13 research outputs found
Neutral lipid metabolism influences phospholipid synthesis and deacylation in Saccharomyces cerevisiae.
No full textEstablishment and maintenance of equilibrium in the fatty acid (FA) composition of phospholipids (PL) requires both regulation of the substrate available for PL synthesis (the acyl-CoA pool) and extensive PL turnover and acyl editing. In the present study, we utilize acyl-CoA synthetase (ACS) deficient cells, unable to recycle FA derived from lipid deacylation, to evaluate the role of several enzymatic activities in FA trafficking and PL homeostasis in Saccharomyces cerevisiae. The data presented show that phospholipases B are not contributing to constitutive PL deacylation and are therefore unlikely to be involved in PL remodeling. In contrast, the enzymes of neutral lipid (NL) synthesis and mobilization are central mediators of FA trafficking. The phospholipid:DAG acyltransferase (PDAT) Lro1p has a substantial effect on FA release and on PL equilibrium, emerging as an important mediator in PL remodeling. The acyl-CoA dependent biosynthetic activities of NL metabolism are also involved in PL homeostasis through active modulation of the substrate available for PL synthesis. In addition TAG mobilization makes an important contribution, especially in cells from stationary phase, to FA availability. Beyond its well-established role in the formation of a storage pool, NL metabolism could play a crucial role as a mechanism to uncouple the pools of PL and acyl-CoAs from each other and thereby to allow independent regulation of each one
Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling
No full textIn 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
Nocturnal energy demand in plants: insights from studying mutants impaired in β-oxidation
No full textFatty acid beta-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core beta-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal beta-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly alpha-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable alpha-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of alpha-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and beta-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal beta-oxidation plays a major role in dark-treated plants after depletion of starch reserves
Investigation of fatty acid transport across cellular and peroxisomal membranes
No full textDer Mechanismus, der den Transport von freien Fettsäuren durch die Plasmamembran vermittelt, ist trotz intensiver Forschung und einer Vielzahl von Publikationen weiterhin unaufgeklärt. Im Rahmen dieser Arbeit sind wir der Frage nachgegangen, ob Acyl-CoA-Synthetasen am Fettsäuretransport in Saccharomyces cerevisiae beteiligt sind. In früheren Studien konnten wir zeigen, dass die kombinierte Deletion der Acyl-CoA-Synthetasen FAA1 und FAA4 in YB332 zu einem Fettsäuresekretions-Phänotyp führt, der durch einen massiven Export von freien Fettsäuren während der exponentiellen Phase und einen Re-Import von freien Fettsäuren während der stationären Phase charakterisiert ist. Für die Durchführung weiterer Transportstudien wurden zusätzlich in der Doppelmutante faa1Δfaa4Δ alle anderen bekannten Acyl-CoA-Synthetasen inaktiviert. Unsere Ergebnisse zeigten, dass der Transport durch die Plasmamembran ohne jegliche Acyl-CoA-Synthetase-Aktivität stattfinden kann. Die Richtung des Transportes von freien Fettsäuren ist umkehrbar und wird durch den metabolischen Zustand der Zellen aktiv reguliert. Dabei existiert anscheinend ein Kontrollmechanismus, der bei einer drastischen Änderung der Zusammensetzung des Fettsäure-Pools in den Zellen einen aktiven Export der Fettsäuren initiiert. Hingegen wird der Import von exogenen Fettsäuren durch das Fehlen anderer Kohlenstoffquellen, also einem Hungersignal, im Stadium der stationären Phase ausgelöst. Im Gegensatz zum Fettsäuretransport durch die Plasmamembran ist der Transport von Fettsäuren in das peroxisomale Lumen im Detail besser verstanden. In Hefen und Pflanzen wurden peroxisomale ABC-Transporter identifiziert, die eine essentielle Funktion bei der Aufnahme von Fettsäuren im Zuge der β-Oxidation haben. Trotz vergleichbarer Komponenten scheint sich der Mechanismus des Fettsäure-Imports in Peroxisomen der Pflanzen grundlegend von dem in S. cerevisiae zu unterscheiden. