Mitochondrially Translated Research Articles

Translation Fidelity and Respiration Deficits in CLPP-Deficient Tissues: Mechanistic Insights from Mitochondrial Complexome Profiling.

The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5'-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.

Chronic Inhibition of Mitochondrial Translation Alters Metabolism and Stemness in AML

Abstract 1364Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in AML and AML stem cells over normal hematopoietic cells through the inhibition of mitochondrial translation. This heightened sensitivity was due to increased mitochondrial mass and reliance on oxidative metabolism in the AML cells compared to normal hematopoietic cells. Here, we sought to better understand the mechanisms of sensitivity and resistance to inhibitors of mitochondrial translation.To establish cells resistant to tigecycline, we exposed TEX leukemia cells to increasing concentrations of tigecycline over 4 months and selected a population of TEX cells resistant to tigecycline (RTEX+TIG) with an IC50 > 24 μM (versus an IC50 of 2.8 + 0.4 μM in wild type cells). We then profiled oxidative metabolism in the resistant cells. In RTEX+TIG cells, levels of Cox-1 and Cox-2, subunits of respiratory complex IV in the electron transport chain that are translated by mitochondrial ribosomes, were undetectable. In contrast, Cox-4 that is part of the same respiratory chain, but translated in the cytoplasm, was only slightly reduced. RTEX+TIG cells also had undetectable levels of oxygen consumption and increased rates of glycolysis compared to wild type cells. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia, thus demonstrating the functional importance to the change in their metabolic status. RTEX+TIG cells also had reduced mitochondrial membrane potential by 44.4 + 7.2% and reduced mitochondrial mass compared to wild type cells. Morphologically, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures.To understand the molecular basis for the metabolic changes in the RTEX+TIG cells, we performed RNA sequencing of the RTEX+TIG cells and wild type TEX cells. Unbiased analysis, by two independent approaches, of the promoter sequences of transcripts upregulated 1.5-fold or greater in RTEX+TIG cells demonstrated a significant over-representation of binding sites for the hypoxia-inducible factor 1 alpha HIF1α :HIF1β transcription factor complex. Specifically, a subset of HIF1α target genes involved in energy balance and cellular metabolism were coordinately upregulated in RTEX+TIG cells, corresponding with our phenotypic observations of the metabolic state of these cells. We validated the upregulation of HIF1α mRNA and protein by Q-RTPCR and immunoblotting.Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels. However, HIF1α remained elevated. Upon re-treatment with tigecycline, the cells remained resistant and the glycolytic phenotype was re-established.TEX cells display features of leukemia stem cells, including differentiation, self-renewal and hierarchical organization. Interestingly, RTEX+TIG cells were more differentiated and had reduced stemness compared to the wild type TEX cells. By immunohistochemistry, RTEX+TIG had increased non-specific esterase activity (NSE). In addition, RTEX+TIG cells had reduced clonogenic growth and ability to engraft immune deficient mice compared to wild type cells. Moreover, RNA sequencing data showed reduced expression of stem cell maintenance genes in RTEX+TIG cells.Depletion of mitochondrial DNA via prolonged exposure of parental cell lines to cationic lipophilic agents such as ethidium bromide produces rho-zero cells that have irreversibly lost mitochondrially translated proteins. These cells lack a functional respiratory chain and cannot derive energy from oxidative phosphorylation. Instead, these cells rely on glycolysis for their energy supply. Here, we have produced a reversible rho-zero like metabolic phenotype by sustained inhibition of mitochondrial translation. This work, therefore, highlights mechanisms of metabolic adaption to inhibition of oxidative phosphorylation. Finally, these data suggest a unique role for metabolism in differentiation and stemness in AML. Disclosures:No relevant conflicts of interest to declare.

Mutations in MTFMT Underlie a Human Disorder of Formylation Causing Impaired Mitochondrial Translation

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.

Respiratory chain-linked NADH dehydrogenase. Mechanisms of assembly.

The assembly of mitochondrially and cytoplasmically translated subunits of NADH dehydrogenase in the inner mitochondrial membrane was studied in rat hepatoma cultures. A polyclonal antibody to the purified bovine heart holoenzyme, which reacted with comigrating proteins of both rat liver and hepatoma mitochondria on immunoblots, precipitated 25-30 [35S]methionine-labeled proteins from hepatoma cell lysates. Six of these were sensitive to an inhibitor of mitochondrial translation (chloramphenicol), resistant to an inhibitor of cytosolic translation (cycloheximide), and were not present in cytochrome oxidase. By these criteria, six NADH dehydrogenase subunits are identified as being translated on mitochondrial ribosomes. The metabolic properties of the three most prominent of these at 51, 43, and 11 kDa were studied in more detail. Mitochondrial and nuclear-coded polypeptides assemble into NADH dehydrogenase at different rates as measured by incorporation of pulse-labeled proteins into immunoprecipitable enzyme. Nuclear-coded, imported polypeptides appear immediately after a pulse with [35S]methionine and retain constant stoichiometry. Mitochondrially coded proteins, although rapidly translated, appear at peak levels at different times between 0 and 12 h of chase in the immunoprecipitated enzyme. Ongoing synthesis and import of nuclear-coded proteins is necessary for mitochondrially coded proteins to be assembled. Excess, unassembled mitochondrially translated subunits are degraded in an oligomycin-sensitive manner. These data are consistent with a model in which a scaffold of imported proteins forms the inner core of the enzyme, and later arriving mitochondrially translated proteins attach to the scaffold in a time-dependent manner.

