Integration of porin synthesized in vitro into outer mitochondrial membranes

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).     

N-terminal deletions and His-tag fusions dramatically affect expression of cytochrome p450 2C2 in bacteria

Mitochondria generate reactive oxygen species as by-products of oxidative metabolism. Since ascorbic acid can scavenge such destructive species, we studied the ability of mitochondria from rat liver and muscle to take up, recycle, and oxidize ascorbate. Freshly prepared mitochondria contain ascorbate, as do mitoplasts that lack the outer mitochondrial membrane. Both mitochondria and mitoplasts rapidly take up oxidized ascorbate as dehydroascorbic acid and reduce it to ascorbate. Ascorbate concentrations in mitochondria and mitoplasts rise into the low millimolar range during dehydroascorbic acid uptake, although uptake and reduction is opposed by ascorbate efflux. Mitochondrial dehydroascorbic acid reduction depends mainly on GSH, but mitochondrial thioredoxin reductase may also contribute. Reactive oxygen species generated within mitochondria oxidize ascorbate more readily than they do GSH and alpha-tocopherol. These results show that mitochondria can recycle ascorbate, which in turn might help to prevent deleterious effects of oxidant stress in the organelle.     

The inhibition of mitochondrial DNA replication in vitro by the metabolites of benzene, hydroquinone and p-benzoquinone

Rat liver mitochondria incubated with the metabolites of benzene, p-benzoquinone or 1,2,4-benzenetriol, showed a dose-dependent inhibition of [3H]dTTP incorporation into mtDNA with median inhibitory concentrations of 1 mM for each compound. Benzene and the metabolites phenol, catechol and hydroquinone did not inhibit at concentrations up to 10 mM. Similarly, incubation of p-benzoquinone or hydroquinone with rabbit bone marrow mitochondria showed a dose-dependent inhibition of mtDNA synthesis with 50% inhibition at 1 mM and 10 mM, respectively. That these metabolites inhibit mitochondrial replication was evidenced by the fact that [3H]dTTP incorporation into characteristic 38S, 27S and 7S mitochondrial replication intermediates was decreased by the quinones, as analyzed on 5-20% neutral sucrose velocity gradients. p-Benzoquinone, hydroquinone and 1,2,4-benzenetriol inhibited the activity of partially purified rat liver mtDNA polymerase gamma using either activated calf thymus DNA or poly(rA) X p(dT)12-18 as primer/template, with 50% inhibitory concentrations of 25 microM, 25 microM and 180 microM, respectively. Preincubation of the metabolites with polymerase gamma or primer/template, followed by removal of the unreacted metabolite by gel filtration, indicated that inhibition resulted from interaction of the metabolites with the enzyme, rather than with the template. Binding appeared to involve a sulfhydryl residue on the enzyme since the binding of [14C]hydroquinone was prevented by N-ethylmaleimide. The ability of hydroquinone or p-benzoquinone to inhibit binding of [14C]hydroquinone to the enzyme suggests that the compounds bind to a common site or are converted to a common intermediate. Inhibition of, or changes in, replication in mitochondria of bone marrow cells by hydroquinone and p-benzoquinone may explain the changes in the mitochondrial genome observed in marrow stem cells in acute myelogenous leukemia and may suggest a mechanism for benzene leukemogenesis.     

ATP浓度和缺氧暴露对大鼠脑线粒体RNA和蛋白质体外合成的影响

本文探讨介质中ATP浓度和急,慢性缺氧暴露对大鼠脑线粒体内RNA和蛋白质合成的影响。用差速离心法分离正常和低压舱模拟4000m高原急性连续缺氧暴露3d和慢性连续缺氧暴露40d大鼠脑线粒体,用体外无细胞（cell-free in vitro)^3H-UTP和^3H-Leucine掺入法分别测定线粒体RNA和蛋白质合成活性,结果显示,大鼠急性缺氧暴露后大脑皮质线粒体RNA体外合成活性降低40%,蛋白质合成活性降低60%;慢性缺氧暴露后线粒体RNA和蛋白质合成活性分别为对照的72%和76%;ATP对正常大鼠脑线粒体RNA以及蛋白质的体外合成活性的影响均呈双相性,大于或小于1mmol/L均可产生不同程度的抑制效应,结果提示,缺氧可在转录和翻译两个水平上影响脑线粒体mtDNA的表达,而慢性缺氧暴露时,线粒体半自主性功能的改善可能是机体对缺氧适应的细胞机制之一;ATP对脑线粒体内转录和释放活性的调节是一种经济有效的反馈调节方式。     

