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.     

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.     

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.     

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.     

Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p.

In Saccharomyces cerevisiae, beta-oxidation of fatty acids is confined to peroxisomes. The acetyl-CoA produced has to be transported from the peroxisomes via the cytoplasm to the mitochondrial matrix in order to be degraded to CO(2) and H(2)O. Two pathways for the transport of acetyl-CoA to the mitochondria have been proposed. The first involves peroxisomal conversion of acetyl-CoA into glyoxylate cycle intermediates followed by transport of these intermediates to the mitochondria. The second pathway involves peroxisomal conversion of acetyl-CoA into acetylcarnitine, which is subsequently transported to the mitochondria. Using a selective screen, we have isolated several mutants that are specifically affected in the second pathway, the carnitine-dependent acetyl-CoA transport from the peroxisomes to the mitochondria, and assigned these CDAT mutants to three different complementation groups. The corresponding genes were identified using functional complementation of the mutants with a genomic DNA library. In addition to the previously reported carnitine acetyl-CoA transferase (CAT2), we identified the genes for the yeast orthologue of the human mitochondrial carnitine acylcarnitine translocase (YOR100C or CAC) and for a transport protein (AGP2) required for carnitine transport across the plasma membrane.     

Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-CoA in rat heart

Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonyl-CoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were (13)C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the (13)C labeling of malonyl-CoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the (13)C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal beta-oxidation forming C(12) and C(14) acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal beta-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.     

The sensitivity of acetyl-coenzyme A carboxylase to citrate stimulation in a homogenate of rat liver containing subcellular particles

1. Although citrate is known to activate purified preparations of acetyl-CoA carboxylase, it had no stimulatory effect on the incorporation of [¹⁴C]acetate into long-chain fatty acids in a whole homogenate of rat liver (S_0.7) under conditions in which the activity of acetyl-CoA carboxylase was rate-limiting for fatty acid synthesis. 2. The rate of incorporation of acetyl carbon into fatty acids was estimated in S_0.7 preparations incubated with [¹⁴C]acetate, by measuring the specific radioactivity of the acetyl carbon of acetyl-CoA and the incorporation of ¹⁴C into fatty acids. These estimates were compared with estimates of acetyl-CoA carboxylase activity in the S_0.7 preparation obtained by direct assay in conditions in which the enzyme was in the fully activated state. 3. In the absence of citrate, incorporation of acetyl carbon into fatty acids was about 75% of the value expected if the acetyl-CoA carboxylase in the S_0.7 preparation were in the fully activated state. 4. Incorporation of acetyl carbon into fatty acids in the S_0.7 preparation was stimulated by citrate, but the effect was many times less than the stimulation of [¹⁴C]acetate incorporation by citrate in particle-free preparations. 5. When the mitochondria and microsomes were removed from the S_0.7 preparation, [¹⁴C]acetate incorporation into fatty acids fell to a negligible value and the preparation became highly sensitive to stimulation by citrate. 6. It is suggested that in the presence of mitochondria and microsomes, and in the intact liver cell, the degree of activation of acetyl-CoA carboxylase is such that citrate activation may not be of physiological significance.     

Serum factors that stimulate fatty acid oxidation: physiological specificity

In the accompanying paper (Wice et al., 1986) we reported that serum from chickens contains small molecular weight compounds that stimulate long-chain fatty acid oxidation ten fold or more in HeLa cells. Here we show that this response is not limited to specific sera or to specific target cells. The specificity of the metabolic response to these factors was also investigated. They had no effect on the following major pathways of HeLa cell metabolism: 1) the oxidation of the medium-chain fatty acid, octanoic acid, 2) the rate of glycolysis of glucose, 3) the flux of glucose carbon through the oxidative arm of the pentose cycle, 4) the entry of pyruvate into the citrate cycle, 5) the oxidation of glutamine carbon, 6) the utilization rate of oxygen or 7) the rate of fatty acid synthesis. Furthermore, the increased oxidation of long-chain fatty acids was not a result of an increased uptake into the cells. Thus, the serum factors appear to be very specific for the oxidation of long-chain fatty acids for energy. Since carnitine also stimulates long-chain fatty acid oxidation in these cells, it seems likely that these compounds either facilitate the activity of carnitine or provide the same function--presumably the transport of long-chain fatty acid into and out of the mitochondria.     

Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short chain fatty acids

Short chain fatty acids such as acetic, propionic, and butyric acids can be synthesized into polyhydroxyalkanoates (PHAs) by Ralstonia eutropha. Metabolic carbon fluxes of the acids in living cells have significant effect on the yield, composition, and thermomechanical properties of PHA bioplastics. Based on the general knowledge of central metabolism pathways and the unusual metabolic pathways in R. eutropha, a metabolic network of 41 bioreactions is constructed to analyze the carbon fluxes on utilization of the short chain fatty acids. In fed-batch cultures with constant feeding of acid media, carbon metabolism and distribution in R. eutropha were measured involving CO2, PHA biopolymers, and residual cell mass. As the cells underwent unsteady state metabolism and PHA biosynthesis under nitrogen-limited conditions, accumulative carbon balance was applied for pseudo-steady-state analysis of the metabolic carbon fluxes. Cofactor NADP/NADPH balanced between PHA synthesis and the C3/C4 pathway provided an independent constraint for solution of the underdetermined metabolic network. A major portion of propionyl-CoA was directed to pyruvate via the 2-methylcitrate cycle and further decarboxylated to acetyl-CoA. Only a small amount of propionate carbon (<15% carbon) was directly condensed with acetyl-CoA for 3-hydroxyvalerate. The ratio of glyoxylate shunt to TCA cycle varies from 0 to 0.25, depending on the intracellular acetyl-CoA level and acetic acid in the medium. Malate is the node of the C3/C4 pathway and TCA cycle and its decarboxylation to dehydrogenation ranges from 0.33 to 1.28 in response to the demands on NADPH and oxaloacetate for short chain fatty acids utilization.     

Probing the link between citrate and malonyl-CoA in perfused rat hearts

Little is known about the sources of cytosolic acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of fatty acid oxidation in the heart. We tested the hypothesis that citrate provides acetyl-CoA for malonyl-CoA synthesis after its mitochondrial efflux and cleavage by cytosolic ATP-citrate lyase. We expanded on a previous study where we characterized citrate release from perfused rat hearts (Vincent G, Comte B, Poirier M, and Des Rosiers C. Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846-E856, 2000). In the present study, we show that citrate release rates, ranging from 6 to 22 nmol/min, can support a net increase in malonyl-CoA concentrations induced by changes in substrate supply, at most 0.7 nmol/min. In experiments with [U-(13)C](lactate + pyruvate) and [1-(13)C]oleate, we show that the acetyl moiety of malonyl-CoA is derived from both pyruvate and long-chain fatty acids. This (13)C-labeling of malonyl-CoA occurred without any changes in its concentration. Hydroxycitrate, an inhibitor of ATP-citrate lyase, prevents increases in malonyl-CoA concentrations and decreases its labeling from [U-(13)C](lactate + pyruvate). Our data support at least a partial role of citrate in the transfer from the mitochondria to cytosol of acetyl units for malonyl-CoA synthesis. In addition, they provide a dynamic picture of malonyl-CoA metabolism: even when the malonyl-CoA concentration remains constant, there appears to be a constant need to supply acetyl-CoA from various carbon sources, both carbohydrates and lipids, for malonyl-CoA synthesis.     

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.     

Promoting lipid utilization with l-carnitine to improve oocyte quality

Successful embryo and fetal development is dependent on the quality of the oocyte from which it was derived. Several studies to date have demonstrated the link between appropriate metabolism and sufficient ATP production with oocyte quality and preimplantation embryo development. Metabolism of fatty acids for the purpose of synthesizing ATP occurs within mitochondria via β-oxidation and entry of fatty acids into this organelle is the rate-limiting step in this process. Transport of activated fatty acids into mitochondria is catalyzed by carnitine palmitoyl transferase-I (CPTI) which also requires the metabolite carnitine. Once inside the mitochondrial matrix, fatty acids are broken down into acetyl CoA molecules which are further metabolized via the TCA cycle and electron transport chain to produce ATP. The potential to improve oocyte quality by modulating fatty acid metabolism and β-oxidation with carnitine in culture media formulations or via dietary supplementation has received little attention. This review summarizes studies to date investigating the developmental importance of β-oxidation through the use of metabolic inhibitors and whether regulation by carnitine, in vitro or in vivo, has beneficial effects on oocyte and embryo development. Overall, there is little evidence to date that dietary carnitine can improve oocyte quality or female fertility; however inclusion of l-carnitine to in vitro oocyte maturation and embryo growth media improves embryo outcomes, most likely by supplying the oocyte and embryo with an essential co-factor required to utilize fatty acids.     

The effect of metabolites of the propionate pathway on the oxidative activity of liver mitochondria

Methylmalonate and propionate, the major metabolites of the propionate pathway of fatty and amino acid metabolism used at 1-4 mM cause selective inhibition of succinate and palmitoyl carnitine oxidation in liver mitochondria. Methylmalonate is more specific towards succinate, whereas propionate--towards palmitoyl carnitine oxidation. Methylmalonate is transported to mitochondria at a high rate with no effect on succinate transport. Being injected intramusculary methylmalonate has no inhibiting effect on the oxidative activity of mitochondria but is able to activate succinate and palmitoyl carnitine oxidation. The inhibiting effect of propionate on palmitoyl carnitine oxidation is a long-term one. Injections of these metabolites precursors, isoleucine, methionine and valine, produce an activating effect on succinate oxidation. Thus, propionate pathway metabolites may participate in the regulation of lipid-carbohydrate metabolism.     

