Expression of the mammalian mitochondrial genome. Role for membrane potential in the production of mature translation products

Protein synthesis was investigated in isolated mitochondria under conditions which either inhibited electron transport or uncoupled oxidative phosphorylation. In a medium containing an exogenous source of ATP and oligomycin, an inhibitor of the ATP synthase complex, incorporation of [35S]methionine into proteins is stimulated in the presence of inhibitors of the electron transport chain; substituting uncouplers of oxidative phosphorylation for the latter leads, in contrast, to a decrease in the rate of incorporation of the labeled amino acid into mitochondrial translation products. Studies on the metabolic stability of mitochondrial translation products revealed that "mature" polypeptides made in isolated mitochondria are stable as indicated by the absence of degradation during a 50 min "chase" period. Under conditions which reduce or dissipate the membrane potential, 50-60% of the newly made polypeptides (pulse) are degraded within 50 min. The kinetics of the degradation process for individual mitochondrial gene products reveal that the largest proportion of polypeptides degraded to an acid-soluble form during the chase period are abnormal proteins, likely the result of premature chain termination. Emerging as a common denominator in these studies is a role for a transmembrane potential across the inner membrane in the production of mature "stable" mitochondrial gene products.     

Correlation between the rate of proteolysis of mitochondrial translation products and fluidity of the mitochondrial inner membrane in Saccharomyces cerevisiae yeast. Alteration of the rate of proteolysis under glucose repression.

Our previous results [Kalnov, Novikova, Zubatov & Luzikov (1979) FEBS Lett. 101, 355-358; Biochem. J. 182, 195-202] suggested that in yeast the mitochondrial translation products localized in the mitochondrial inner membrane are rapidly broken down by a proteolytic system inherent in the membrane. In the present work, it is demonstrated that, on glucose repression in undividing cells of Saccharomyces cerevisiae, there is no proteolysis of the mitochondrial translation products. This effect is not likely to be associated with lower activity of the proteolytic system of the mitochondrial inner membrane. Nor is the cessation of proteolysis due to qualitative changes in the composition of mitochondrial translation products. What repression does cause is a considerable alteration in the physical state (i.e. structure of the lipid bilayer) of the mitochondrial inner membrane; this was established by experiments involving lipid-soluble spin probes. The conclusion is reached that the rate of proteolysis of mitochondrial translation products in the mitochondrial inner membrane depends on the physical state of the membrane, which in its turn is controlled by the relative content of unsaturated fatty acid chains in the mitochondrial phospholipids.     

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 micrograms/100 g body wt) of triiodothyronine (T3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T3 increases the numbers of functional respiratory chain units. T3 also induces mitochondrial protein synthesis by 50-100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T3 belong to two subsets, which were either induced or repressed by hormone. Thus, T3 stimulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.     

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).     

Effect of ethionine on the in vitro synthesis and degradation of mitochondrial translation products in yeast

The effect of ethionine, an amino acid analog of methionine, has been studied in Saccharomyces cerevisiae in relation to cell growth, oxygen consumption, in vitro protein synthesis of mitochondrial translation products (MTPs) and the degradation of those mitoribosomally made proteins by an ATP-dependent process present within the organelle. Ethionine was found to increase the generation time of those cells already committed to cell division and to abolish the initiation of new cell cycles. Oxygen consumption of cultures grown in the presence of the analog was drastically reduced. Ethionine was also found to impair the incorporation of methionine and leucine into mitochondrial translation products, however the synthesis of proteins was not totally blocked and, apparently, mitochondria utilized ethionine as a precursor amino acid. MTPs synthesized by isolated mitochondria in the presence of ethionine were rapidly degraded inside the organelle at a faster rate compared with the normal proteins synthesized under identical conditions in the mitochondria. It is also shown that these in vitro synthesized proteins are degraded by an ATP-stimulated proteolytic system, as has been previously established.     

Incorporation of labeled formate in rat liver mitochondrial proteins and initiation of protein synthesis

During incubation of rat liver mitochondria in vitro labeled formate is incorporated into TCA-insoluble substance during mitochondrial translation. Data from hydrolysis with CNBr (after methionine residues) or with 0.5 N HCl (deformylation of amino acid N-formyl derivatives) suggest that about half of the total protein radioactivity is incorporated in formate groups of N-terminal methionine. Labeling of growing polypeptides with formate (but not with phenylalanine or methionine) oscillates with a period of about 13 min. The potential initiation capacity is unchangeable and exceeds that observed experimentally by one order of magnitude. The data obtained are consistent with the previously proposed hypothesis no synchronization of mitochondrial protein synthesis which cannot be induced by the steps preceding the formation of the first peptide bound.     

