Maturation of cellular Fe-S proteins: an essential function of mitochondria.

Iron-sulfur (Fe-S) cluster-containing proteins perform important tasks in catalysis, electron transfer and regulation of gene expression. In eukaryotes, mitochondria are the primary site of cluster formation of most Fe-S proteins. Assembly of the Fe-S clusters is mediated by the iron-sulphate cluster assembly (ISC) machinery consisting of some ten proteins.

The yeast mitochondrial carrier Leu5p and its human homologue Graves' disease protein are required for accumulation of coenzyme A in the matrix.

The transport of metabolites, coenzymes, and ions across the mitochondrial inner membrane is still poorly understood. In most cases, membrane transport is facilitated by the so-called mitochondrial carrier proteins. The yeast Saccharomyces cerevisiae contains 35 members of the carrier family, but a function has been identified for only 13 proteins. Here, we investigated the yeast carrier Leu5p (encoded by the gene YHR002w) and its close human homologue Graves' disease protein. Leu5p is inserted into the mitochondrial inner membrane along the specialized import pathway used by carrier proteins. Deletion of LEU5 (strain Deltaleu5) was accompanied by a 15-fold reduction of mitochondrial coenzyme A (CoA) levels but did not affect the cytosolic CoA content. As a consequence, the activities of several mitochondrial CoA-dependent enzymes were strongly decreased in Deltaleu5 cells. Our in vitro and in vivo analyses assign a function to Leu5p in the accumulation of CoA in mitochondria, presumably by serving as a transporter of CoA or a precursor thereof. Expression of the Graves' disease protein in Deltaleu5 cells can replace the function of Leu5p, demonstrating that the human protein represents the orthologue of yeast Leu5p. The function of the human protein might not be directly linked to the disease, as antisera derived from patients with active Graves' disease do not recognize the protein after expression in yeast, suggesting that it does not represent a major autoantigen. The two carrier proteins characterized herein are the first components for which a role in the subcellular distribution of CoA has been identified.

Effect of insulin and glucagon on the uptake of carnitine by perfused rat liver.

The possible direct effects of insulin and glucagon on carnitine uptake by perfused rat liver were studied with L-[3H]carnitine of an initial concentration of 50 microM in the perfusate. Insulin (10 nM) did not significantly affect the uptake by livers from fed animals. However, insulin could reverse the stimulated transport by livers from 24-h fasted animals, reducing the uptake rate from 852 +/- 54.1 to 480 +/- 39.9 (mean +/- S.E.), P less than 0.01 (rates are expressed as nmol per h per 100 g body wt). Glucagon (50 nM) stimulated the uptake rate when livers were either from fed (551 +/- 40.1 vs. 915 +/- 55.3, P less than 0.01) or from fasted animals (852 +/- 54.1 vs. 1142 +/- 88.1, P less than 0.02). Based on these and earlier observations, we propose that the carnitine concentration in rat liver is controlled by insulin and glucagon via cellular transport processes.

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.

Release of carnitine from the perfused rat liver.

Perfused rat liver was shown to be the proper model for studies on hepatic cellular transport of carnitine. During recirculating perfusion the livers kept equilibrium with 45 nmol/ml total carnitine in perfusate, exhibited concentrative uptake and there was no sign of artificial leakage. The release side of the carnitine transport was characterized by utilizing outflow perfusions. The livers from fed rats exported daily 9.93 mumol per 100 g body weight total carnitine. This release rate is 4- or 10-fold higher than the estimated daily turnover in vivo or the measured urinary excretion. Therefore, the major part of the released carnitine has to re-enter the liver. The outward carnitine transport does not depend on energy or the Na+-K+ pump, since it did not respond to metabolic poisons and ouabain. However, the release rate was strongly inhibited by mersalyl and showed saturability in function of tissue carnitine levels. The Vmax of the saturable outward transport system was 2.47 nmol . min-1 . g-1 liver, the apparent Km was 0.27 mM tissue level (both as compared to total carnitine). These data showed the outward transport of carnitine from the liver to be protein mediated. The contribution of a diffusion (nonsaturable) component was estimated to be 20-25% in the range of tissue levels occurring in vivo. The rate of carnitine release from the liver decreased as an effect of 24 h starvation from the daily 9.92 mumol release to 6.55 mumol on 100 g body weight basis. This decrease is more pronounced when the release rates are expressed on the basis of tissue carnitine levels. The resulting value can be called rate constant (at the linear part of the saturation curve, Fig. 5) and it decreased to 5.00 min-1 from 8.41 min-1 as an effect of starvation. We have concluded that the altered parameters of carnitine transport across the liver cell is decisive in developing the higher hepatic carnitine concentration in the fasted state.

