Membrane-bound glutathione peroxidase-like activity in mitochondria

A membrane-bound glutathione peroxidase-like activity has been detected in liver and cardiac mitochondrial membrane. This enzyme activity differs from the cytosol and mitochondrial matrix selenium-dependent glutathione peroxidase in that it is membrane bound, sensitive to sonication and triton-X-100, and is unaffected by prolonged feeding of a selenium-free diet. This mitochondrial membrane-bound enzyme activity differs from the glutathione-S-transferases which exhibit glutathione peroxidase activity in that it is capable of utilizing both cumene hydroperoxide and hydrogen peroxide as substrates. Digitonin fractionation studies indicate that this enzyme is not located in either inner or outer mitochondrial membrane but rather within inter-membrane space. This newly described membrane-bound enzyme activity may play an important role in the maintenance of cardiac mitochondrial integrity in that mitochondrial matrix does not contain glutathione peroxidase.     

Superoxide radical-initiated apoptotic signalling pathway in selenite-treated HepG(2) cells: mitochondria serve as the main target

The exact role of superoxide radicals (O(2)(*)(-)) in apoptosis is still a matter of debate. The main objective of the present study is to evaluate the apoptotic signalling pathway initiated by O(2)(*)(-). The reductive reaction of sodium selenite with glutathione was used as the intracellular O(2)(*)(-)-generating system. When cells were exposed to 5 to 25 microM selenite, a temporal pattern of apoptotic events was observed following the elevation of O(2)(*)(-), in which cytochrome c release and mitochondrial depolarization preceded caspase-3 activation and DNA fragmentation. The simultaneous treatment with N-acetylcysteine and 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl markedly reduced O(2)(*)(-) level and suppressed the mitochondrial changes and the downstream apoptotic events. Moreover, pretreatment with cyclosporin A plus trifluoperazine, two mitochondrial permeability transition (MPT) inhibitors, was capable of attenuating O(2)(*)(-)-mediated cytochrome c release and mitochondrial depolarization, and subsequently inhibiting apoptosis. Thus, the present results provide convincing evidence that O(2)(*)(-) generated from the reductive reaction of selenite with GSH is capable of triggering a mitochondria-dependent apoptotic pathway. Such knowledge may not only help to obtain a better understanding of the apoptotic effect of selenite per se, but of the role of O(2)(*)(-) in initiation and execution of apoptosis.     

Ceramide channels and their role in mitochondria-mediated apoptosis

A key, decision-making step in apoptosis is the release of proteins from the mitochondrial intermembrane space. Ceramide can self-assemble in the mitochondrial outer membrane to form large stable channels capable of releasing said proteins. Ceramide levels measured in mitochondria early in apoptosis are sufficient to form ceramide channels in the outer membrane. The channels are in dynamic equilibrium with non-conducting forms of ceramide in the membrane. This equilibrium can be strongly influenced by other sphingolipids and Bcl-2 family proteins. The properties of ceramide channels formed in a defined system, planar phospholipid membranes, demonstrate that proteins are not required for channel formation. In addition, experiments in the defined system reveal structural information. The results indicated that the channels are barrel-like structures whose staves are ceramide columns that span the membrane. Ceramide channels are good candidates for the protein release pathway that initiates the execution phase of apoptosis.     

Contribution of inorganic polyphosphate towards regulation of mitochondrial free calcium

BackgroundCalcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, from cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. This calcium buffering system involves the participation of inorganic phosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how calcium-orthophosphate interactions, which normally lead to formation of insoluble precipitates, are capable to dynamically regulate free calcium concentration. Here we test the hypothesis that inorganic polyphosphate, which is a polymerized form of orthophosphate, is capable to from soluble complexes with calcium, playing a significant role in the regulation of the mitochondrial free calcium concentration.MethodsWe used confocal fluorescence microscopy to measure the relative levels of mitochondrial free calcium in cultured hepatoma cells (HepG2) with variable levels of inorganic polyphosphate (polyP).ResultsThe depletion of polyP leads to the significantly lower levels of mitochondrial free calcium concentration under conditions of pathological calcium overload. These results are coherent with previous observations showing that inorganic polyphosphate (polyP) can inhibit calcium-phosphate precipitation and, thus, increase the amount of free calcium.ConclusionsInorganic polyphosphate plays an important role in the regulation of mitochondrial free calcium, leading to its significant increase.General significanceInorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system.     

