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PubMed2002

RNA splicing in higher plant mitochondria: determination of functional elements in group II intron from a chimeric cox II gene in electroporated wheat mitochondria.

Higher plant mitochondria mainly contain group II introns presenting a secondary structure with six helical domains linked to a central hub. Experimental evidence of functional elements in higher plant mitochondria introns is limited since they are unable to undergo self-splicing and the definition of functional domains is based on data obtained from yeast autocatalytic introns. Here we study the role of putative functional elements required for the splicing reaction. The exon-binding and intron-binding sites (EBS and IBS, respectively), and the domain 6, which is involved in lariat formation, were analysed by site-directed mutagenesis and transient expression in electroporated mitochondria. The data presented here demonstrate the role of EBS1-IBS1 and EBS2-IBS2 interactions and reveal a new secondary-structure interaction. The role of the C to U editing conversion in the IBS1 motif is discussed.

Base Sequence↗
PubMed2001

Presence of mitochondria-type ribosomes outside mitochondria in germ plasm of Drosophila embryos.

Mitochondrially encoded large and small ribosomal RNAs (mtlrRNA and mtsrRNA) are transported out of mitochondria to polar granules, the distinctive organelles of germ plasm in Drosophila. Reduction of the extramitochondrial mtlrRNA amount leads to the failure of embryos to form the germ-line progenitors, or pole cells, suggesting that mtlrRNA, along with mtsrRNA, functions on the polar granules to specify the germ line. In this study, we provide several lines of evidence showing that there are mitochondria-type ribosomes on the polar granules during a short period before pole cell formation. Our ultrastructural analysis reveals that these ribosomes include both mitochondrial rRNAs and at least two mitochondrial ribosomal proteins (S12 and L7/L12). Furthermore, these ribosomes are integrated into well developed polysomes on the surface of polar granules. We propose that translation dependent on mitochondria-type ribosomes is an important mechanism underlying germ-line formation.

Animals↗
PubMed2003

Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria.

To better understand the functional role of the mitochondrial network in shaping the Ca2+ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca2+ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca(2+)-sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca2+ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca2+ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca2+ release channels (and thus responsible for the fast uptake of Ca2+ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.

Aequorin↗
PubMed2006

Non-structural protein 4A of Hepatitis C virus accumulates on mitochondria and renders the cells prone to undergoing mitochondria-mediated apoptosis.

Non-structural protein 4A (NS4A) of Hepatitis C virus (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the endoplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome c into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells.

Apoptosis↗
PubMed1984

[Isolation of the RNA-poly A+ fraction from mitochondria of Drosophila melanogaster embryos. Incorporation of methionine-35S into newly-synthesized proteins in whole and lysed mitochondria].

The poly A+ RNA fraction of embryos mitochondria of Drosophila melanogaster was prepared by fractionation by oligo dT-cellulose chromatography. It accounted for 5% of total insoluble RNA. 15 bands were identified by electrophoresis, from 500 to 2,000 base pairs. This poly A+ RNA fraction hybridizes with mitochondrial DNA clones. It contains mitochondrial transcripts: messenger RNA and the large ribosomal RNA. The sum of the transcripts (29 kb) exceed the coding capacities of the mitochondrial DNA. Some of the transcripts may be precursors, greater in size, of the mRNA corresponding to a gene. Our results are compared to those previously published (9, 11, 12). The relationship between mitochondrial transcripts and proteins was studied in whole and lysed isolated mitochondria. In the former, incubated in the presence of 35S-methionine, 20 to 30 radioactive bands were identified (M. Wt. 100 to 16 kd). This incorporation was totally inhibited by chloramphenicol. When the mitochondria were opened in the presence of detergent (lysis). 35S-methionine incorporation was markedly enhanced. In all cases the number of proteins identified was greater than the number of potential genes (12). This finding is discussed.

Animals↗
PubMed1983

Import of proteins into mitochondria. Isolated yeast mitochondria and a solubilized matrix protease correctly process cytochrome c oxidase subunit V precursor at the NH2 terminus.