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 μg/100 g body wt) of triiodothyronine (T 3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T 3 increases the numbers of functional respiratory chain units. T 3 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 T 3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T 3 belong to two subsets, which were either induced or repressed by hormone. Thus, T 3 timulated 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.

No unique mitochondrial translation products in respiratory chain-linked NADH dehydrogenase

Antibody prepared against beef heart mitochondrial NADH dehydrogenase immunoprecipitated 26 polypeptides from detergent solubilized beef heart mitochondria. All 26 polypeptides co-migrated with those present in the dehydrogenase antigen when resolved side by side on sodium dodecyl sulfateurea polyacrylamide gels. From mixed rat liver-[ 35S]methionine pulsed hepatoma mitochondria the antibody immunoprecipitated 24 stained liver polypeptides and 19 radio-labelled hepatoma polypeptides. The translation of three of the labelled polypeptides was resistent to inhibition by cycloheximide, indicating these are translated on mitochondrial ribosomes. These same polypeptides, however, wre previously identified as cytochrome c oxidase subunits; and, apparently, non-specifically co-precipitate with dehydrogenase associated polypeptides. We conclude that there are no mitochondrially translated polypeptides specifically associated with NADH dehydrogenase.

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII.

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.

Mitochondrial translation products of yeast mutants resistant to mucidin.

Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the mitochondrially translated polypeptides of mucidin-resistant mutantsmuc1, muc2 andmuc3 failed to reveal any modification in the electrophoretic mobility of apocytochromeb coded for by sequences of mitochondrial DNA comprising these mutations.

Synthesis of cytochrome oxidase in isolated rat hepatocytes

1. The synthesis of cytochrome oxidase was studied in isolated rat hepatocytes labeled in vitro. Labeled whole cells, isolated mitochondria, microsomes and the post microsomal supernatant were treated with antisera to rat liver holo-cytochrome oxidase, and the subunits were adsorbed onto Sepharoseprotein A. 2. Seven peptides, corresponding to subunits of rat liver cytochrome oxidase, were immunoabsorbed from mitochondria isolated from cells labeled in the absence of inhibitors. Two peptides, corresponding to subunits I (45 500 daltons) and II (26 000 daltons), were labeled in mitochondria isolated from cycloheximide-treated cells. Labeling of these peptides was inhibited by chloramphenicol. Peptides I and II correspond to the two most heavily labeled mitochondrial translation products found in submitochondrial particles. Possible explanations for the lack of labeling of a third mitochondrially translated subunit are discussed. Labeling of the five smallest peptides was inhibited by cyclohexamide but not by chloramphenicol. 3. Peptide I appears in the holoenzyme later than the other six peptides after a pulse-chase. It is not labeled in the immunoabsorbed cytochrome oxidase after a 30 min pulse with [ 35S]-methionine, but appears after a 3 h chase with unlabeled methionine. Labeling of the other subunits showed no further increase after the chase. 4. Antibodies to cytochrome oxidase specifically absorb two high molecular weight microsomal peptides which do not correspond to subunits of the holoenzyme. These peptides turnover rapidly after a pulse chase, but no concomitant increase in the cytoplasmically translated subunits were observed. A second band (50 000–55 000) was also absorbed from isolated microsomes, but in our hands is an artifact due to electrophoresis. Thus, we have not been able to identify a high molecular weight polyprotein precursor to the cytoplasmically translated subunits of cytochrome oxidase.

Assembly of the mitochondrial membrane system: nuclear suppression of a cytochrome b mutation in yeast mitochondrial DNA.

In a previous study, a mitochondrial mutant expressing a specific enzymatic deficiency in co-enzyme QH2-cytochrome c reductase was described (TZAGO-LOFF, FOURY and AKAI 1976). Analysis of the mitochondrially translated proteins revealed the absence in the mutant of the mitochondrial product corresponding to cytochrome b and the presence of a new low molecular weight product. The premature chain-termination mutant was used to obtain suppressor mutants with wild-type properties. One such revertant strain was analyzed genetically and biochemically. The revertant was determined to have a second mutation in a nuclear gene that is capable of partially suppressing the original mitochondrial cytochrome b mutation. Genetic data indicate that the nuclear mutation is recessive and is probably in a gene coding for a protein involved in the mitochondrial translation machinery.

Identification of the structural gene for yeast cytochrome c oxidase subunit II on mitochondrial DNA.