Effects of chronic ethanol consumption on the synthesis of polypeptides encoded by the hepatic mitochondrial genome

Liver mitochondria from rats fed ethanol chronically demonstrate an impaired ability to incorporate [35S]methionine into polypeptide products in vitro. This ethanol-induced effect on mitochondrial translation in vitro could not be attributed to significant differences in the methionine precursor pool sizes of ethanol and control mitochondria or to the acute effects of residual ethanol. The observed reduction of radiolabeled methionine incorporation into mitochondrial gene products of ethanol mitochondria in vitro reflects a decrease in the synthesis of all the mitochondrial gene products. However, the percentage of total radiolabel incorporated into each gene product is unaffected by ethanol, suggesting an ethanol-induced coordinate depression of mitochondrial protein synthesis. Moreover, SDS-PAGE and densitometry of submitochondrial particles from ethanol-fed and control rats demonstrated that the steady-state concentration of each of the mitochondrial gene products is decreased in ethanol-fed rats. This reduction of the steady-state concentration of the mitochondrial gene products may be related to the observed depressions of oxidative phosphorylation activities associated with hepatic mitochondria from ethanol-fed rats.     

Partial purification of mitochondrial ribosomes from broad bean and identification of proteins encoded by the mitochondrial genome

An approach towards the identification at the protein level of the ribosomal proteins encoded by the mitochondrial genome of broad bean (Vicia faba) has been developed. After Triton X-100 treatment of isolated mitochondria, a fraction enriched in mitochondrial ribosomes was obtained by successive centrifugation, first onto a sucrose cushion, and then in a sucrose gradient. Mitochondrial translation products were labelled in isolated mitochondria with [³⁵S]methionine and added to the enriched mitochondrial ribosomal proteins before separation by two-dimensional gel electrophoresis. Six spots, identified both by Coomassie blue staining and autoradiography, were analysed by protein micro-sequencing. Two of these were shown to correspond to ribosomal proteins S10 and S12. We conclude that these two proteins are encoded by the mitochondrial genome of broad bean and that the method described here can be used to identify other proteins encoded by the mitochondrial genome. Received: 4 September 1996 / Accepted: 30 November 1996     

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPR^mt) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPR^mt, suggesting that the UPR^mt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPR^mt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPR^mt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPR^mt and longevity.     

Proteolysis of the products of mitochondrial protein synthesis in yeast mitochondria and submitochondrial particles.

Degradation of mitochondrial translation products in Saccharomyces cerevisiae mitochondria was studied by selectively labelling these entities in vivo in the presence of cycloheximide and following their fate in isolated mitochondria. One-third to one-half of the mitochondrial translation products are shown to be degraded, depending on the culture growth phase, with an approximate half-life of 35 min. This process is shown to be ATP-dependent, enhanced in the presence of puromycin and inhibited by chloramphenicol. Further, the proteolysis is suppressed by detergents and is insensitive to antisera against yeast proteinases A and B when measured in mitochondria or 'inside-out' submitochondrial particles. It is concluded that the breakdown of mitochondrial translation products is most probably due to the action of endogenous proteinase(s) associated with the mitochondrial inner membrane. This proteinase is inhibited by phenylmethanesulphonyl fluoride, leupeptin, antipain and chymostatin.     

Effect of methionine dietary supplementation on mitochondrial oxygen radical generation and oxidative DNA damage in rat liver and heart

Methionine restriction without energy restriction increases, like caloric restriction, maximum longevity in rodents. Previous studies have shown that methionine restriction strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative damage to mitochondrial DNA, lowers membrane unsaturation, and decreases five different markers of protein oxidation in rat heart and liver mitochondria. It is unknown whether methionine supplementation in the diet can induce opposite changes, which is also interesting because excessive dietary methionine is hepatotoxic and induces cardiovascular alterations. Because the detailed mechanisms of methionine-related hepatotoxicity and cardiovascular toxicity are poorly understood and today many Western human populations consume levels of dietary protein (and thus, methionine) 2–3.3 fold higher than the average adult requirement, in the present experiment we analyze the effect of a methionine supplemented diet on mitochondrial ROS production and oxidative damage in the rat liver and heart mitochondria. In this investigation male Wistar rats were fed either a L-methionine-supplemented (2.5 g/100 g) diet without changing any other dietary components or a control (0.86 g/100 g) diet for 7 weeks. It was found that methionine supplementation increased mitochondrial ROS generation and percent free radical leak in rat liver mitochondria but not in rat heart. In agreement with these data oxidative damage to mitochondrial DNA increased only in rat liver, but no changes were observed in five different markers of protein oxidation in both organs. The content of mitochondrial respiratory chain complexes and AIF (apoptosis inducing factor) did not change after the dietary supplementation while fatty acid unsaturation decreased. Methionine, S-AdenosylMethionine and S-AdenosylHomocysteine concentration increased in both organs in the supplemented group. These results show that methionine supplementation in the diet specifically increases mitochondrial ROS production and mitochondrial DNA oxidative damage in rat liver mitochondria offering a plausible mechanism for its hepatotoxicity.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Larger precursors of mitochondrial translation products in Neurospora crassa. Indications for a precursor of subunit 1 of cytochrome c oxidase