The cytoplasmically made subunit V was isolated from enzymically active yeast cytochrome c oxidase and its NH2-terminal amino acid sequence was determined to be (formula; see text) In order to exclude that this NH2 terminus had been generated by proteolysis during the lengthy isolation of the subunit, subunit V was directly immunoprecipitated from yeast cells that had been pulse-labeled with [35S]methionine; radiochemical sequencing revealed methionine at position 12, in agreement with the sequence given above. When the precursor to subunit V was synthesized in vitro in the presence of either [35S]methionine, [3H]leucine, or [3H]histidine and then incubated either with isolated yeast mitochondria or the partially purified matrix protease (Böhni, P. C., Daum, G., and Schatz, G. (1983) J. Biol. Chem. 258, 4937-4943), it was converted to a polypeptide co-migrating with mature subunit V on dodecyl sulfate-polyacrylamide gels. Radiochemical sequence analysis of the processed in vitro product showed that it contained histidine, leucine, and methionine in positions 4, 6, and 12, respectively, exactly as the authentic mature protein. In contrast, the unprocessed precursor contained methionine only at position 9, but not at position 12; thus, the precursor has a NH2 terminus different from the mature polypeptide. Similarly, if the in vitro synthesized cytochrome b2 precursor is incubated with isolated mitochondria, it is converted to a polypeptide which co-migrates with mature cytochrome b2 and, like the latter, contains leucine and methionine in positions 4 and 6, respectively. These data show that isolated yeast mitochondria convert the precursors to polypeptides which have the NH2 terminus of the authentic mature polypeptides. In the case of cytochrome c oxidase subunit V, correct NH2-terminal processing was also demonstrated with the purified matrix protease.

Amino Acid Sequence↗
PubMed1982

Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria.

Import of in vitro-synthesized cytochrome b2 (a soluble intermembrane space enzyme) was studied wih isolated yeast mitochondria. Import requires an electrochemical gradient across the inner membrane and is accompanied by cleavage of the precursor to the corresponding mature form. This conversion proceeds via an intermediate form of cytochrome b2, which can be detected as a transient species when mitochondria are incubated with the cytochrome b2 precursor for short times or at low temperatures. Conversion of the precursor to the intermediate form is energy-dependent and catalyzed by an o-phenanthroline-sensitive protease located in the soluble matrix. The intermediate is subsequently converted to mature cytochrome b2 in a reaction which is o-phenanthroline-insensitive and requires neither an energized inner membrane nor a soluble component of the intermembrane space. Whereas mature cytochrome b2 is soluble, the intermediate formed by isolated mitochondria is membrane-bound and exposed to the intermembrane space. The same intermediate is detected as a transient species during cytochrome b2 maturation in intact yeast cells (Reid, G. A., Yonetani, T., and Schatz, G (1982) J. Biol. Chem. 257, 13068-13074). The in vitro studies reported here suggest that a part of the cytochrome b2 precursor polypeptide chain is transported to the matrix where it is cleaved to a membrane-bound intermediate form by the same protease that processes polypeptides destined for the matrix space or for the inner membrane. In a second reaction, the cytochrome b2 intermediate is converted to mature cytochrome b2 which is released into the intermembrane space. The binding of heme is not necessary for converting the intermediate to the mature polypeptide.

Biological Transport↗
PubMed1994

Rps3 and rpl16 genes do not overlap in Oenothera mitochondria: GTG as a potential translation initiation codon in plant mitochondria?

Characterization of the Oenothera mitochondrial ribosomal gene cluster rps19-rps3-rpl16 shows the two genes rps3 and rpl16 to be separated by 9 nucleotides. The first codon of rpl16 is a GTG codon for valine and the only potential translational start. This GTG codon is conserved at the same position in maize, Petunia and Marchantia mitochondria, while sequences diverge upstream. These observations suggest that GTG at least at this position may act as translation initiation codon in plant mitochondria. Analysis of RNA editing suggests both genes to code for functional ribosomal proteins in Oenothera mitochondria. A duplication/recombination event at a decanucleotide in the intron of rps3 created a pseudogene missing part of the intron and the 3' exon.

Amino Acid Sequence↗
PubMed1967

Properties of Higher Plant Mitochondria. II. Effects of DNP, m-Cl-CCP, and Oligomycin on Respiration of Mung Bean Mitochondria.