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der ABC-Transporter Pat1p-Pat2p aus Hefe nicht durch den pflanzlichen ABC-Transporter PXA1 funktional zu ersetzen ist. Erst die kombinierte Expression der pflanzlichen Proteine PXA1 und LACS7 führte zu einer erfolgreichen Komplementation der Doppelmutante pat1Δfaa2Δ. Der Mechanismus des Fettsäure-Imports in Peroxisomen der Pflanzen scheint sich demnach grundlegend von dem in S. cerevisiae zu unterscheiden. Zusätzliche Erkenntnisse über den Ablauf von Transport der Fettsäuren durch die peroxisomale Membran und anschließender Metabolisierung durch die β-Oxidation haben wir durch die Manipulation des peroxisomalen Acyl-CoA-Pools in S. cerevisiae gewonnen. Die kombinierte Deletion der peroxisomalen Acyl-CoA-Thioesterase TES1 und der peroxisomalen Acyl-CoA-Synthetase FAA2 in YB332 führte zu einem deutlichen Phänotyp. Bei dieser Mutante wurde weder auf Minimalmedium mit Ölsäure noch auf Minimalmedium ohne Ölsäure Wachstum nachgewiesen. Außerdem wurde ein drastisches Absinken der Konzentration des zellulären Acyl-CoA-Pools beobachtet. Unsere Daten belegen somit ein Zusammenwirken von Tes1p und Faa2p, die gemeinsam das Verhältnis von freiem CoA zu Acyl-CoA im Peroxisom zu regulieren scheinen. Interessanterweise konnte durch die zusätzliche Deletion des peroxisomalen ABC-Transporters PAT1 der Phänotyp teilweise aufgehoben werden. Demnach wird eine Destabilisierung des CoA/Acyl-CoA-Verhältnisses durch die Verhinderung des Imports von Acyl-CoA in die Peroxisomen unterbunden. Unsere Daten zeigen somit erstmals, dass ein fehlgeleiteter peroxisomaler Fettsäurestoffwechsel dramatische Auswirkungen auf den Metabolismus der gesamten Zelle erlangen kann. Ein weiterer Aspekt des peroxisomalen Fettsäurestoffwechsels wurde in Pflanzen untersucht. Über die Funktion des ABC-Transporters PXA1 in Arabidopsis thaliana während der vegetativen Wachstumsphase ist wenig bekannt. In dieser Arbeit wurde ein durch eine verlängerte Dunkelphase induzierter Phänotyp der pxa1-Mutante untersucht. Eine Verlängerung der Dunkelphase führte bei diesen Pflanzen zum vollständigen Absterben, während die Wildtyp-Pflanzen zu diesem Zeitpunkt keine Symptome zeigten. Längere Dunkelphasen führten zu massiven Beschädigungen der Membransysteme. Eine massive Welke setzte trotz ausreichender Wasserversorgung ein. Unsere Studien zeigten, dass TAG unter den Bedingungen einer langanhaltenden Dunkelphase offensichtlich als Depot für Fettsäuren dient, die letztendlich für den Abbau durch die β-Oxidation vorgesehen sind. Die Kombination von β-Oxidation und TAG-Synthese führt dementsprechend zu einem konstant niedrigen Fettsäurespiegel im Wildtyp. In der pxa1-Mutante entfällt der Abbau der Fettsäuren via β-Oxidation und es erfolgt ein deutlicher Anstieg der Konzentration der freien Fettsäuren. Der Detergens-Charakter der freien Fettsäuren führt zu gravierende strukturelle Schäden der Chloroplasten und anschließendem Zelltod. Da dieser Phänotyp durch Zugabe exogener Saccharose unterdrückt werden kann, postulieren wir, dass die Freisetzung von Fettsäuren als Kompensationsmechanismus bei Engpässen der Energieversorgung während langanhaltender Dunkelheit dient. Demnach spielt die β-Oxidation in adulten Pflanzen eine essentielle Rolle für die Aufrechthaltung der Energieversorgung bei einer verlängerten Dunkelphase.Despite intensive research and numerous publications the precise mechanism by which free fatty acids cross the plasma membrane is still controversial. Within this work we addressed the question whether acyl-CoA synthetases are involved in lipid transport in Saccharomyces cerevisiae. In our previous studies we could show that the combined deletion of the acyl-CoA synthetases FAA1 and FAA4 in YB332 leads to a fatty acid secretion phenotype which is characterized by a massive export of free fatty acids during the exponential phase and a re-import of free fatty acids during the stationary phase. In order to carry out