Mammalian Mitochondrial Protein Synthesis Research Articles

Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes

N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNAMet by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essential for mitochondrial protein synthesis but is critical for efficient synthesis of several mitochondrially encoded peptides and for OXPHOS complex stability and assembly into supercomplexes.

Initiation and elongation of mammalian mitochondrial protein synthesis

Initiation and elongation of mammalian mitochondrial protein synthesis

Using mitoribosomal profiling to investigate human mitochondrial translation.

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA Phe whilst in humans it is mt-tRNA Val. We have previously shown that when a mutation in mt-tRNA Val causes very low steady state levels, there is preferential recruitment of mt-tRNA Phe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA Phe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA Phe containing mitoribosomes with those of controls that integrate mt-tRNA Val revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5' leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA Val cell line suggest that despite mt-tRNA Phe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

Using mitoribosomal profiling to investigate human mitochondrial translation

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA Phe whilst in humans it is mt-tRNA Val. We have previously shown that when a mutation in mt-tRNA Val causes very low steady state levels, there is preferential recruitment of mt-tRNA Phe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA Phe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA Phe containing mitoribosomes with those of controls that integrate mt-tRNA Val revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA Val cell line suggest that despite mt-tRNA Phe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

Using mitoribosomal profiling to investigate human mitochondrial translation

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNAPhe whilst in humans it is mt-tRNAVal. We have previously shown that when a mutation in mt-tRNAVal causes very low steady state levels, there is preferential recruitment of mt-tRNAPhe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNAPhe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNAPhe containing mitoribosomes with those of controls that integrate mt-tRNAVal revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNAVal cell line suggest that despite mt-tRNAPhe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

The antibiotic chloramphenicol may be an effective new agent for inhibiting the growth of multiple myeloma.

Chloramphenicol is an old antibiotic that also inhibits mammalian mitochondrial protein synthesis. Our studies demonstrated that chloramphenicol is highly cytotoxic to myeloma cells, acting in a dose- and time-dependent manner. Chloramphenicol sharply suppressed ATP levels in myeloma cells at concentrations ≥ 25 μg/mL. Colorimetric and clonogenic assays indicate that chloramphenicol inhibits growth of myeloma cell lines at concentrations ≥ 50 μg/mL, and inhibits primary myeloma cell growth at concentrations ≥ 25 μg/mL. Flow cytometry and Western blotting showed that chloramphenicol induces myeloma cell apoptosis at concentrations ≥ 50 μg/mL. Chloramphenicol increased levels of cytochrome c, cleaved caspase-9 and cleaved caspase-3, suggesting that myeloma cell apoptosis occurs through the mitochondria-mediated apoptosis pathway. It thus appears chloramphenicol is not only an old antibiotic, it is also a potential cytotoxic agent effective against myeloma cells. This suggests chloramphenicol may be an effective “new” drug for the treatment of myeloma.

High throughput gene complementation screening permits identification of a mammalian mitochondrial protein synthesis (ρ−) mutant

To identify nuclear DNA (nDNA) oxidative phosphorylation (OXPHOS) gene mutations using cultured cells, we have developed a complementation system based on retroviral transduction with a full length cDNA expression library and selection for OXHOS function by growth in galactose. We have used this system to transduce the Chinese hamster V79-G7 OXPHOS mutant cell line with a defect in mitochondrial protein synthesis. The complemented cells were found to have acquired the cDNA for the bS6m polypeptide of the small subunit of the mitochondrial ribosome. bS6m is a 14kDa polypeptide located on the outside of the mitochondrial 28S ribosomal subunit and interacts with the rRNA. The V79-G7 mutant protein was found to harbor a methionine to threonine missense mutation at codon 13. The hamster bS6m null mutant could also be complemented by its orthologs from either mouse or human. bS6m protein tagged at its C-terminus by HA, His or GFP localized to the mitochondrion and was fully functional. Through site-directed mutagenesis we identified the probable RNA interacting residues of the bS6m peptide and tested the functional significance of mammalian specific C-terminal region. The N-terminus of the bS6m polypeptide functionally corresponds to that of the prokaryotic small ribosomal subunit, but deletion of C-terminal residues along with the zinc ion coordinating cysteine had no functional effect. Since mitochondrial diseases can result from hundreds to thousands of different nDNA gene mutations, this one step viral complementation cloning may facilitate the molecular diagnosis of a range of nDNA mitochondrial disease mutations. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi.