Enhanced uptake of carnitine by perfused rat liver following starvation.

Previously, the release of carnitine from the perfused rat liver was found to be protein-mediated, dependent on the nutritional state but not on metabolic energy. Further, it was shown to exceed the physiological demand by about 10-fold (Sandor et al. (1985) Biochim. Biophys. Acta 835, 83-91). In the present study the uptake of carnitine by perfused rat liver has been investigated. The liver tissue and the perfusate were in equilibrium when the carnitine concentration in the perfusate was close to 45 microM, physiological in the rat plasma. Under this condition, when no net carnitine transport occurred, an unidirectional uptake of L-[3H]carnitine was observed. Quantitatively, the uptake rate was 355 +/- 60 (S.D.) nmol/h per 100 g body weight at 45-50 microM perfusate concentration. This uptake capacity balances the previously reported excessive release (Sandor et al., op. cit.). On this basis we propose that a futile release/uptake cycle operates in carnitine transport across the liver cell membrane. Liverse of 24-h starved rats took up L-[3H]carnitine at 56% higher rate from the perfusate (75 microM) than livers of fed rats. Kinetic analysis revealed that fasting caused a decrease in Km value from 4.22 mM to 2.59 mM, whereas Vmax remained practically unchanged, average 0.95 mumol/min per 100 g body weight. D-[3H]Carnitine was transported at the same rate as L-carnitine and underwent the effect of fasting as well. The uptake was partially inhibited by 1 mM 2,4-dinitrophenol and 5 mM KCN, showing its dependency on metabolic energy. If Li+ replaced Na+ a strong inhibitory effect (to 20% of control) was observed, which suggests a co-transport of carnitine with Na+. Mersalyl, an SH reagent, had no effect on the uptake, whereas it practically abolished the release of carnitine from the perfused livers. This observation suggests that the inward and outward transport of carnitine are mediated by two different proteins.

Identification of a human mitochondrial ABC transporter, the functional orthologue of yeast Atm1p.

We have sequenced the entire coding region of the human ABC transporter ABC7. The protein represents a 'half-transporter' and displays high sequence similarity to the mouse ABC7 protein and to the mitochondrial ABC transporter Atm1p of Saccharomyces cerevisiae. As shown by immunostaining using a specific antibody, the human ABC7 protein (hABC7) is a constituent of mitochondria. The N-terminus of hABC7 contains the information for targeting and import into the organelles. When synthesised in yeast cells defective in Atm1p (strain delta atm1/hABC7), hABC7 protein can revert the strong growth defect observed for delta atm1 cells to near wild-type behaviour. The known phenotypical consequences of inactivation of the ATM1 gene are almost fully amended by expression of hABC7 protein. delta atm1/hABC7 cells harbour wild-type levels of cytochromes and extra-mitochondrial heme-containing proteins, they contain normal levels of mitochondrial iron, and the cellular content of glutathione is substantially reduced relative to the high levels detected in delta atm1 cells. Our results suggest that hABC7 is a mitochondrial protein, and represents the functional orthologue of yeast Atm1p.

The ABC transporter Atm1p is required for mitochondrial iron homeostasis.

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Delta atm1 cells are deficient in the holoforms, but not the apoforms of heme-carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Delta atm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30-fold higher levels of free iron as compared to wild-type organelles, i.e. three-fold more than mitochondria deficient in frataxin, the protein mutated in Friedreich's ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme-containing proteins in delta atm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

Opposite regulation of uncoupling protein 1 and uncoupling protein 3 in vivo in brown adipose tissue of cold-exposed rats.

