scholarly journals Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N-myristoylation and cellular lipid metabolism.

1994 ◽  
Vol 127 (3) ◽  
pp. 751-762 ◽  
Author(s):  
D R Johnson ◽  
L J Knoll ◽  
D E Levin ◽  
J I Gordon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Carbon Source ◽  
Vegetative Growth ◽  
Fatty Acid Synthetase ◽  
Acid Activation ◽  
Beta Oxidation ◽  
Fatty Acid Activation ◽  
Long Chain

Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyl-transferase (Nmt1p), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faa1p, although Faa4p contributes to this process. Cells with nmt181p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faa1p and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plb1p, a nonessential lysophospholipase/phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181. None of the four known FAAs is exclusively responsible for targeting imported fatty acids to peroxisomal beta-oxidation pathways. Introduction of a peroxisomal assembly mutation, pas1 delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles revealed that when Fas is inhibited, peroxisomes contribute to myristoylCoA pools used by Nmt1p. When Fas is active, a fraction of cellular myristoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myristate, palmitate, or oleate as the sole carbon source--indicating that S. cerevisiae contains at least one other FAA which directs fatty acids to beta-oxidation pathways.

scholarly journals Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N-myristoylation.

1992 ◽  
Vol 117 (3) ◽  
pp. 515-529 ◽  
Author(s):  
R J Duronio ◽  
L J Knoll ◽  
J I Gordon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
De Novo ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Single Copy ◽  
Synthetase Activity ◽  
Acid Activation ◽  
The Right ◽  
Long Chain

Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.

scholarly journals Identification of the fatty acid activation site on human ClC-2

2017 ◽  
Vol 312 (6) ◽  
pp. C707-C723 ◽  
Author(s):  
John Cuppoletti ◽  
Kirti P. Tewari ◽  
Jayati Chakrabarti ◽  
Danuta H. Malinowska
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Kinase Inhibitor ◽  
Intracellular Camp ◽  
Acid Activation ◽  
Cellular Mechanisms ◽  
T84 Cells ◽  
Fatty Acid Activation ◽  
Current Activation ◽  
Activation Site

Fatty acids (including lubiprostone and cobiprostone) are human ClC-2 (hClC-2) Cl− channel activators. Molecular and cellular mechanisms underlying this activation were examined. Role of a four-amino acid PKA activation site, RGET691, of hClC-2 was investigated using wild-type (WT) and mutant (AGET, RGEA, and AGAA) hClC-2 expressed in 293EBNA cells as well as involvement of PKA, intracellular cAMP concentration ([cAMP]i), EP2, or EP4 receptor agonist activity. All fatty acids [lubiprostone, cobiprostone, eicosatetraynoic acid (ETYA), oleic acid, and elaidic acid] caused significant rightward shifts in concentration-dependent Cl− current activation (increasing EC50s) with mutant compared with WT hClC-2 channels, without changing time and voltage dependence, current-voltage rectification, or methadone inhibition of the channel. As with lubiprostone, cobiprostone activation of hClC-2 occurred with PKA inhibitor (myristoylated protein kinase inhibitor) present or when using double PKA activation site (RRAA655/RGEA691) mutant. Cobiprostone did not activate human CFTR. Fatty acids did not increase [cAMP]i in hClC-2/293EBNA or T84 cells. Using T84 CFTR knockdown cells, cobiprostone increased hClC-2 Cl− currents without increasing [cAMP]i, while PGE2 and forskolin-IBMX increased both. Fatty acids were not agonists of EP2 or EP4 receptors. L-161,982, a supposed EP4-selective inhibitor, had no effect on lubiprostone-activated hClC-2 Cl− currents but significantly decreased T84 cell barrier function measured by transepithelial resistance and fluorescent dextran transepithelial movement. The present findings show that RGET691 of hClC-2 (possible binding site) plays an important functional role in fatty acid activation of hClC-2. PKA, [cAMP]i, and EP2 or EP4 receptors are not involved. These studies provide the molecular basis for fatty acid regulation of hClC-2.

scholarly journals Syringomycin E Inhibition of Saccharomyces cerevisiae: Requirement for Biosynthesis of Sphingolipids with Very-Long-Chain Fatty Acids and Mannose- and Phosphoinositol-Containing Head Groups

2000 ◽  
Vol 44 (5) ◽  
pp. 1174-1180 ◽  
Author(s):  
Stephen D. Stock ◽  
Hiroko Hama ◽  
Jeffrey A. Radding ◽  
Debra A. Young ◽  
Jon Y. Takemoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Mutant Allele ◽  
Polar Head Group ◽  
Long Chain Fatty Acids ◽  
Syringomycin E ◽  
Yeast Genes ◽  
Long Chain ◽  
Chain Fatty Acids

ABSTRACT Syringomycin E is an antifungal cyclic lipodepsinonapeptide that inhibits the growth of Saccharomyces cerevisiae by interaction with the plasma membrane. A screen conducted to find the yeast genes necessary for its fungicidal action identified two novel syringomycin E response genes, SYR3 and SYR4. Asyr3 mutant allele was complemented by ELO2 andELO3. These genes encode enzymes that catalyze the elongation of sphingolipid very long chain fatty acids. Tetrad analysis showed that SYR3 was ELO2. Strains with deletions of SYR3/ELO2 and ELO3 were resistant to syringomycin E, and lipid analyses of both mutants revealed shortened fatty acid chains and lower levels of sphingolipids.SYR4 was identified by Tn5 inactivation of genomic library plasmids that complemented a syr4 mutant allele. SYR4 was found to be identical to IPT1, which encodes the terminal sphingolipid biosynthetic enzyme, mannosyl-diinositolphosphoryl-ceramide synthase. Deletion Δsyr4/ipt1 strains were viable, were resistant to syringomycin E, did not produce mannosyl-diinositolphosphoryl-ceramide, and accumulated mannosyl-inositolphosphoryl-ceramide. Accumulation of mannosyl-inositolphosphoryl-ceramide was not responsible for resistance since a temperature-sensitive secretory pathway mutant (sec14-3ts ) accumulated this sphingolipid and was sensitive to syringomycin E. Finally, Δcsg1/sur1 and Δcsg2 strains defective in the transfer of mannose to inositolphosphoryl-ceramide were resistant to syringomycin E. These findings show that syringomycin E growth inhibition of yeast is promoted by the production of sphingolipids with fully elongated fatty acid chains and the mannosyl and terminal phosphorylinositol moieties of the polar head group.

scholarly journals The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions

1970 ◽  
Vol 119 (2) ◽  
pp. 221-242 ◽  
Author(s):  
E. D. Saggerson ◽  
A. L. Greenbaum
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Fatty Acyl ◽  
Acetyl Coa Carboxylase ◽  
Tissue Concentrations ◽  
Adipose Tissues ◽  
Long Chain

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO2 and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [14C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.

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