Peroxisomal and mitochondrial beta-oxidation of monocarboxylyl-CoA, omega-hydroxymonocarboxylyl-CoA and dicarboxylyl-CoA esters in tissues from untreated and clofibrate-treated rats

In control rats, long-chain monocarboxylyl-CoA, omega-hydroxymonocarboxylyl-CoA, and dicarboxylyl-CoA esters were substrates for hepatic, renal, and myocardial peroxisomal beta-oxidation. The latter enzyme system could not be detected in skeletal muscle. Clofibrate treatment resulted in an enhancement of peroxisomal beta-oxidizing capacity in various tissues. Intact mitochondria from control rat liver and kidney cortex incubated in the presence of L-carnitine were capable of oxidizing long-chain monocarboxylyl-CoAs and omega-hydroxymonocarboxylyl-CoAs but not dicarboxylyl-CoAs. However, control rat liver mitochondria permeabilized by digitonin oxidized dodecanedioyl-CoA indicating that the liver mitochondrial beta-oxidation system can act on dicarboxylyl-CoA esters even if the overall intact mitochondrial system is inactive on these substrates. Intact liver mitochondria from clofibrate-treated animals rapidly oxidized lauroyl-CoA and 12-hydroxylauroyl-CoA but not dodecanedioyl-CoA. These mitochondria were active on hexadecanedioyl-CoA and this activity amounted to 20-25% of that measured with palmitoyl-CoA and 16-hydroxypalmitoyl-CoA as substrates. No mitochondrial dicarboxylyl-CoA oxidation could be detected in kidney cortex from animals receiving clofibrate in their diet. Heart and skeletal muscle intact mitochondria from untreated and clofibrate-treated rats were capable of oxidizing each type of acyl-CoA as a substrate. Dicarboxylyl-CoA synthetase and carnitine dicarboxylyltransferase activities were detected in various tissues from untreated and clofibrate-treated rats with the exception of carnitine dodecanedioyltransferase reaction in livers from untreated and clofibrate-treated rats. In skeletal muscle, the acyl-CoA synthetase activities could be detected only in the presence of detergents.     

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa.

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).     

Proteolysis in mitochondrial preparations and in lysosomal preparations derived from rat liver

A method of preparing rat liver mitochondria with low residual contamination by lysosomal proteases is described. Preparations of mitochondria are divided into two equal portions, one of which is supplemented with a lysosomal fraction. The addition of the lysosomal fraction causes an increase in proteolysis of between 26- and 56-fold at pH 5.0 in four similar experiments. This increase matches the increase in the lysosomal marker beta-glucuronidase and indicates that all proteolysis at pH 5.0 is due to enzymes of the lysosomal fraction. Above pH 7.0, the addition of a lysosomal supplement increases proteolysis by 1.5- to 5-fold only, suggesting that in the absence of a lysosomal supplement very little of the observed proteolysis is due to enzymes of lysosomal origin. A method of calculating the contribution to total proteolysis of enzymes of the lysosomal fraction or of the mitochondrial fraction is described. The calculations show that at pH 7.0 and above, more than 93% of the observed proteolysis is due to enzymes originating in the mitochondrial fraction. The results support the view of other workers that rat liver mitochondria contain an endogenous neutral proteolytic system capable of degrading mitochondrial proteins to acid-soluble products.     

Mitochondrial morphology and dynamics in <Emphasis Type="Italic">Triticum aestivum</Emphasis> roots in response to rotenone and antimycin A

Mitochondria are dynamic organelles, capable of fusion and fission as a part of cellular responses to various signals, such as the shifts in the redox status of a cell. The mitochondrial electron transport chain (ETC.) is involved in the generation of reactive oxygen species (ROS), with complexes I and III contributing the most to this process. Disruptions of ETC. can lead to increased ROS generation. Here, we demonstrate the appearance of giant mitochondria in wheat roots in response to simultaneous application of the respiratory inhibitors rotenone (complex I of mitochondrial ETC.) and antimycin A (complex III of mitochondrial ETC.). The existence of such megamitochondria was temporary, and following longer treatment with inhibitors mitochondria resumed their conventional size and oval shape. Changes in mitochondrial morphology were accompanied with a decrease in mitochondrial potential and an unexpected increase in oxygen consumption. Changes in mitochondrial morphology and activity may result from the fusion and fission of mitochondria induced by the disruption of mitochondrial ETC. Results from experiments with the inhibitor of mitochondrial fission Mdivi-1 suggest that the retarded fission may facilitate plant mitochondria to appear in a fused shape. The processes of mitochondrial fusion and fission are involved in the regulation of the efficacy of the functions of the respiratory chain complexes and ROS metabolism during stresses. The changes in morphology of mitochondria, along with the changes in their functional activity, can be a part of the strategy of the plant adaptation to stresses.     

