Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.     

Contributions of Carnitine Acetyltransferases to Intracellular Acetyl Unit Transport in Candida albicans

Transport of acetyl-CoA between intracellular compartments is mediated by carnitine acetyltransferases (Cats) that reversibly link acetyl units to the carrier molecule carnitine. The genome of the opportunistic pathogenic yeast Candida albicans encodes several (putative) Cats: the peroxisomal and mitochondrial Cat2 isoenzymes encoded by a single gene and the carnitine acetyltransferase homologs Yat1 and Yat2. To determine the contributions of the individual Cats, various carnitine acetyltransferase mutant strains were constructed and subjected to phenotypic and biochemical analyses on different carbon sources. We show that mitochondrial Cat2 is required for the intramitochondrial conversion of acetylcarnitine to acetyl-CoA, which is essential for a functional tricarboxylic acid cycle during growth on oleate, acetate, ethanol, and citrate. Yat1 is cytosolic and contributes to acetyl-CoA transport from the cytosol during growth on ethanol or acetate, but its activity is not required for growth on oleate. Yat2 is also cytosolic, but we were unable to attribute any function to this enzyme. Surprisingly, peroxisomal Cat2 is essential neither for export of acetyl units during growth on oleate nor for the import of acetyl units during growth on acetate or ethanol. Oxidation of fatty acids still takes place in the absence of peroxisomal Cat2, but biomass formation is absent, and the strain displays a growth delay on acetate and ethanol that can be partially rescued by the addition of carnitine. Based on our results, we present a model for the intracellular flow of acetyl units under various growth conditions and the roles of each of the Cats in this process.     

Carnitine Acetyltransferase: Candidate for the Transfer of Acetyl Groups Through the Mitochondrial Membrane of Yeast

On the basis of its specific activity and its affinity for acetyl-coenzyme A, carnitine acetyltransferase appears to be the most likely candidate for acetyl group transfer out of yeast mitochondria.     

Purification and properties of carnitine acetyltransferases from bovine spermatozoa and heart

To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of M_r 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-l-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of l-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme.     

Intracellular acetyl unit transport in fungal carbon metabolism

Acetyl coenzyme A (acetyl-CoA) is a central metabolite in carbon and energy metabolism. Because of its amphiphilic nature and bulkiness, acetyl-CoA cannot readily traverse biological membranes. In fungi, two systems for acetyl unit transport have been identified: a shuttle dependent on the carrier carnitine and a (peroxisomal) citrate synthase-dependent pathway. In the carnitine-dependent pathway, carnitine acetyltransferases exchange the CoA group of acetyl-CoA for carnitine, thereby forming acetyl-carnitine, which can be transported between subcellular compartments. Citrate synthase catalyzes the condensation of oxaloacetate and acetyl-CoA to form citrate that can be transported over the membrane. Since essential metabolic pathways such as fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle are physically separated into different organelles, shuttling of acetyl units is essential for growth of fungal species on various carbon sources such as fatty acids, ethanol, acetate, or citrate. In this review we summarize the current knowledge on the different systems of acetyl transport that are operational during alternative carbon metabolism, with special focus on two fungal species: Saccharomyces cerevisiae and Candida albicans.     

Isolation and characterization of carnitine acetyltransferase from S. cerevisiae

Carnitine acetyltransferase was isolated from yeast Saccharomyces cerevisiae with an apparent molecular weight of 400,000. The enzyme contains identical subunits of 65,000 Da. The Km values of the isolated enzyme for acetyl-CoA and for carnitine were 17.7 microM and 180 microM, respectively. Carnitine acetyltransferase is an inducible enzyme, a 15-fold increase in the enzyme activity was found when the cells were grown on glycerol instead of glucose. Carnitine acetyltransferase, similarly to citrate synthase, has a double localization (approx. 80% of the enzyme is mitochondrial), while acetyl-CoA synthetase was found only in the cytosol. In the mitochondria carnitine acetyltransferase is located in the matrix space. The incorporation of 14C into CO2 and in lipids showed a similar ratio, 2.9 and 2.6, when the substrate was [1-14C]acetate and [1-14C]acetylcarnitine, respectively. Based on these results carnitine acetyltransferase can be considered as an enzyme necessary for acetate metabolism by transporting the activated acetyl group from the cytosol into the mitochondrial matrix.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

Localization and solubilization of a rat liver microsomal carnitine acetyltransferase.

