limitation. We then focused on the Deltasnf2 disruptant due to its higher lipid content and its function as a regulator of phospholipid synthesis. Lipid class analysis indicated that triacylglycerol and free fatty acids contributed to the increase in total lipids of the Deltasnf2 disruptant. The addition of exogenous fatty acids was not so effective at increasing the lipid content in the Deltasnf2 disruptant as it was in the wild type. It should be noticed that exogenous free linoleic acid was much higher in the Deltasnf2 disruptant than in the wild type, as in the case of endogenous free fatty acids. In addition, the incorporation of exogenous fatty acids into cells increased in the disruptant, suggesting that fatty acid transporters were regulated by SNF2. The results suggest that metabolic fluxes into storage lipids, which are activated in the Deltasnf2 disruptant, is repressed by the incorporation of exogenous fatty acids. They provide new insight into the biosynthesis of storage lipids in yeast.     

The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p

The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta-king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings.     

Biogenesis of mitochondria. The effects of altered membrane lipid composition on cation transport by mitochondria of Saccharomyces cerevisiae

1. The fatty acid composition of the membrane lipids of a fatty acid desaturase mutant of Saccharomyces cerevisiae was manipulated by growing the organism in a medium containing defined fatty acid supplements. 2. Mitochondria were obtained whose fatty acids contain between 20% and 80% unsaturated fatty acids. 3. Mitochondria with high proportions of unsaturated fatty acids in their lipids have coupled oxidative phosphorylation with normal P/O ratios, accumulate K(+) ions in the presence of valinomycin and an energy source, and eject protons in an energy-dependent fashion. 4. If the unsaturated fatty acid content of the mitochondrial fatty acids is lowered to 20%, the mitochondria simultaneously lose active cation transport and the ability to couple phosphorylation to respiration. 5. The loss of energy-linked reactions is accompanied by an increased passive permeability of the mitochondria to protons. 6. Free fatty acids uncouple oxidative phosphorylation in yeast mitochondria and the effect is reversed by bovine serum albumin. 7. The free fatty acid contents of yeast mitochondria are unaffected by depletion of unsaturated fatty acids, and free fatty acids are not responsible for the uncoupling of oxidative phosphorylation in organelles depleted in unsaturated fatty acids. 8. It is suggested that the loss of energy-linked reactions in yeast mitochondria that are depleted in unsaturated fatty acids is a consequence of the increased passive permeability to protons, and is caused by a change in the physical properties of the lipid phase of the inner mitochondrial membrane.     

Eukaryotic release factor 1 phosphorylation by CK2 protein kinase is dynamic but has little effect on the efficiency of translation termination in Saccharomyces cerevisiae

Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Delta mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.