Earlier we reported a 14-fold increase of glycogen in the brown adipose tissue (BAT) in rats when the animals were placed back from cold to neutral temperature. To elucidate the mechanism, here we compared the level of glucose transporter 4 (GLUT4) protein, uncoupling protein (UCP) 1 and UCP3 mRNA and protein expressions in the BAT under the same conditions. We found that the increased GLUT4 level in cold was maintained during the reacclimation. After 1 week cold exposure the mRNA and protein content of UCP1 increased parallel, while the protein level of UCP3 decreased, contrary to its own mRNA level.

Enhanced ADP-ribosylation and its diminution by lipoamide after ischemia-reperfusion in perfused rat heart.

Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD+ content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 +/- 8%) increase in ROS production determined by the dihydrorhodamine 123 method and significantly increased the malondialdehyde production (from < 1 to 23 +/- 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 +/- 7% to 23.1 +/- 5%), and NAD+ catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 +/- 2.4 nmol/ml, abolished DNA damage, and reduced NAD+ catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD+ catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.

Mitochondrial Isa2p plays a crucial role in the maturation of cellular iron-sulfur proteins.

The assembly of iron-sulfur (Fe/S) clusters in a living cell is mediated by a complex machinery which, in eukaryotes, is localised within mitochondria. Here, we report on a new component of this machinery, the protein Isa2p of the yeast Saccharomyces cerevisiae. The protein shares sequence similarity with yeast Isa1p and the bacterial IscA proteins which recently have been shown to perform a function in Fe/S cluster biosynthesis. Like the Isa1p homologue, Isa2p is localised in the mitochondrial matrix as a soluble protein. Deletion of the ISA2 gene results in the loss of mitochondrial DNA and a strong growth defect. Simultaneous deletion of the ISA1 gene does not further exacerbate this growth phenotype suggesting that the Isa proteins perform a non-essential function. When Isa2p was depleted by regulated gene expression, mtDNA was maintained, but cells grew slowly on non-fermentable carbon sources. The maturation of both mitochondrial and cytosolic Fe/S proteins was strongly impaired in the absence of Isa2p. Thus, Isa2p is a new member of the Fe/S cluster biosynthesis machinery of the mitochondrial matrix and may be involved in the binding of an intermediate of Fe/S cluster assembly.

Mitochondrial ABC transporters.

In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane. The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p. In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria. The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol. Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). MTABC3 may be a candidate gene for the lethal neonatal syndrome. The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

Is placental tissue protein 17b/TIP47 a new factor in cervical cancer genesis?

We identified novel members of the placental tissue protein 17 (PP17) protein family which consists of different-size variants of PP17; cDNAs of PP17a and PP17b variants have also been cloned and sequence analyzed. By Western-blot analysis in cervical carcinoma tissues we found overexpression of PP17b. Compared to healthy controls a mean five-fold increase in the amount of PP17b was also detected in the sera of untreated cervical carcinoma patients, which declined after radical operations. In our recent findings, in sera of inoperable cervical carcinoma patients, we also found elevated PP17b levels which did not change after irradiation. By Northern-blot analyses we confirmed PP17b overexpression in cervical carcinoma tissues and also the alternative splicing of PP17 mRNAs in various normal human tissues. Presently, the amino acid sequence of TIP47--a mannose-6-phosphate receptor cargo selection device--turned out to be identical to that of PP17b. Due to its oncodevelopmental function, PP17b/TIP47 is more than likely to be connected to HSV-2 infection, which is probably one of the main etiopathogenic agents of cervical carcinoma along with the HPV virus, and may open a new trend in the research of pathological processes in human uterine cervical cancer.

Synthesis and antibacterial study of unsaturated Mannich ketones.

Several Mannich ketones of 2-arylmethylenecycloalkanones were synthesised using the classical acid-catalysed Mannich reaction. Antibacterial activity of these new water-soluble compounds was reported against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus saprophyticus, Micrococcus luteus and Bacillus subtilis standard strains. Human cell line cytotoxicity of our new compounds was evaluated against HeLa cell lines. Some compounds showed low cytotoxicity (41.52 nM mL(-1) for 14 and 46.60 nM mL(-1) for 18) and proved to be efficient antibacterial agents against the Gram-positive strains. Minimum inhibitory concentrations varied from 1.56 to 100 microg mL(-1). The mechanism of action was examined, too.