Carnitine and derivatives in rat tissues

1. Free carnitine, acetylcarnitine, short-chain acylcarnitine and acid-insoluble carnitine (probably long-chain acylcarnitine) have been measured in rat tissues. 2. Starvation caused an increase in the proportion of carnitine that was acetylated in liver and kidney; at least in liver fat-feeding had the same effect, whereas a carbohydrate diet caused a very low acetylcarnitine content. 3. In heart, on the other hand, starvation did not cause an increase in the acetylcarnitine/carnitine ratio, whereas fat-feeding caused a decrease. The acetylcarnitine content of heart was diminished by alloxan-diabetes or a fatty diet, but not by re-feeding with carbohydrate. 4. Under conditions of increased fatty acid supply the acid-insoluble carnitine content was increased in heart, liver and kidney. 5. The acylation state of carnitine was capable of very rapid change. Concentrations of carnitine derivatives varied with different methods of obtaining tissue samples, and very little acid-insoluble carnitine was found in tissues of rats anaesthetized with Nembutal. In liver the acetylcarnitine (and acetyl-CoA) content decreased if freezing of tissue samples was delayed; in heart this caused an increase in acetylcarnitine. 6. Incubation of diaphragms with acetate or dl-β-hydroxybutyrate caused the acetylcarnitine content to become elevated. 7. Perfusion of hearts with fatty acids containing an even number of carbon atoms, dl-β-hydroxybutyrate or pyruvate resulted in increased contents of acetylcarnitine and acetyl-CoA. Accumulation of these acetyl compounds was prevented by the additional presence of propionate or pentanoate in the perfusion medium; this prevention was not due to extensive propionylation of CoA or carnitine. 8. Perfusion of hearts with palmitate caused a severalfold increase in the content of acid-insoluble carnitine; this increase did not occur when propionate was also present. 9. Comparison of the acetylation states of carnitine and CoA in perfused hearts suggests that the carnitine acetyltransferase reactants may remain near equilibrium despite wide variations in their steady-state concentrations. This is not the case with the citrate synthase reaction. It is suggested that the carnitine acetyltransferase system buffers the tissue content of acetyl-CoA against rapid changes.     

Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells

The tricarboxylic acid (TCA) cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoint glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.     

Metabolic and Developmental Effects Resulting from Deletion of the citA Gene Encoding Citrate Synthase in Aspergillus nidulans

Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsAΔ citAΔ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.There has been increased interest in primary carbon metabolism in fungi in recent years. There are two main reasons for this. As fungal pathogens establish infection they must adapt their utilization of carbon sources to the substrates present in the new environment of the host cells (reviewed in reference 6). With many of the fungal genomes available, the number of genes encoding enzymes and transporters potentially involved in central metabolism has become apparent and is greater than might have been anticipated (for example, see reference 16). Deciphering this complexity requires not only genome-wide studies but also detailed studies of individual genes encoding these proteins in order to determine their regulation and the cellular localization of the proteins, as well as their roles in metabolism and development. Here we report molecular genetic analysis of the citA gene encoding citrate synthase (EC 4.1.3.7), a central enzyme of carbon metabolism, in the filamentous ascomycete Aspergillus nidulans.Citrate synthase is required for the formation of citrate from acetyl-coenzyme A (CoA) and oxaloacetate in the tricarboxylic acid (TCA) cycle and is therefore necessary for respiratory growth as well as for the generation of intermediates for biosynthetic reactions. Together with aconitase, malate dehydrogenase, isocitrate lyase, and malate synthase, it is also an essential enzyme in the glyoxylate cycle, which is necessary for growth on carbon sources such as acetate, ethanol, and fatty acids which are catabolized via acetyl-CoA (reviewed in reference 26).In Saccharomyces cerevisiae the mitochondrial Cit1 is the major citrate synthase of the TCA cycle. An additional enzyme, Cit2, is peroxisomally localized via a C-terminal peroxisomal targeting sequence (PTS1) (29). In response to mitochondrial dysfunction CIT2 is upregulated via the retrograde response mediated by RTG1, -2, and -3, while mitochondrial respiratory deficiency results in RTG-dependent expression of CIT1 as well as that of aconitase (ACO1) and isocitrate dehydrogenase (IDH1 and IDH2), all enzymes necessary for 2-oxoglutarate formation and hence the synthesis of glutamate required for amino acid biosynthesis (9, 15, 30). In addition a third gene, CIT3, encodes a mitochondrial enzyme with citrate synthase activity. This enzyme has greater activity with propionyl-CoA, forming methylcitrate, and is necessary for the mitochondrial methylcitrate cycle involved in the metabolism of propionate (24). Cit2 has also been proposed to have methylcitrate synthase activity (17).In S. cerevisiae Cit2 also plays a role in the transfer of acetyl-CoA generated in peroxisomes by β-oxidation of fatty acids or by ethanol and acetate metabolism in the cytoplasm to the mitochondria for metabolism via the TCA cycle. There are two alternative pathways: transfer as acetyl-carnitine formed by the peroxisomal/mitochondrial carnitine acetyltransferase Cat2, together with the cytoplasmic Yat1 and Yat2 carnitine acetyltransferases, or transfer via citrate formed by Cit2 (45, 51, 52). Only disruption of both pathways (e.g., by deletion of CAT2 and CIT2) results in a growth defect on fatty acids. The fact that deletion of CIT2 is not essential for utilization of carbon sources metabolized via acetyl-CoA indicates that mitochondrial citrate synthase activity can replace the peroxisomal activity in the glyoxylate cycle. In contrast, in the pathogenic yeast Candida albicans, there is a single gene for citrate synthase and it is mitochondrial, and acetyl-CoA transport to mitochondria is solely dependent on the carnitine pathway (43, 57). In the plant Arabidopsis thaliana, there are five genes encoding citrate synthase enzymes. Two are peroxisomal (CSY2 and CSY3) and required for fatty acid respiration and seed germination, indicating that carnitine acetyltransferases are not required for shuttling acetyl units to the mitochondria (37).The filamentous ascomycete Aspergillus nidulans has both citrate synthase-encoding and methylcitrate synthase-encoding genes, citA and mcsA, respectively (8, 36). In both A. nidulans and Aspergillus fumigatus it has been shown that McsA is mitochondrial and has both methylcitrate and citrate synthase activities and is required for propionyl-CoA metabolism (8, 22, 31). Cell fractionation studies have shown that citrate synthase activity colocalizes with the mitochondrial fraction (35), and an N-terminal mitochondrial targeting sequence is predicted by the gene sequence (36). However, CitA has a putative C-terminal peroxisomal targeting sequence (PTS1 AKL), and genes in some filamentous ascomycetes also have potential PTS1 sequences (see below). The role of peroxisomal citrate synthase activity is not at all clear. The acuJ-encoded peroxisomal/mitochondrial carnitine acetyltransferase is required for growth on both fatty acids and acetate, while the facC-encoded cytoplasmic enzyme is required for growth on acetate (1, 20, 42). Therefore, like C. albicans, the carnitine shuttle is absolutely required for acetyl-CoA intracellular transport.Because of our interest in the role of peroxisomes in fatty acid and acetate metabolism in A. nidulans (21), we have investigated phenotypes resulting from deletion of the citA gene. Our results indicate that loss of CreA-mediated carbon repression allows expression of mcsA, resulting in the restoration of sufficient citrate synthase activity to suppress growth and developmental defects resulting from citAΔ. We have also investigated the role of peroxisomal localization of CitA and found this is at most extremely low and does not play a major role.     

Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.     

Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism

Carnitine plays an essential role in mitochondrial fatty acid β-oxidation as a part of a cycle that transfers long-chain fatty acids across the mitochondrial membrane and involves two carnitine palmitoyltransferases (CPT1 and CPT2). Two distinct carnitine acyltransferases, carnitine octanoyltransferase (COT) and carnitine acetyltransferase (CAT), are peroxisomal enzymes, which indicates that carnitine is not only important for mitochondrial, but also for peroxisomal metabolism. It has been demonstrated that after peroxisomal metabolism, specific intermediates can be exported as acylcarnitines for subsequent and final mitochondrial metabolism. There is also evidence that peroxisomes are able to degrade fatty acids that are typically handled by mitochondria possibly after transport as acylcarnitines. Here we review the biochemistry and physiological functions of metabolite exchange between peroxisomes and mitochondria with a special focus on acylcarnitines.     

Intramitochondrial factors controlling hepatic fatty acid oxidation at weaning in the rat

Fatty acid oxidation was studied in isolated liver mitochondria of rats during the suckling-weaning transition. The oxidation rate of oleyl-CoA and palmitoylcarnitine was reduced 2.5-fold in rats weaned on a high-carbohydrate diet compared to suckling rats, when acetyl-CoA produced by beta-oxidation was directed towards ketone-body synthesis. Weaning on a high-fat diet minimized this change. Channeling of acetyl-CoA towards citrate synthesis doubled the oxidation rate of both substrates in HC-weaned rats. Thus, in addition to changes in carnitine palmitoyltransferase I activity, the beta-hydroxymethylglutaryl-CoA synthase pathway is also involved in the decreased fatty acid oxidation at weaning. This was confirmed by measurement of beta-hydroxymethylglutaryl-CoA synthase pathway activity.