Exploration of the roles of IF3mt in mammalian mitochondrial translation

The initiation of mammalian mitochondrial protein synthesis is poorly understood. Two initiation factors with homology to bacterial IF2 and IF3 have been identified. IF3mt is a 29 kDa protein with an internal region having homology to prokaryotic IF3. This homology region consists of an N‐terminal domain, a linker and a C‐terminal domain. The homology domains are preceded and followed by short extensions. No information is currently available on the regions of IF3mt important for its activity. Based on homology models of IF3mt, mutations were designed in the N‐terminal, C‐terminal, and linker domains to identify the functions of these regions. Mutation of residues 66–70 and 121–124 in the N‐terminal domain and residues 194–197 in the C‐terminal domain had no effect on the ability of IF3 to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 143–147 in the linker region and residues 184–186 in the C‐terminal domain caused a significant reduction in activity indicating an important role for this region for initiation complex formation. Mutation of histidine 170 and aspartic acid 171 to alanine caused a nearly complete loss of activity. However, sucrose density centrifugation indicates that this variant can bind to 28S subunits. Mutation of the nearby region (residues 175–177) also resulted in a significant loss of activity. The causes for these defects are under investigation.

Inhibition of Mammalian Mitochondrial Protein Synthesis by Oxazolidinones

The effects of a variety of oxazolidinones, with different antibacterial potencies, including linezolid, on mitochondrial protein synthesis were determined in intact mitochondria isolated from rat heart and liver and rabbit heart and bone marrow. The results demonstrate that a general feature of the oxazolidinone class of antibiotics is the inhibition of mammalian mitochondrial protein synthesis. Inhibition was similar in mitochondria from all tissues studied. Further, oxazolidinones that were very potent as antibiotics were uniformly potent in inhibiting mitochondrial protein synthesis. These results were compared to the inhibitory profiles of other antibiotics that function by inhibiting bacterial protein synthesis. Of these, chloramphenicol and tetracycline were significant inhibitors of mammalian mitochondrial protein synthesis while the macrolides, lincosamides, and aminoglycosides were not. Development of future antibiotics from the oxazolidinone class will have to evaluate potential mitochondrial toxicity.

Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu

The bovine liver mitochondrial protein synthesis elongation factor Tu · Ts complex (EF-Tu · Ts mt) has been purified and partial peptide sequence information has been obtained for EF-TU mt. A complete cDNA has been obtained encoding bovine EF-Tu mt and a nearly complete cDNA has been obtained for human EF-Tum mt. The bovine cDNA has a 5′ untranslated leader, an open reading frame of 1356 nucleotides and a 3′ untranslated region of 189 base pairs. NH 2-terminal sequencing of the mature protein indicates that the transit peptide for the mitochondrial localization of this protein is 43 amino acids in length. The human and bovine factors are 95% identical. The deduced protein sequences show considerable identity to bacterial and organellar EF-Tu sequences. At least two genes for EF-Tu mt are present in the bovine system. Northern analysis indicates that EF-Tu mt is synthesized in all tissues but that the level of expression varies over a wide range. EF-Tu mt has been expressed in E. coli as a His-tagged protein and purified to near homogeneity. The expressed form of the factor is active in the poly(U)-directed polymerization of phenylalanine although it is less active than the native EF-Tu · Ts mt complex.

Mechanism of mRNA binding to bovine mitochondrial ribosomes

The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.

Chloramphenicol

Chloramphenicol was introduced into medical practice in 1949. At therapeutic concentrations of 10 to 20 micrograms drug per ml, the drug inhibits bacterial ribosomal and, to a lesser extent, mammalian mitochondrial protein synthesis but concentrations above 60 micrograms drug per ml induce progressive reduction of oxygen-dependent cellular metabolism. Some adverse reactions (e.g. bone marrow suppression and the "gray baby syndrome") reflect these effects. The pathogenesis of chloramphenicol-induced aplastic anemia remains unclear. Chloramphenicol is most bioavailable after oral administration and has a remarkable ability to diffuse into body fluids and tissues. However, there are wide interindividual variations in its metabolism and elimination, particularly in newborns. Chloramphenicol is indicated for invasive ampicillin-resistant H. influenzae infections; for patients allergic to penicillin with pneumococcal, meningococcal or H. influenzae meningitis; in patients under 8 years of age with Rocky Mountain spotted fever; and for the treatment of brain abscess and other severe anaerobic infections (excluding endocarditis) due to B. fragilis. Other indications include selected patients with Salmonella meningitis or carditis, rickettsioses and intraocular infections. Unravelling the pathogenesis of chloramphenicol-induced aplastic anemia is critical to more widespread application of this remarkable antimicrobial.