SLONIMSKI From the Centre de Gkn6tique Moldculaire du Centre National de la Recherche Scientifique, F-91190 Gif-sur-Yvette, France The mitochondrially translated polypeptides were ana- lyzed in yeast mutants which specifically lacked cytochrome c oxidase as a result of a mutation in mitochondrial DNA. In an earlier study, the mutations have been mapped in one of three distinct loci designated OXI 1, OX1 2, and OX1 3. The OX1 1 locus is now shown to code for the mitochon- drially translated Subunit II by the following criteria: (a) in two OX1 1 mutants, cytochrome c oxidase Subunit II is replaced by a shorter mitochondrially made polypeptide which is not detected in the wild type parent; these “new” polypeptides can be identified as fragments of Subunit II by their weak, but significant cross-reaction with an antiserum against Subunit II from wild type cells. (b) When these two OX1 1 mutants are reverted by an additional mutation of mitochondrial DNA, Subunit II regains its strong interac- tion with anti-Subunit II serum, but its electrophoretic mobility on dodecyl sulfate-polyacrylamide gels is increased over that of wild type Subunit II. Electrophoresis in the presence of the cationic detergent, cetyltrimethylammo- nium bromide, determination of the free electrophoretic mobility, and peptide mapping by limited proteolysis indi- cate that the increased mobility of Subunit II in the revert- ants does not merely reflect abnormal binding of dodecyl sulfate. We conclude that the OX1 1 locus contains the structural gene for cytochrome oxidase Subunit II. Mitochondria contain a genetic system that participates in their own formation (l-4). The main function of this system appears to be the synthesis of at least nine hydrophobic polypeptides which are tightly associated with the mitochon- drial inner membrane. Three of these polypeptides are associ- * This work was supported by Grant 3.2350.74 from the Swiss National Science Foundation, a fellowship from the Sandoz Foun- dation, and Grant 75-7-0748 from DQl&$tion G&&ale B la Re- cherche Scientifique. Int&actions Mol&ulaires en Biologie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address, National Institutes of Health, Building 37, Bethesda, Md. 20014. § To whom correspondence should be addressed. ated with cytochrome c oxidase, two to four (depending on the organism) with the ATPase complex, and at least one cytochrome b (1, 2, 4). The function of the remaining polypep- tides is still unknown. While it is firmly established that these polypeptides are translated on mitochondrial ribosomes, no structural gene for any of these polypeptides has yet been identified (2-4). For many years it was even debated whether these structural genes were localized on nuclear rather than mitochondrial DNA (2). Recent experiments with cell-free systems pro- grammed with mitochondrial DNA (5) or RNA (6) strongly suggest, however, that at least some of the mitochondrial translation products are coded by mitochondrial DNA. This is also implied by studies which exploited the differences in electrophoretic mobility of several mitochon- drial translation products from different yeast strains (7). However, none of these efforts have led to the identification of a gene coding for a functionally defined mitochondrially made polypeptide. The present study is based on the recent demonstration (8, 9) that mutations in three loci on yeast mitochondrial DNA can lead to the selective loss of cytochrome oxidase. These loci (termed OX1 1, 2, and 3) have been mapped (9), but it remained unclear whether they were structural or regula- tory genes for cytochrome oxidase. We now report evidence which indicates that the OX1 1 locus is the structural gene for cytochrome oxidase Subunit II.

Partial independence of synthesis and membrane attachment of mitochondrial and cytoplasmic precursors of electron transfer complexes III and IV in adapting Bakers' yeast

When anaerobically grown Saccharomyces cerevisiae are aerated under conditions which may deplete them of cytoplasmically translated, mitochondrial inner membrane enzyme precursors, they show no immediate decrease in in vivo mitochondrial translational activity compared with cells which have not been so depleted. Similarly, cells depleted of mitochondrially translanted precursors show no immediate decrease in their cytoplasmic translation of mitochondrial inner membrane proteins. These experiments suggest that the synthesis and nonspecific membrane attachment of mitochondrially and cytoplasmically translated inner membrane proteins are not stringently delimited by a prior depletion of inner membrane precursors elaborated by the “other” genetic system. It is thus possible to demonstrate a degree of uncoupling of the activities of the two genetic systems. The oxygen inductions of reduced CoQ cytochrome c reductase (complex III) and of cytochrome c oxidase (complex IV) activities in cells which have been sequentially exposed first to cycloheximide and then to chloramphenicol, or first to chloramphenicol and then to cycloheximide reflect the levels to which specifically integrated, mitochondrial and cytoplasmic precursors of these complexes can accumulate in the absence of concomitant translational activity by the “other” genetic system. These data again suggest the degree to which the translational activities of the two genetic systems can be uncoupled. A detailed study of the inductions of these two complexes in cells exposed first to chloramphenicol shows that the modes of induction of the two complexes are different. Complex III develops approximately 50% of its activity as an expression of a precursor (presumably mitochondrially translated) which is already present in the anaerobic cells, but which requires oxygen-induced cytoplasmic translation for its expression. The remainder of the induced complex III activity appears to require oxygen-induced mitochondrial translation for its expression. There was no analogous anaerobically present component evident during complex IV induction.

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: Dependence upon a mitochondrially translated repressor

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in in vivo [ 14C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome c reductase (complex III) and of cytochrome c oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.