further transport studies all additional acyl-CoA synthetases were inactivated in the background of the double mutant faa1Δfaa4Δ. Our results could show that transport through the plasma membrane can take place in the absence of any acyl-CoA synthetase activity. The direction of free fatty acid transport is reversible and can be actively regulated by the metabolic state of the cell. Obviously, a specific control mechanism initiates an active export of fatty acids upon a drastic alteration in the composition of the fatty acid pool of the cell. In contrast, shortage of carbon sources, namely a starvation signal, triggers the import of exogenous fatty acids during the stationary phase. In contrast to fatty acid transport across the plasma membrane, transport of fatty acids across the peroxisomal membrane is understood in more detail. In both yeast and plants peroxisomal ABC-transporters with an essential function in the uptake of fatty acids during β-oxidation have been identified. Despite the existence of comparable elements, the mechanism of fatty acid import in plant peroxisomes appears to differ fundamentally from that of S. cerevisiae. In this work, it could be shown, that the ABC-transporter Pat1p-Pat2p from yeast cannot be functionally replaced by the plant ABC-transporter PXA1. Only the combined expression of the plant proteins PXA1 and LACS7 resulted in successful complementation of the double mutant pat1Δfaa2Δ. Therefore, the mechanism of fatty acid import in plant peroxisomes appears to be significantly different from that of S. cerevisiae. In addition, it was possible to obtain insights on the process of fatty acid transport across the peroxisomal membrane and subsequent metabolisation by β-oxidation through the manipulation of the peroxisomal acyl-CoA pool in S. cerevisiae. The combined deletion of the acyl-CoA thioesterase TES1 and the peroxisomal acyl-CoA synthetase FAA2 in YB332 led to a distinct phenotype. This mutant did not exhibit growth in minimal medium in the presence or absence of oleic acid. In addition, a drastic reduction of the cellular acyl-CoA pool was observed. Our data support the hypothesis of a tight interaction of Tes1p and Faa2p, which in combination appear to regulate the ratio of free CoA to acyl-CoA in the peroxisomes. Interestingly, the additional deletion of the peroxisomal ABC-transporter PAT1 could partly suppress the phenotype. Thus, inhibition of the import of acyl-CoA into the peroxisomes can prevent the destabilization of the CoA/acyl-CoA-ratio. Our data indicate for the first time that a degenerated peroxisomal fatty acid metabolism is able to impact the metabolism of the entire cell. Another aspect of fatty acid metabolism was investigated in plants. Very little is known regarding the function of the ABC-transporter PXA1 in Arabidopsis thaliana during the vegetative growth phase. In this work, the phenotype of the pxa1-mutant induced by a phase of prolonged darkness was investigated. An extension of the dark phase resulted in lethality for these plants, while wild-type plants showed no symptoms. Extended dark conditions led to massive damages to the membrane system. Extensive wilting was observed despite sufficient water supply. Our studies showed that under conditions of prolonged darkness, TAG functions as a transient buffer for fatty acids that can finally be released by β-oxidation. The combination of β-oxidation and TAG-synthesis resulted in constant low levels of fatty acids in the wild-type. In the pxa1-mutant, the degradation of fatty acids via β-oxidation is impaired leading to a distinct increase in the concentration of free fatty acids. The detergent-like properties of free fatty acids resulted in severe structural damage of chloroplasts and subsequent cell death. As this phenotype can be suppressed by providing exogenous sucrose, we propose that the release of fatty acids serves as a mechanism to compensate for shortage of energy during extended darkness. It can be concluded that β-oxidation plays an essential role in energy maintenance in adult plants during a phase of prolonged darkness
Impact of SE and TAG synthesis and degradation on FFA homeostasis.