Effects of inhibitors of phosphorylation on the oxidation of succinate and of l-malate were investigated with tightly coupled mitochondria isolated from mung bean hypocotyls. When mitochondria were incubated with 2,4-dinitrophenol, or carbonyl cyanide m-chlorophenylhydrazone prior to the addition of substrate, the uncoupling effects of these chemicals were relatively small. This is probably caused by relative lack in these mitochondria of endogenous substrates, ATP, and/or "high-energy intermediates". The action of uncoupling agents is, therefore, revealed in a more striking manner when they are introduced during the second state 4. Of the 2 uncoupling agents tested, malate oxidation consistently required 1.5 to 2 times higher concentration of the agents for the half-maximal effects than succinate oxidation. From the comparison of the degree of uncoupling it is concluded that 2,4-dinitrophenol is a better uncoupler of succinate oxidation, whereas carbonyl cyanide m-chlorophenylhydrazone functions as a more complete uncoupler of malate oxidation.Oligomycin does not inhibit state 4 rates, while the increment of respiration due to added ADP is completely inhibited by this antibiotic. Identical half-maximal effects are observed with the same concentration of oligomycin in both succinate and l-malate oxidation. The oligomycin effect depends on the mitochondrial concentration employed. The concentration of this chemical required for the half-maximal effect is 55 to 80 mmumoles per mg mitochondrial protein. It is suggested that this inhibitor of phosphorylation binds all of the phosphorylation sites regardless of whether the sites are functional or not.

Journal Article↗
PubMed1970

The Respiratory Chain of Plant Mitochondria: VII. Kinetics of Flavoprotein Oxidation in Skunk Cabbage Mitochondria.

The oxidation kinetics of the two high potential flavo-proteins, one (Fp(hf)) fluorescent and the other (Fp(ha)) nonfluorescent, in mitochondria from skunk cabbage (Symplocarpus foetidus) spadices have been measured by combined spectrophotometry and fluorimetry. In the absence of respiratory inhibitors, both flavoproteins are oxidized at nearly the same rate with half-times between 120 and 160 milliseconds at 24 C. When slight differences in rate are observed, it is Fp(ha) which consistently has the shorter half-time. The presence of 0.3 millimolar KCN has no perceptible effect on the oxidation rate of either component. Antimycin A (2 nanomoles per milligram of protein) increases the oxidation half-time of Fp(ha) about 3-fold, but it has no effect on the oxidation half-time of Fp(hf). In contrast to these two inhibitors, m-chlorobenzhydroxamic acid-an inhibitor specific to the cyanide insensitive, alternate oxidase pathway in these mitochondria-increases the oxidation half-time of Fp(hf) 10-fold to about 2 seconds, while increasing that of Fp(ha) only some 20%. This result implies that the flavoprotein Fp(hf) mediates electron transport to the alternate oxidase from the region of the mitochondrial respiratory chain encompassing Fp(ha), ubiquinone, and the cytochromes b. The oxidation rate of cytochrome b(557) is unaffected by either m-chlorobenzhydroxamic acid or cyanide but is strongly inhibited by antimycin A. This result implies that cytochrome b(557) plays no direct role in the respiratory pathway to the alternate oxidase and is different from cytochrome b(7) found in mitochondria from the spadices of Arum maculatum.

Journal Article↗
PubMed1971

The respiratory chain of plant mitochondria: x. Oxidation-reduction potentials of the flavoproteins of skunk cabbage mitochondria.

The oxidation-reduction potentials of the flavoproteins of skunk cabbage (Symplocarpus foetidus) mitochondria have been measured under anaerobic conditions by means of a combined spectrophotometric or fluorimetric-potentiometric method. Five components were resolved whose oxidation-reduction reactions corresponded to two-electron changes, as expected for flavoproteins. The midpoint potentials at pH 7.2 are as follows, listed in order of increasingly negative potential: +170 millivolts, +110 millivolts, +20 millivolts, -70 millivolts, and -155 millivolts. The most negative component was highly fluorescent; the other components could only be identified by their characteristic absorbance changes. In addition to these components, which are mitochondrial, variable amounts of a very highly fluorescent flavoprotein with a midpoint potential of -215 millivolts was found. This component appears to be extra-mitochondrial. The same midpoint potential values at pH 7.2 were obtained with mitochondria in the uncoupled state as in mitochondria energized with ATP in the absence of phosphate.

Journal Article↗
PubMed1971

The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria.

Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P(i)) and ATP are present. No reduction occurs in the absence of P(i), even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.In the absence of P(i) and presence of oligomycin, oxidation of succinate by the alternative cyanide-insensitive oxidase pathway, in the presence of sulfide to inhibit cytochrome oxidase, does not reduce endogenous NAD, either in the aerobic steady state or in anaerobiosis. Under these conditions, only the reversed electron transport pathway from succinate to endogenous NAD is active and ATP cannot interact with the respiratory chain. The source of energy for NAD reduction must come from the respiratory chain, and this result shows that oxidation of succinate through the alternate pathway does not provide this energy.