Control of yeast and mammalian mitochondrial protein synthesis by cytoplasmic factors

The biogenesis of the mitochondrial inner membrane requires the synthesis of proteins at 2 intracellular sites: 1 cytoplasmic; 1 mitochondrial. A mechanism must exist to coordinate the synthesis of proteins at the 2 distinct intracellular sites such that mitochondrial formation occurs in an orderly manner. Previous studies in yeast using selective inhibitors of cytoplasmic and mitochondrial protein synthesis have suggested that cytoplasmically synthesized proteins may control mitochondrial protein synthesis both in vivo [l-4] and in vitro [3] and that this control may occur by either stimulation of chain initiation or increased formation of mitochondrial mRNAs [5]. In [6] protein synthesis by yeast mitochondria in vitro was reported stimulated by addition of a high-speed cytoplasmic supernatant from yeast [6]. This effect is not species-specific, as protein synthesis in vitro by isolated yeast mitochondria could be stimulated markedly by post-polysomal supernatants from either E. coli, rat liver or yeast [7]. However, stimulation of yeast mitochondrial protein synthesis by all of these high speed supernatants may be due to the presence of non-dialyzable GMP which is converted to GDP (or GTP) during the incubation in the protein-synthesizing medium [8]. We have confirmed that GTP stimulates yeast mitochondrial protein synthesis in vitro, but addition of a post-polysomal supernatant from yeast doubles the rate of mitochondrial protein synthesis in

Differences in the mitochondrially synthesized subunits of human and mouse cytochrome c oxidase

Ten or so proteins synthesized in mammalian mitochondria in vivo can be resolved by electrophoresis in polyacrylamide gels, after solubilization with sodium dodecyl sulphate [ I] . Using interspecific differences in the electrophoretic pattern of these proteins, we analysed the nature of the mitochondrially made proteins which are synthesized in human-mouse somatic cell hybrids. The observed patterns of these proteins in such cells suggested that interspecific variations in mitochondrially synthesized proteins are determined extrachromosomally and therefore possibly by mitochondrial DNA [2] . We have attempted to discover the functions of some of these products of mammalian mitochondrial protein synthesis, and now report that one of them, which shows considerable interspecific variation, appears to be associated with the cytochrome c oxidase (EC 1.9.3.1) complex of mammalian mitochondria.

Initiation of mammalian mitochondrial protein synthesis the effect of methotrexate

Protein synthesis in mammalian cell mitochondria is initiated with fMet-tRNA Met f, the formyl group being most likely donated by N 10-formyltetrahy-drofolate. Methotrexate, which depletes the C 1-folate pools by inhibiting dihydrofolate reductase, might block the formylation reaction, inhibiting mitochondrial protein synthesis and indirectly cell growth. L cells grown in suspension culture are found to grow normally in the presence of 5 · 10 −5 M methotrexate when supplied with metabolites whose synthesis requires the C 1-folate pools. Preincubation for 15 min with 10 −4 M methotrexate does not affect the subsequent formylation of mitochondrial initiator tRNA. Also, there is no effect on the synthesis of N- formylmethionylpuromycin in L cells grown for 160 h in the presence of 5 · 10 −6 M methotrexate, nor in KB cells grown for 72 h in its presence. These results demonstrate that cell growth, the formylation of mitochondrial initiator tRNA and the initiation of mitochondrial protein synthesis are not inhibited by methotrexate when cells are supplied with metabolites whose de novo synthesis requires C 1 -folate pools. The mechanism of continued formylation in the presence of methotrexate is not known.

The interference of the macrolide antibiotics with mitochondrial protein synthesis

1. Protein synthesis by isolated BHK-21 mitochondria was shown to be inhibited by the macrolides erythromycin, tylosin tartrate, spiramycin and carbomycin. Intact mitochondria from rat liver were insensitive to erythromycin, but inhibited by tylosin tartrate, spiramycin and carbomycin. 2. Cytochrome c oxidase formation in BHK-21 cells was unaffected by tylosin tartrate and spiramycin, but strongly inhibited by carbomycin. Mitochondrial protein synthesis in whole BHK-21 cells showed the same insensitivity to tylosin tartrate and spiramycin. 3. Peptidyl transferase activity of isolated rat-liver mitochondrial ribosomes was inhibited by tylosin tartrate, spiramycin and carbomycin, whereas chloramphenicol inhibition was reversed by erythromycin. Rather high antibiotic concentrations (200 μg/ml or higher) were necessary to obtain these effects. For Neurospora crassa mitochondrial ribosomes a similar response to these macrolides was found. Therefore mitochondrial ribosomes from N. crassa are markedly less sensitive to macrolides than yeast mitochondrial ribosomes. 4. It is shown that mammalian mitochondrial protein synthesis is sensitive to erythromycin, tylosin tartrate, spiramycin and carbomycin, but that for the first three antibiotics membrane barriers exist: for erythromycin the mitochondrial membrane is the barrier and for tylosin tartrate and spiramycin the cell membrane. 5. Amino acid incorporation by isolated mitochondria appears much more sensitive to inhibition by tylosin tartrate, spiramycin and carbomycin than the isolated 55-S ribosomes. For carbomycin this can be explained in part by the finding that carbomycin was concentrated by intact rat-liver mitochondria in vitro.