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.     

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.     

The role of inner membrane in the realization of cAMP-dependent activation of mitochondrial enzymes

It was shown that the increase in the activities of transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase after incubation of mitochondria with cAMP is due to the stimulating effect of cAMP on mitochondria, but not to the increased stability of mitochondria to the incubation procedure. Treatment of mitochondria with trypsin prevents the action of cAMP on the both enzymes. The integrity of the inner mitochondrial membrane is necessary for the manifestation of cAMP effect. Pretreatment of mitochondria with the local anesthetic, lidocaine, prevents the activation of NAD(P)(+)-transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase during subsequent incubation of mitochondria with cAMP. It is concluded that the role of the inner mitochondrial membrane consists in the reception of the cAMP signal for the internal compartment of mitochondria, i.e. for mitoplasts. Peripheral protein(s) on the external side of the inner mitochondrial membrane seems to play a role in cAMP reception.     

Influence of the conformational state on the isoelectric points of rat brain synaptosomes, mitochondria and mitoplasts

The isoelectric points of rat brain synaptosomes, mitochondria and mitoplasts have been determined by using different charged two-phase systems containing dextran and poly(ethylene glycol). The cross-partition diagrams of these organelles show isoelectric points at pH 4.1, 4.5 and 4.7, respectively. The influence of the conformational state of mitochondrial membranes upon their partition in two-phase systems has been studied. Shrunk mitoplasts showed a large change in their partition behavior as reflected by an increased affinity for the lower dextran phase, while shrinkage of mitochondria did not affect their partition. Shrunk mitoplasts showed the same isoelectric point of pH 4.7 as swollen mitoplasts, which indicates that no charge changes occurred on the outer side of the inner mitochondrial membrane during shrinkage of mitoplasts.     

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.     

Separation of outer and inner membranes of mitochondria from the brown adipose tissue of infant rats.

Digitonin treatment and the swelling-shrinkage-sonication procedure as used to separate mitochondria membranes were applied to mitochondria from the brown adipose tissue (BAT) of infant rats. Digitonin at a concentration of 0.15 mg/mg mitochondrial protein produced disruption of the outer membrane of BAT mitochondria and a complete release of adenylate kinase. However, fragments of the outer membrane remained firmly attached to the inner membrane-matrix particles (mitoplasts) and sedimented at 10 000 g, as indicated by the activity of monoamine oxidase in the pellet. Only at 0.5 mg digitonin/mg protein did outer membrane become almost entirely separated. Oxidation of external cytochrome c by mitoplasts was only 50% of the total cytochrome oxidase at 0.5 mg digitonin/mg protein, indicating an incomplete exposure of the inner membrane to the external medium. Ultrastructural studies revealed that a large proportion of mitoplasts retained the orthodox configuration under these conditions. Outer membrane fragments obtained by the swelling-shrinkage-sonication procedure were of buoyant density corresponding to 20–30% (weight/vol) sucrose. After a 10 sec sonication of mitochondria, a relatively pure outer membrane fraction could be obtained with a yield not exceeding 20%. Longer sonication increased the yield, but also increased the degree of contamination by inner membrane fragments. Optimum conditions for the separation of outer and inner membranes from brown adipose tissue mitochondria are described.     

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.     

A carboxyl-terminal hydrophobic region of yeast cytochrome c1 is necessary for functional assembly into complex III of the respiratory chain