Journal Article↗
PubMed1974

The Respiratory Chain of Plant Mitochondria: XVII. Flavoprotein-Cytochrome b(562) Interaction in Antimycin-treated Skunk Cabbage Mitochondria.

During the transition from the aerobic steady state with succinate as substrate to anaerobiosis, in suspensions of skunk cabbage (Symplocarpus foetidus) mitochondria treated with antimycin A, cytochrome b(562) becomes reoxidized to the extent of about 20%, synchronously with the reduction of cytochrome c(549). This reoxidation occurs in both the absence and presence of m-chlorobenzhydroxamic acid, a specific inhibitor for the alternate terminal oxidase of plant mitochondria. A flavoprotein component, amounting to 13% to 15% of the total nonfluorescent mitochondrial flavoprotein, undergoes reduction synchronously with the oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid. This flavoprotein component remains reduced in the presence of cyanide. The half-time for reduction of the flavoprotein component and cytochrome c(549) and for oxidation of cytochrome b(562) during the aerobic to anaerobic transition with succinate as substrate in the presence of both antimycin A and m-chlorobenzhydroxamic acid is 2 seconds. The half-times for oxidation of cytochrome c(549) and the flavoprotein component are 2.1 and 170 milliseconds, respectively, during the anaerobic to aerobic transition induced by addition of 14 mum O(2) to the mitochondrial suspensions. The half-time for reduction of cytochrome b(562) under these conditions is 150 milliseconds, synchronous with the flavoprotein component. The synchrony of the flavoprotein oxidation and of the cytochrome b(562) reduction at a rate much slower than that of cytochrome c(549) oxidation implies that, in antimycin-treated plant mitochondria, the state of the cytochrome b(562)/antimycin complex is regulated by the redox state of this flavoprotein component, rather than by cytochrome c(549). It is tentatively suggested that these two components are not part of the main sequence of the respiratory chain, but may be part of a multienzyme complex active in the hydroxylation reactions required for ubiquinone biosynthesis in the inner mitochondrial membrane.

Journal Article↗
PubMed1981

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons : II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA.

THERE ARE TWO TYPES OF MITOCHONDRIA PRESENT IN IMBIBED PEANUT COTYLEDONS: a light type (density 1.182 grams per cubic centimeter) and a heavy type (density 1.205 grams per cubic centimeter). The membrane fractions from these two types can be distinguished using sucrose density gradient analysis, and differences in membrane density between the light and heavy types are reflected in differences in their protein N and phospholipid P composition. With increasing time after imbibition, there is a substantial increase in the amount and activity of the light type of mitochondria due to their de novo synthesis. The membrane density of the light mitochondrial fraction declines over 5 days after the start of imbibition as the phospholipid P to protein N ratio increases. The heavy mitochondrial fraction declines during the first 3 days after the start of imbibition, and then it remains at a low, but constant, level thereafter. Even during the decline, however, there is synthesis of proteins comparable to that into light mitochondria. The mitochondrial biogenesis that has been observed in peanut cotyledons is of the light type, the function and physiological importance of the minor heavy type is not known.

Journal Article↗
PubMed1982

Phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria, and from Escherichia coli: substrate specificity relationship with regard to ATP analogs.

Twelve structural analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria and E. coli. Three compounds are substrates for all five phenylalanyl-tRNA synthetase, three are completely inactive, while the other ATP analogs show differing properties with the different enzymes. Their Km, Ki and V values have been determined. The importance of the amino group in Position 6, the nitrogen in Position 7 and an unsubstituted Position 8 of the purine moiety as well as the supposed anti-conformation of the glycosidic bond and coordination of the magnesium cation to N-7 appear to be conserved through evolution. Bulky substituents on the 2' and 3' of the ribose moiety are generally not tolerated. Graduation of substrate properties of some analogs are similar for the intracellular heterotopic isoenzymes from yeast and hen liver.

Adenosine Triphosphate↗
PubMed1993

Thapsigargin causes Ca2+ release and collapse of the membrane potential of Trypanosoma brucei mitochondria in situ and of isolated rat liver mitochondria.

Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (> 10 microM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells.

Animals↗
PubMed1992

Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria.

The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.

Animals↗