Carnitine acetyltransferase activity had been previously shown to occur in peroxisomes, mitochondria, and a membranous fraction of rat and pig hepatocytes. When components of this third subcellular fraction (plasma membranes, components of the Golgi apparatus, and microsomes) were further separated, carnitine acetyltransferase fractionated with the microsomes. Microsomes isolated by three different methods (isopycnic sucrose density zonal centrifugation, high-speed differential centrifugation, and aggregation with Ca²⁺ followed by low-speed differential centrifugation) all contained carnitine acetyltransferase activity. The lability of carnitine acetyltransferase in microsomes isolated by different methods and in different isolation media is reported.When total microsomes were subfractionated into rough and smooth components, carnitine acetyltransferase activity was found to the same extent in both and was tightly associated with the microsomal membrane. The microsomal enzyme was rapidly inactivated in 0.25 m sucrose or 0.1 m phosphate, but was stable for at least 2 weeks in 0.4 m KCl. Extensive treatment with high ionic strength salt solutions, 1% Triton X-100, or a combination of the two was used to solubilize microsomal carnitine acetyltransferase activity.Carnitine octanoyltransferase activity was also found in the microsomal fractions isolated by three different methods, but no carnitine palmitoyltransferase was detected in the microsomal fractions. It is proposed that microsomal carnitine acetyl- and octanoyltransferases could be involved in the transfer of acyl groups across the microsomal membrane, thereby providing a source of acetyl and other acyl CoA's at sites of acetylation reactions and synthesis.     

DL-aminocarnitine and acetyl-DL-aminocarnitine. Potent inhibitors of carnitine acyltransferases and hepatic triglyceride catabolism

DL-Aminocarnitine (3-amino-4-trimethylaminobutyric acid) and acetyl-DL-aminocarnitine (3-acetamido-4-trimethylaminobutyric acid) have been synthesized and the interactions of these compounds with carnitine acetyltransferase and carnitine palmitoyltransferase investigated. As anticipated from the low group transfer potential of amides, carnitine acetyltransferase catalyzes the transfer of acetyl groups from CoASAc to aminocarnitine (Km = 3.8 mM) but does not catalyze detectable transfer from acetylaminocarnitine to CoASH. Acetyl-DL-aminocarnitine is, however, a potent competitive inhibitor of carnitine acetyltransferase (Ki = 24 microM) and is bound to carnitine acetyltransferase about 13-fold more tightly than is acetylcarnitine, with which it is isosteric. DL-Aminocarnitine and, to a lesser extent, acetyl-DL-aminocarnitine are also inhibitors of the carnitine palmitoyltransferase activity of detergent-lysed rat liver mitochondria; in the presence of 1 mM L-carnitine, 5 microM aminocarnitine inhibits palmitoyl transfer by 64%. Significant acylation of aminocarnitine by palmitoyl-CoA was not observed. Neither aminocarnitine nor acetylaminocarnitine is significantly catabolized by mice; aminocarnitine is converted to acetylaminocarnitine in vivo. Both compounds are excreted in the urine. Mice given acetylaminocarnitine catabolize [14C]acetyl-L-carnitine and [14C]palmitate to 14CO2 more slowly than do control animals. Mice given acetylaminocarnitine and then starved are found to reversibly accumulate triglycerides in their livers; mice given the inhibitor but not starved do not show this effect.     

Oxidation of acetate,acetyl CoA and acetylcarnitine by pea mitochondria

Acetylcarnitine was rapidly oxidised by pea mitochondria. (-)-carnitine was an essential addition for the oxidation of acetate or acetyl CoA. When acetate was sole substrate, ATP and Mg²⁺ were also essential additives for maximum oxidation. CoASH additions inhibited the oxidation of acetate, acetyl CoA and acetylcarnitine. It was shown that CoASH was acting as a competitive inhibitor of the carnitine stimulated O₂ uptake. It is suggested that acetylcarnitine and carnitine passed through the mitochondrial membrane barrier with ease but acetyl CoA and CoA did not. Carnitine may also buffer the extra- and intra-mitochondrial pools of CoA. The presence of carnitine acetyltransferase (EC 2.3.1.7) on the pea mitochondria is inferred.     

Role of carnitine acetyltransferases in acetyl coenzyme A metabolism in Aspergillus nidulans

The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while β-oxidation of fatty acids generates acetyl-CoA in peroxisomes. The acetyl-carnitine shuttle in which acetyl-CoA is reversibly converted to acetyl-carnitine by carnitine acetyltransferase (CAT) enzymes is important for intracellular transport of acetyl units. In the filamentous ascomycete Aspergillus nidulans, a cytoplasmic CAT, encoded by facC, is essential for growth on sources of cytoplasmic acetyl-CoA while a second CAT, encoded by the acuJ gene, is essential for growth on fatty acids as well as acetate. We have shown that AcuJ contains an N-terminal mitochondrial targeting sequence and a C-terminal peroxisomal targeting sequence (PTS) and is localized to both peroxisomes and mitochondria, independent of the carbon source. Mislocalization of AcuJ to the cytoplasm does not result in loss of growth on acetate but prevents growth on fatty acids. Therefore, while mitochondrial AcuJ is essential for the transfer of acetyl units to mitochondria, peroxisomal localization is required only for transfer from peroxisomes to mitochondria. Peroxisomal AcuJ was not required for the import of acetyl-CoA into peroxisomes for conversion to malate by malate synthase (MLS), and export of acetyl-CoA from peroxisomes to the cytoplasm was found to be independent of FacC when MLS was mislocalized to the cytoplasm.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.     