No full text<p>Displayed are the total FFA in YB526 cells and YB526 cells additionally deficient for enzymes of SE and TAG synthesis and degradation. Cells were grown to late stationary phase (136 h) in YPR media. Mean values of at least three independent experiments. Error bars correspond to standard deviation. Asterisks indicate significantly different values between the reference strain YB526 and the individual mutant strain (P≤0.05).</p
Impact of TAG metabolism on FFA homeostasis.
No full text<p>Given are the content of total FFA in YB526 cells and YB526 cells additionally deficient for combinations of the DAG acyltransferases <i>LRO1</i> and <i>DGA1</i>, TAG lipases and phospholipases B. Cells were grown to late stationary phase (136 h) in YPR media. Mean values of at least three independent experiments. Error bars correspond to standard deviation. Asterisks indicate significantly different values between the reference strain YB526 and the individual mutant strain (P≤0.05).</p
Overview of lipid metabolism in yeast and the enzymes deleted within this work.
No full text<p>Formation of lyso-PA from dihydroxyacetone phosphate (DHAP) is mediated by synthesis and reduction of 1-acyl-DHAP. Compounds in alphabetic order: Acyl-CoA, acyl-coenzymeA; CDP-DAG, cytidinediphosphate-diacylglycerol; DAG, diacylglycerol; DHAP, dihydroxyacetone phosphate; FFA, free fatty acid; Glycerol-3-P, glycerol-3-phosphate; Lyso-PA, lyso-phosphatidic acid; Lyso-PL, lyso-phospholipid; PA, phosphatidic acid; PL, phospholipid; SE, steryl ester; TAG, triacylglycerol. Enzymatic activities in alphabetic order: Are1,2, acyl-CoA:sterol acyltransferase 1 and 2; Dga1, diacylglycerol acyltransferase 1; Faa1,2,3,4 and Fat1, fatty acid activation 1 to 4 and fatty acid transporter 1 (acyl-CoA synthetases); Lro1, phospholipid:diacylglycerol acyltransferase (PDAT); Nte1, phosphatidylcholine phospholipase B; Plb1,2,3, phospholipase B 1 to 3; Tgl1,Yeh1,Yeh2, steryl ester hydrolases; Tgl2,3,4,5, triacylglycerol lipase 2 to 5.</p
Relative FA composition (in percentage) of specific lipid classes.
No full text<p>14∶0 is included in the total but is not presented in the table. Cells were grown in YPR to late stationary phase (136 h), lipid classes were separated by TLC and their FA composition determined by GC after transmethylation. The mean values correspond to three independent experiments; standard deviation is shown within parentheses.</p
Changes in lipid class composition of mutant strains.
No full text<p>Given are the contents of PC (A), PE (B), TAG (C), DAG (D) and SE (E) in YB526 cells and YB526 cells additionally deficient in TAG or SE metabolism. Cells were grown to late stationary phase (136 h) in YPR, lipid classes were separated by TLC and subjected to transmethylation. The resulting FA methyl esters were quantified by GC. Error bars represent the standard deviation in three independent experiments. Asterisks indicate significantly different values between the reference strain YB526 and the individual mutant strain (P≤0.05).</p
Yeast strains.
No full text<p>The selection marker used for each deletion is indicated next to the targeted gene. Posterior removal of the marker is indicated by the deleted gene followed by no marker description.</p