Cytochrome c1 is an amphiphilic protein which binds to the mitochondrial inner membrane, presumably through a hydrophobic region near the carboxyl (C)-terminus. In the preceding study (Hase, T., et al. (1987) J. Biochem. 102, 401-410), two cytochrome c1 mutations were constructed: delta 1 and delta 2 cytochromes c1, in which the C-terminal segments of 17 and 71 residues were replaced by foreign sequences of 20 and 15 residues, respectively. delta 2 cytochrome c1 had lost the putative membrane anchor. The two cytochrome c1 mutants were localized in mitochondria, but succinate-cytochrome c1 reductase activity was detected only in the mitochondria containing delta 1 cytochrome c1. The membrane association of the two mutant molecules as well as that of authentic cytochrome c1 was investigated. These three molecules were firmly attached to mitochondrial membranes and not solubilized on either sonication or sodium carbonate (pH 11) treatment. However, when the membranes were solubilized with Triton X-100, both the delta 1 and authentic cytochromes c1 were extracted from the membranes more easily than delta 2 cytochrome c1. By fractionating cholate extracts of mitochondrial membranes with ammonium sulfate, delta 1 cytochrome c1 was cofractionated with the enzymatic activity of complex III, but delta 2 cytochrome c1 was clearly separated from the complex III fraction. Trypsin treatment of mitochondria and mitoplasts showed that delta 2 cytochrome c1 was exposed to the intermembrane space, with such a topology that its trypsin susceptibility became much higher than that of the authentic molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

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).     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

ADP-ribosyl transferase and NAD glycohydrolase activities in rat liver mitochondria

ADP-ribosyl transferase and NAD glycohydrolase activities have been estimated in mitochondria in mitoplasts as well as in other submitochondrial fractions. A high activity of these two enzymes was present in mitoplasts as compared to the outer membrane preparation or intermembrane compartment. Inhibitor studies provide strong evidence for the involvement of ADP-ribosyl transferase in the process of ADP-ribosylation of mitochondrial proteins. When NAD glycohydrolase was blocked by nicotinamide or 3-aminobenzamide, the incorporation of ADP-ribose into mitochondrial proteins still occurs. ADP-ribosyl transferase activity could also be detected when NAD glycohydrolase was separated by hydroxylapatite chromatography. The protein-linked ADP-ribose moiety appears to be an oligomer in mitochondria.     

Multiple forms of isocitrate lyase in the matrix of Turbatrix aceti mitochondria

The localization of five separate forms of isocitrate lyase (EC 4.1.3.1) in the free-living nematode Turbatrix aceti was determined by analyzing their distribution among subcellular fractions with continuous (Tris-acetate, pH 7.5) polyacrylamide gel electrophoresis. Enzyme activity on gels was detected either by substrate-dependent Schiff's-aldehyde staining or absorbance of phenylhydrazone at 324 nm. Following rate sedimentation of worm homogenates, the highest specific activity for isocitrate lyase was recovered in the pellet containing intact mitochondria. Glyoxysomes (microbodies) were not observed by electron microscopy in this or any other fraction. Selective removal of mitochondrial outer membranes (and hence components in the intermembrane space) was accomplished by two different procedures: (1) passage of mitochondria in a hypersomotic medium through a French pressure cell at 1500 psi, or (2) treatment with 0.6 mg digitonin mg protein. Electron microscopy revealed essentially complete removal for the former procedure, but only partial removal following digitonin treatment. Equilibrium centrifugation on sucrose gradients was necessary to strip the residual outer membranes from the digitonin-treated mitochondria. Mitoplasts resulting from the two procedures were subfractionated into matrix and inner membrane components by high-pressure disruption (14,000 psi) and subsequent rate sedimentation (144,000g, 60 min). Identical electrophoretic patterns were found using both slab and disc gels, whether stained with Schiff's reagent or scanned at 324 nm, in samples taken from clarified homogenates, intact mitochondria, mitoplasts, or matrix fractions. The results indicate that all five forms of the enzyme occur together in the mitochondrial matrices. Their individual functions are not yet known, but they may be involved in the regulation of isocitrate metabolism common to the tricarboxylic acid and glyoxylate cycles occurring within the same mitochondria.     

Asymmetrical distribution of thiol groups involved in ATP-32Pi exchange on mitochondrial membranes.

The use of mitoplasts, that is mitochondria devoid of outer membrane oriented as normal mitochondria, and of sonicated vesicles, the membrane of which is inside-out has shown that the thiol groups involved in the process of ATP synthesis are on the matrix face of the mitochondrial membrane: carboxypyridine disulfide (CPDS) a thiol reagent that cannot penetrate across hydrophobic membranes does not inhibit the ATP-³²P_i exchange catalyzed by mitoplasts, while 5,5′-dithio-$\text{bis}$-(2-nitrobenzoate), which penetrates more readily, can completely inhibit this exchange. In contrast, both reagents react similarly with inside-out vesicles. The nature of the component of the ATPase-ATP synthase complex to which this thiol group may belong is discussed.     