Carnitine acetyltransferase activity in oleaginous yeasts

Abstract The highest activities of carnitine acetyltransferase (CAT) were found in non-oleaginous yeasts ( Candida utilis and Saccharomyces cerevisiae ); lower activities, ranging from 50% down to 3% of the highest values, were found in various strains of oleaginous yeasts ( Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum ). Supply of acetyl units into the cytosol of the latter, but not of the former yeasts, was therefore necessarily reliant on the action of ATP: citrate lyase (ACL), which was present in all oleaginous yeasts. There was no correlation, however, between the amount of lipid in the oleaginous yeasts and the specific activities of either CAT or ACL. Activity of CAT was increased up to 30-fold by growing yeasts on a triacylglycerol.     

Acetyl-l-carnitine as a precursor of acetylcholine

Synthesis of [³H]acetylcholine from [³H]acetyl-l-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [³H] and [¹⁴C] labeled acetylcholine were produced when [³H]acetyl-l-carnitine andd-[U-¹⁴C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-l-carnitine to acetylcholine was dependent on concentration of acetyl-l-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-l-carnitine in the treatment of age-related cholinergic deficits.     

The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions.

We investigated how NADH generated during peroxisomal beta-oxidation is reoxidized to NAD+ and how the end product of beta-oxidation, acetyl-CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysates. In addition, mdh3-disrupted cells were unable to grow on oleate whereas growth on other non-fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta-oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.     

ATP: citrate lyase and carnitine acetyltransferase activity in a citric-acid-producing Aspergillus niger strain

Summary Two enzymes are responsible for the transport of acetyl groups across the mitochondrial membrane, ATP:citrate lyase and carnitine acetyltransferase. At the beginning of the fermentation process an increase in the specific activities of both enzymes was observed, followed by a drop after 2–3 days of fermentation. Under citric-acid-accumulating conditions this drop was much more pronounced, especially for ATP:citrate lyase activity. After the first day the carnitine acetyltransferase activity was higher than that of ATP:citrate lyase. Offsprint requests to: K. Jernejc     

THE SYNTHESIS OF ACETYLCHOLINE FROM ACETYL-CoA, ACETYL-DEPHOSPHO-CoA AND ACETYLPANTETHEINE PHOSPHATE BY CHOLINE ACETYLTRANSFERASE

Abstract— The synthesis of ACh by choline acetyltransferase (ChAc) has been examined using acetyl-CoA, acetyl-dephospho-CoA and acetylpantetheine phosphate. At pH 7.5 K_m values of 25.7 μ m for acetyl-CoA, 54.8 μ m for acetyl-dephospho-CoA and 382 μ m for acetylpantetheine phosphate were obtained and are similar to those at pH 6.0. This indicates that the 3-phosphate may not be required for binding the substrate to the enzyme unlike carnitine acetyltransferase.
Inhibitor constants ( K_i ) for CoA, dephospho-CoA and pantetheine phosphate were also measured and when considered with the K_m values obtained for the acetyl derivatives it is concluded that acetyl-dephospho-CoA could be a successful acetyl donor in the synthesis of ACh.
Acetyl-dephospho-CoA was found to be less satisfactory as a substrate for citrate synthase.     

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.     

Aspects of carnitine ester metabolism in sheep liver

1. Carnitine acetyltransferase (EC 2.3.1.7) activity in sheep liver mitochondria was 76nmol/min per mg of protein, in contrast with 1.7 for rat liver mitochondria. The activity in bovine liver mitochondria was comparable with that of sheep liver mitochondria. Carnitine palmitoyltransferase activity was the same in both sheep and rat liver mitochondria. 2. The [free carnitine]/[acetylcarnitine] ratio in sheep liver ranged from 6:1 for animals fed ad libitum on lucerne to approx. 1:1 for animals grazed on open pastures. This change in ratio appeared to reflect the ratio of propionic acid to acetic acid produced in the rumen of the sheep under the two dietary conditions. 3. In sheep starved for 7 days the [free carnitine]/[acetylcarnitine] ratio in the liver was 0.46:1. The increase in acetylcarnitine on starvation was not at the expense of free carnitine, as the amounts of free carnitine and total acid-soluble carnitine rose approximately fivefold on starvation. An even more dramatic increase in total acid-soluble carnitine of the liver was seen in an alloxan-diabetic sheep. 4. The [free CoA]/[acetyl-CoA] ratio in the liver ranged from 1:1 in the sheep fed on lucerne to 0.34:1 for animals starved for 7 days. 5. The importance of carnitine acetyltransferase in sheep liver and its role in relieving ;acetyl pressure' on the CoA system is discussed.