[Mitochondrial DNA deletion in hereditary cardio-encephalo-myopathy]. / Mitokondriális DNS deléció herediter cardio-encephalo-myopathiában.

The case of a female patient with cardio-encephalo-myopathy who died of her illness at one year of age, similarly to her three sisters, is reported. In autopsy samples, like muscle, heart, liver and cerebellum activities of several mitochondrial enzymes were determined. In the skeletal muscle serious decrease of carnitine acetyltransferase was observed (from the normal 4.8 U/g to 0.08 U/g wet weight), while in other tissues this activity was normal. In the muscle activities of several other mitochondrial enzymes were also decreased (cytochrome oxidase, NADH cytochrome C oxidoreductase, citrate synthase), while in other tissues there were no similar changes. Serious distortion was observed in the structure of the majority of mitochondria of muscle and heart by electronmicroscopy. The number of the Purkinje-cells in the cerebellum decreased, and the cells were shrunken, their axons were fragmented and disoriented. Also the structure of the mitochondria was abnormal in the Purkinje-cells, while it was normal in other areas of the cerebrum. In te tissues of the patient normal and deleted mitochondrial DNA coexisted as which could explain the genetic background of this disease at molecular level.

[Screening for carrier state of Haemophilia B using indirect genomic detection]. / Konduktorszúrés indirekt géndiagnosztika alkalmazásával B haemophiliában.

The authors report the first data having applied the indirect genomic diagnosis in carrier screening in Hungary. 22 patients with haemophilia B and female family members of 14 out of them were examined by PCR based restriction fragment length polymorphism analysis. The combined use of 3 intra- and 1 extragenic polymorphisms have been examined at the same population. DNA fragments, containing the single nucleotide change polymorphic site (Xmnl, Hhal, Taql), or the 50 bp insertion/deletion element (Dde) were amplified. The products were digested by the appropriate restriction digestion enzyme and were detected on agarose gel following ethidium-bromide staining. 20 siblings were interested in the determination of their carrier-state. 15 (75%) of them could get definite diagnosis. The carrier-state was established in 7 cases, excluded in 8 subjects. For the remaining 5 participants studied, the absence of the parental DNA sample caused uncertainty, while in 2 cases (10%) none of the analyzed RFLP was informative. The heterozygosity rate, the gene and haplotype frequency were also recorded and compared with the international data. The indirect methods have proved to be sufficient and well suitable for routine carrier testing. The results provide the basis of the subsequent prenatal diagnosis.

Studies on yeast peroxisomal citrate synthase.

Peroxisomal (nonmitochondrial) citrate synthase (CS2) has been purified from a Saccharomyces cerevisiae strain in which the gene for the mitochondrial citrate synthase (CS1) had been disrupted and no CS1 protein is produced. The enzyme, CS2, the sequence of which had been previously determined from its DNA, behaved differently from CS1 in its purification, kinetics, stability, and binding to the inner surface of mitochondrial inner membranes.

Isolation and characterization of 3-hydroxyacyl coenzyme A dehydrogenase-binding protein from pig heart inner mitochondrial membrane.

3-Hydroxyacyl coenzyme A (CoA) dehydrogenase-binding protein was solubilized from inner mitochondrial membrane by using taurodeoxycholate at high ionic strength. The binding protein was isolated from the suspension using 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The protein eluted from the affinity column had a molecular weight of approximately 150,000, as determined by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protein is a dimer consisting of 69,000 and 71,000 molecular weight subunits. The enzyme binding capacity of this protein was tested with a polyethylene glycol precipitation method: 0.5 mg of enzyme could be precipitated together with 1 mg of binding protein, showing that 1 mol of binding protein binds 1 mol of enzyme. This protein had no affinity toward malic dehydrogenase, citrate synthase, and fumarase. The approximately 2-fold increase in the 3-hydroxyacyl-CoA dehydrogenase activity when it was measured in the presence of the binding protein is additional evidence of enzyme-binding protein interaction. When incorporated into liposomes, the binding protein retained its ability to bind 3-hydroxyacyl-CoA dehydrogenase, but did not bind malic dehydrogenase, citrate synthase, and fumarase. These results suggest that the protein isolated by us has a specific function in anchoring a beta-oxidation enzyme to the matrix surface of the mitochondrial membrane.

Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria.

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.