Absence of expression from RNA internalised into electroporated mammalian mitochondria

Transfection of mammalian mitochondria has proved to be notoriously difficult. Whilst there have been sporadic reports of import of foreign nucleic acids into isolated organelles, these imported nucleic acids have never been demonstrated to be functional. Inability to manipulate mitochondrial gene expression has hampered our understanding of RNA processing, maturation and translation in mitochondria. In an attempt to establish a model system for mt-RNA expression, we have electroporated rat liver mitochondria and mitoplasts in the presence of various RNA constructs built around the mitochondrial reporter gene mt-luciferase. Following electroporation, a fraction of the RNA was shown to be stably maintained, mitochondria remained coupled for oxidative phosphorylation and intramitochondrial protein synthesis was unaffected. In no case, however, was this RNA translated.     

Submitochondrial distribution of cAMP during incubation with rat liver mitochondria

Labeled cAMP incubated with rat liver mitochondria penetrates not only through outer mitochondrial membranes, but also into mitoplasts, where it is accumulated mainly in the matrix. Damage of mitochondrial membranes caused by single freezing-thawing treatment promotes no influx, but efflux of cAMP from mitoplasts. cAMP molecules penetrate inside mitochondria largely in an unchanged state in all submitochondrial fractions, as was demonstrated by the TLC method. cAMP transport into mitochondria can serve as a reason for: 1) stimulation of mitochondrial function by hormones whose effects are realized through activation of cytoplasmic adenylate cyclase and by extramitochondrial (cytosolic) cAMP; 2) existence of cAMP-dependent protein kinase and cAMP-phosphodiesterase in mitochondria.     

Translocation of proteins into rat liver mitochondria. Existence of two different precursor polypeptides of liver fumarase and import of the precursor into mitochondria

Two different putative precursor polypeptides of rat liver fumarase were synthesized when RNA prepared from rat liver were translated in vitro using the rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P1) was synthesized as a larger molecular mass than the mature subunit of fumarase (45,000 daltons) by about 5,000 daltons and the other (P2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30 degrees C, P1 and the 35S-labeled mature size fumarase were associated with the mitochondria. Of these, the 35S-labeled mature size fumarase was resistant to externally added protease, but P1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P1, which was energy-dependently imported and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased proportionally to the duration of incubation, while the amount of P1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of the incubation. 2) Only P1 could bind with the mitochondrial outer membrane at 0 degrees C even in the presence of an uncoupler of the oxidative phosphorylation but P2 did not. 3) P1 bound to the mitochondrial outer membrane was imported into the matrix, when the mitochondria binding only P1 at 0 degrees C was reisolated and incubated at 30 degrees C in the presence of an energy-generating system. The specific receptor was involved in the binding of P1 to mitochondria, since a high concentration of NaCl did not interfere with the binding of P1 to the membrane and did not discharge P1 bound onto the membrane. It was shown that P1 formed an aggregate composed of 6 to 8 molecules and P2 was a dimer in the cell-free translation mixture and that P1 and P2 were enzymatically inactive. These results suggest that the precursor for the mitochondrial enzyme has a larger molecular weight than that of the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme.     