Regulation of mitochondrial transhydrogenase activity by catecholamines

Administration of catecholamines to rats or their addition to liver and heart homogenates activates (by 30-50%) mitochondrial transhydrogenase in the direction of hydride-ion transfer NADPH----NAD+ via beta-adrenoreceptors and cAMP. Glucagon administration also increases by 48% the transhydrogenase activity of liver mitochondria. cAMP (1 microM) incubated with both liver homogenates and mitochondria exerts an independent activating effect on transhydrogenase. The effect of cAMP is specific and is expressed as an increase of V. The integrity of mitochondrial membranes is crucial for the manifestation of cAMP effect. Possible mechanisms of cAMP action on the transhydrogenase activity and the significance of this regulation for mitochondrial energetics are discussed.     

Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ

A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.     

A sulphate metabolizing centre in Euglena mitochondria.

We have previously shown that a sulphate activating system is present on the outside of the inner mitochondrial membrane of Euglena gracilis Klebs. var. bacillaris Cori, but efforts to couple this system to ATP produced from oxidative phosphorylation were unsuccessful. In the present work we show that the concentration of Pi ordinarily used to support oxidative phosphorylation in these mitochondria (10 mM) inhibits sulphate activation completely; by reducing the concentration of Pi 10-fold, both processes proceeded normally. Sulphate activation under these conditions is inhibited nearly completely by the uncouplers of oxidative phosphorylation dinitrophenol (0.1 mM) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (0.2 microM). Sulphate reduction to form free cysteine, most of which appears outside the organelle, and in the cysteine of mitochondrial protein can be demonstrated in the same preparations, is membrane-bound and is inhibited by chloramphenicol (100 micrograms/ml), NaN3 (5 mM), KCN (100 microM); dinitrophenol (0.1 mM) or CCCP (0.2 microM). Digitonin fractionation of the mitochondria into mitoplasts, outer membranes and an intermembrane fraction show that reduction of 35SO4(2-) to form free cysteine and cysteine of protein is located on the mitoplasts; adenosine 5'-phosphosulphate sulphotransferase, the first enzyme of sulphate reduction, is found in the same location. Sulphate activation is highly enriched in the mitochondrial fraction of Euglena; the small amount found in the chloroplast fraction can be attributed to mitochondrial contamination. Thus, in Euglena, sulphate activation and reduction are contained in a sulphate metabolizing centre on the outside of the mitochondrial inner membrane; this centre appears to supply the mitochondrion and the rest of the cell with the products of sulphate activation as well as with reduced sulphur in the form of cysteine. Mitochondria from wild-type Euglena cells and from W10BSmL, a mutant lacking plastids completely, appear to be similar in the properties studied.     

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.     

Localization of the NADH kinase in the inner membrane of yeast mitochondria

The bulk of NADH kinase of Saccharomyces cerevisiae was recovered in the mitochondrial fraction prepared from spheroplasts. Most of the NADH kinase was localized in the inner membrane fraction, which was separated from other mitochondrial components by the combined swelling, shrinking, and sonication procedure. Treatment of mitoplasts with antiserum against the NADH kinase caused inactivation of the enzyme. On the contrary, no influence was observed upon the same treatment of intact mitochondria. p-Chloromercuribenzoate and eosin-5-maleimide inactivated the enzyme without affecting the matrix ATPase. The NADH kinase was enzymatically iodinated in mitoplasts, but not in the intact mitochondria. These results support the conclusion that NADH kinase is localized and functions at the intermembrane space side of the mitochondrial inner membrane. It is evident that the NADH kinase is encoded by nuclear gene(s) because it is synthesized in the presence of chloramphenicol or acriflavine, and a significant amount of the enzyme was detected in mitochondrial DNA-deficient mutants.