Specific labeling in vivo of the formylated N termini of mitochondrial translation products revealed that some mitochondrially synthesized proteins were not labeled this way. As a consequence, it was worthwhile considering that larger precursor proteins of mitochondrial translation products exist. Although we used a rapid isolation procedure, only after 2-h of labeling in the presence of cycloheximide, could three additional mitochondrial translation products (molecular mass 45, 36, and 25 kilodaltons) be detected. Preincubation with cycloheximide indicated that these proteins might be larger precursors which were no longer processed due to the prolonged presence of cycloheximide. To prevent processing of the precursors during isolation, cells of the slime mutant were directly lysed in boiling sodium dodecyl sulphate solution. In this way, the same three additional mitochondrial translation products were detected after a pulse-labeling of 1 min. These proteins behave in a precursor-like fashion. Labeling at 9 degrees C resulted in a partial accumulation of the three additional proteins. Finally protein blots treated with antibodies and 125I-labeled protein A, support the idea that the 45-kDa protein is a precursor of subunit 1 of cytochrome c oxidase; 50-80% of this precursor could be detected in the post-mitochondrial supernatant, indicating that this polypeptide is not tightly bound to the membrane.     

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene

The A3243G mutation in the human mitochondrial tRNA^Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA^Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA^Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.     

Overexpression of mitochondrial methionine sulfoxide reductase B2 protects leukemia cells from oxidative stress-induced cell death and protein damage

According to the mitochondrial theory of aging, mitochondrial dysfunction increases intracellular reactive oxidative species production, leading to the oxidation of macromolecules and ultimately to cell death. In this study, we investigated the role of the mitochondrial methionine sulfoxide reductase B2 in the protection against oxidative stress. We report, for the first time, that overexpression of methionine sulfoxide reductase B2 in mitochondria of acute T-lymphoblastic leukemia MOLT-4 cell line, in which methionine sulfoxide reductase A is missing, markedly protects against hydrogen peroxide-induced oxidative stress by scavenging reactive oxygen species. The addition of hydrogen peroxide provoked a time-gradual increase of intracellular reactive oxygen species, leading to a loss in mitochondrial membrane potential and to protein carbonyl accumulation, whereas in methionine sulfoxide reductase B2-overexpressing cells, intracellular reactive oxygen species and protein oxidation remained low with the mitochondrial membrane potential highly maintained. Moreover, in these cells, delayed apoptosis was shown by a decrease in the cleavage of the apoptotic marker poly(ADP-ribose) polymerase-1 and by the lower percentage of Annexin-V-positive cells in the late and early apoptotic stages. We also provide evidence for the protective mechanism of methionine sulfoxide reductase B2 against protein oxidative damages. Our results emphasize that upon oxidative stress, the overexpression of methionine sulfoxide reductase B2 leads to the preservation of mitochondrial integrity by decreasing the intracellular reactive oxygen species build-up through its scavenging role, hence contributing to cell survival and protein maintenance.     

Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [³⁵S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [³⁵S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots.     

The transport of proteins into yeast mitochondria. Kinetics and pools

By double isotope pulse-labeling of yeast cells, we determined the kinetics of labeling at 9 degrees C of total mitochondrial membrane, mitochondrial matrix, and cytosolic proteins, the alpha, beta, and gamma subunits of F1 ATPase, and glyceraldehyde-3-phosphate dehydrogenase. We find that none of the mitochondrial proteins show a lag in the incorporation of label compared to cytosolic proteins. These results argue against the existence in the cytosol of large pools of mitochondrial proteins awaiting transport into the organelle. Cycloheximide addition during the pulse stops [35S]methionine incorporation into mitochondrial membrane and cytosolic proteins rapidly (approximately 1 min) and with identical kinetics. Compared to cytosolic protein, however, there is a persistent incorporation of label into mitochondria after a chase with cold methionine (t1/2 approximately 1.5 min at 9 degrees C) which cannot be accounted for solely by chain completion. We conclude that this continued incorporation reflects some transport process in addition to a completion of a round of translation. When cells are labeled during a synchronous "restart" of protein synthesis, where ribosome run-off from mRNA was first induced either by incubating cells for 4 h at 0 degrees C or by treatment with 5 mM aurintricarboxylic acid, the initial rate of incorporation of label into mitochondrial protein now lags behind that of cytosolic proteins. From these results and those in the accompanying report (Ades, I.Z., and Butow, R.A. (1980) J. Biol. Chem. 255, 9918-9924) we propose that the translation of mRNA specific for mitochondrial proteins takes place in the cytoplasm and that at least a portion of the polysomes are then transported and bind to the outer mitochondrial membrane, followed by completion of translation and transfer of the newly synthesized polypeptides into the mitochondria. From a consideration of all of the available data on protein transport into mitochondria in yeast, we conclude that cytoplasmic polysomes bound to the outer mitochondrial membrane function in the transport of proteins into mitochondria by a process not necessarily mutually exclusive of post-translational transport.     

Products of mitochondrial protein synthesis in Tetrahymena.

The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57,500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform-methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h.