A 3' splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in Neurospora crassa

We showed previously that the cyt-21+ gene of Neurospora crassa encodes a mitochondrial ribosomal protein homologous to Escherichia coli ribosomal protein S-16 (Kuiper, M. T. R., Akins, R. A., Holtrop, M., de Vries, H., and Lambowitz, A. M. (1988) J. Biol. Chem. 263, 2840-2847). A mutation in this gene, cyt-21-1, results in deficiency of mitochondrial small ribosomal subunits and small rRNA (Collins, R. A., Bertrand, H., LaPolla, R. J., and Lambowitz, A. M. (1979) Mol. Gen. Genet. 177, 73-84). In the present work, cloning and sequencing of the cyt-21-1 mutant allele show that it contains a single dG to dA transition at the 3' splice site AG of the first intron in the protein coding region. This mutation leads to inactivation of the normal 3' splice site and activation of a cryptic 3' splice site, 15 nucleotides downstream. The use of this cryptic splice site results in an in-frame deletion of 5 amino acids from the cyt-21 protein. Comparison of mutant and wild-type mitochondrial small ribosomal subunit proteins showed one protein, S-24, with an altered electrophoretic mobility, consistent with the predicted deletion. The mutant ribosomal protein is still capable of binding to mitochondrial small ribosomal subunits, but results in abnormal mitochondrial ribosome assembly.     

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.     

ROLE OF THE MITOCHONDRIAL GENOME DURING EARLY DEVELOPMENT IN MICE : Effects of Ethidium Bromide and Chloramphenicol

The role of the mitochondrial genome in early development and differentiation was studied in mouse embryos cultured in vitro from the two to four cell stage to the blastocyst (about 100 cells). During this period the mitochondria undergo morphological differentiation: progressive enlargement followed by an increase in matrix density, in number of cristae, and in number of mitochondrial ribosomes. Mitochondrial ribosomal and transfer RNA synthesis occurs from the 8 to 16 cell stage on and contributes to the establishment of a mitochondrial protein-synthesizing system. Inhibition of mitochondrial RNA- and protein-synthesis by 0.1 µg/ml of ethidium bromide or 31.2 µg/ml of chloramphenicol permits essentially normal embryo development and cellular differentiation. Mitochondrial morphogenesis is also nearly normal except for the appearance of dilated and vesicular cristae in blastocyst mitochondria. Such blastocysts are capable of normal postimplantation development when transplanted into the uteri of foster mothers. Higher concentrations of these inhibitors have general toxic effects and arrest embryo development. It is concluded that mitochondrial differentiation in the early mouse embryo occurs through the progressive transformation of the preexisting mitochondria and is largely controlled by the nucleocytoplasmic system. Mitochondrial protein synthesis is required for the normal structural organization of the cristae in blastocyst mitochondria. Embryo development and cellular differentiation up to the blastocyst stage are not dependent on mitochondrial genetic activity.     

Raft-like microdomains play a key role in mitochondrial impairment in lymphoid cells from patients with Huntington's disease

Huntington's disease (HD) is a genetic neurodegenerative disease characterized by an exceedingly high number of contiguous glutamine residues in the translated protein, huntingtin (Htt). The primary site of cell toxicity is the nucleus, but mitochondria have been identified as key components of cell damage. The present work has been carried out in immortalized lymphocytes from patients with HD. These cells, in comparison with lymphoid cells from healthy subjects, displayed: i) a redistribution of mitochondria, forming large aggregates; ii) a constitutive hyperpolarization of mitochondrial membrane; and iii) a constitutive alteration of mitochondrial fission machinery, with high apoptotic susceptibility. Moreover, mitochondrial fission molecules, e.g., protein dynamin-related protein 1, as well as Htt, associated with mitochondrial raft-like microdomains, glycosphingolipid-enriched structures detectable in mitochondria. These findings, together with the observation that a ceramide synthase inhibitor and a raft disruptor are capable of impairing the peculiar mitochondrial remodeling in HD cells, suggest that mitochondrial alterations occurring in these cells could be due to raft-mediated defects of mitochondrial fission/fusion machinery.     