Methyl-substitution of benzene and toluene in preparations of human bone marrow

The metabolism of benzene and toluene was investigated in preparations of human bone marrow incubated with S-adenosyl-L-methionine. Benzene undergoes a methyl-substitution reaction to yield toluene as a metabolite. Furthermore, toluene undergoes methyl-substitution in preparations of human bone marrow incubated with S-adenosyl-L-methionine to yield o-xylene, m-xylene, and p-xylene. Metabolites were detected by gas chromatography and mass spectroscopy. No metabolism of either benzene or toluene was detected when a boiled bone marrow preparation was used in the incubation, demonstrating the enzymatic nature of the S-adenosyl-L-methionine dependent methylation of both benzene and toluene.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

Ca2+-dependent inhibition by trifluoperazine of the Na+-Ca2+ carrier in mitoplasts derived from heart mitochondria

The interaction of trifluoperazine and extramitochondrial Ca2+ with the heart mitochondrial Na+-Ca2+ carrier has been investigated. External Ca2+ inhibits the carrier equally in mitochondria and mitoplasts in which the outer membrane is lysed. Sensitivity to Ca2+ is not removed by washing mitoplasts under varied conditions. Trifluoperazine is a potent inhibitor of the carrier in mitoplasts but not in mitochondria. Trifluoperazine inhibition in mitoplasts depends markedly on the presence of extramitochondrial Ca2+ (2 microM).     

Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase

Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).     

Polypeptide synthesis by mitoplasts isolated from mouse liver

Polypeptide synthesis by mouse liver mitochondria was studied by incubating purified mitoplasts (mitochondria treated with digitonin) with [³⁵S]methionine. The products were separated either by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, or by isoelectric focusing, followed by SDS polyacrylamide gel electrophoresis. At least 14 distinct bands with molecular weights (mol. wt) ranging from about 8 000 to about 70 000 were found upon radioautography of the gels. When the samples were incubated in the presence of chloramphenicol, only a single weak band was found, whereas the protein pattern was unaffected by the presence of cycloheximide in the medium. The newly synthesized proteins were all acidic and evidence was obtained that they were hydrophobic in nature. Virtually all the labelled polypeptides were present in the membrane fraction, whereas the matrix showed little radioactivity. The data indicate that the proteins synthesized by mammalian mitochondria, like those in yeast, are components of the inner mitochondrial membrane. One protein of mol. wt 22 000 D was detected in the incubation medium. Since more of this component was present in the medium than in the pelleted mitoplasts and since this protein was not found in the matrix fraction of sonicated mitoplasts, it is believed that it had been excreted from the inner mitochondrial membrane. The finding that the number of proteins synthesized in mitoplasts isolated from mouse liver is considerably higher than that synthesized in yeast mitochondria reflects a most efficient utilization of the mammalian mitochondrial genome.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.     

Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria.

Treatment of isolated yeast mitochondria with high levels (1 mg/ml) of trypsin severely inhibits protein import but does not destroy the integrity of the outer membrane or abolish mitochondrial energy coupling. If the outer membrane of these trypsin-inactivated mitochondria is disrupted by osmotic shock, the resulting mitoplasts are again able to import proteins. Protein import into mitoplasts, like that into intact mitochondria, is energy-dependent; however, whereas import into mitochondria is inhibited by antibody against 45-kd proteins of the outer membrane [Ohba and Schatz, EMBO J., 6, 2109-2115 (1987)], import into mitoplasts not affected by this antibody. Protein import into mitoplasts appears to bypass one or more steps normally occurring at the mitochondrial surface.     

Precursor of a mitochondrial enzyme accumulates in the cytoplasm of preen glands for a specialized function

Malonyl-CoA decarboxylase in the mitochondria of the liver of goose is immunologically identical with the decarboxylase in the cytoplasm of the uropygial gland (Buckner et al. (1978) $\text{Arch. Biochem. Biophys.}$ 186, 152–163). Messenger RNA was isolated from the liver and the uropygial gland and translated in a rabbit reticulocyte system. Specific immunoprecipitation of the translation products with anti malonyl-CoA decarboxylase showed that in both cases the primary translation product was a 50 K dalton peptide identical in size to the cytoplasmic enzyme in the gland. Specific immunoprecipitation of malonyl-CoA decarboxylase from liver slices which had been incubated with [³⁵S]methionine showed that the mature mitochondrial enzyme was a 47 K dalton peptide, 3 K daltons smaller than the primary translation product and the isolated cytoplasmic enzyme. These results suggest that the decarboxylase is proteolytically processed during transport into the mitochondria and that the large amount of the cytoplasmic decarboxylase found in the gland represents accumulation of the unprocessed precursor form of the normally mitochondrial enzyme.