A mutation in yeast mitochondrial DNA results in a precise excision of the terminal intron of the cytochrome b gene

The yeast nuclear gene CBP2 was previously proposed to code for a protein necessary for processing of the terminal intron in the cytochrome b pre-mRNA (McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468). In the present study we describe a mitochondrial mutation capable of suppressing the respiratory deficiency of cbp2 mutants. The mitochondrial suppressor mutation has been shown to be the result of a precise excision of the last intervening sequence from the cytochrome b gene. Strains with the altered mitochondrial DNA have normal levels of mature cytochrome b mRNA and of cytochrome b and exhibit wild type growth on glycerol. These results confirm that CBP2 codes for a protein specifically required for splicing of the cytochrome b intron and further suggest that absence of the intervening sequence does not noticeably affect the expression of respiratory function in mitochondria.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Miro1: New wheels for transferring mitochondria

Mesenchymal stem cells (MSC) are capable of protecting cells harboring mitochondrial damage. This protection is associated with the transfer of mitochondria through tunneling nanotubes (TNT) from MSC to the injured cells. In this issue of The EMBO Journal, the group of Anurag Agrawal shows that mitochondrial transfer is dependent on the levels of Miro1, a mitochondrial Rho‐GTPase that regulates intercellular mitochondrial movement. Miro1 is the first protein shown to accelerate mitochondrial transfer. Amplifying the mitochondrial transfer phenomenon may allow for the study of the mechanisms that regulate it and contribute to our understanding of its role in disease and aging.     

Redox‐dependent lipoylation of mitochondrial proteins in Plasmodium falciparum

Lipoate scavenging from the human host is essential for malaria parasite survival. Scavenged lipoate is covalently attached to three parasite proteins: the H‐protein and the E2 subunits of branched chain amino acid dehydrogenase (BCDH) and α‐ketoglutarate dehydrogenase (KDH). We show mitochondrial localization for the E2 subunits of BCDH and KDH, similar to previously localized H‐protein, demonstrating that all three lipoylated proteins reside in the parasite mitochondrion. The lipoate ligase 1, LipL1, has been shown to reside in the mitochondrion and it catalyses the lipoylation of the H‐protein; however, we show that LipL1 alone cannot lipoylate BCDH or KDH. A second mitochondrial protein with homology to lipoate ligases, LipL2, does not show ligase activity and is not capable of lipoylating any of the mitochondrial substrates. Instead, BCDH and KDH are lipoylated through a novel mechanism requiring both LipL1 and LipL2. This mechanism is sensitive to redox conditions where BCDH and KDH are exclusively lipoylated under strong reducing conditions in contrast to the H‐protein which is preferentially lipoylated under less reducing conditions. Thus, malaria parasites contain two different routes of mitochondrial lipoylation, an arrangement that has not been described for any other organism.     

Non-Mendelian Mutation Allowing Ureidosuccinic Acid Uptake in Yeast

Mutants of Saccharomyces cerevisiae capable of growth on a minimal medium supplemented with ureidosuccinic and glutamic acids have been isolated from a pyrimidineless strain. One of these mutants consistently yielded a non-Mendelian meiotic segregation. Moreover, the mitotic transmission of the mutation was very high. It is suggested that the mutation is nonchromosomal and could be mitochondrial. However, this mutation behaves very differently from other mitochondrial mutations.     

NADH oxidation and NAD+ reduction catalysed by tightly coupled inside-out vesicles from Paracoccus denitrificans.

Tightly coupled inside-out vesicles were prepared from Paracoccus denitrificans cells (SPP, sub-Paracoccus particles) and characterized kinetically. The rate of NADH oxidation, catalysed by SPP, increases 6-8 times on addition of gramicidin. The vesicles are capable of catalysing Delta micro H+-dependent reverse electron transfer from quinol to NAD+. The kinetic parameters of the NADH-oxidase and the reverse electron transfer carried out by membrane-bound P. denitrificans complex I were estimated and compared with those of the mitochondrial enzyme. The data demonstrate that catalytic properties of the dinucleotide-binding site of the bacterial and mitochondrial complex I are almost identical, pointing out similar organization of the site in mammals and P. denitrificans. Inhibition of the bacterial complex I by a specific inhibitor of Q reduction, rotenone, is very different from that of the mitochondrial enzyme. The inhibitor is capable of suppressing the NADH oxidation reaction only at micromolar concentrations, while the activity of mitochondrial enzyme is suppressed by nanomolar concentrations of rotenone. In contrast to the mitochondrial enzyme, rotenone, even at concentrations as high as 10 micro m, does not inhibit the reverse, Delta micro H+-dependent NAD+-reductase reaction on SPP.