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ABSTRACT: Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.
Biochimica et Biophysica Acta 12/2011; 1819(9-10):1035-54. · 4.66 Impact Factor
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ABSTRACT: Alterations in mitochondrial oxidative phosphorylation have long been documented in tumors. Other types of mitochondrial dysfunction, including altered reactive oxygen species (ROS) production and apoptosis, also can contribute to tumorigenesis and cancer phenotypes. Furthermore, mutation and altered amounts of mitochondrial DNA (mtDNA) have been observed in cancer cells. However, how mtDNA instability per se contributes to cancer remains largely undetermined. Mitochondrial transcription factor A (TFAM) is required for expression and maintenance of mtDNA. Tfam heterozygous knock-out (Tfam(+/-)) mice show mild mtDNA depletion, but have no overt phenotypes. We show that Tfam(+/-) mouse cells and tissues not only possess less mtDNA but also increased oxidative mtDNA damage. Crossing Tfam(+/-) mice to the adenomatous polyposis coli multiple intestinal neoplasia (APC(Min/+)) mouse cancer model revealed that mtDNA instability increases tumor number and growth in the small intestine. This was not a result of enhancement of Wnt/β-catenin signaling, but rather appears to involve a propensity for increased mitochondrial ROS production. Direct involvement of mitochondrial ROS in intestinal tumorigenesis was shown by crossing APC(Min/+) mice to those that have catalase targeted to mitochondria, which resulted in a significant reduction in tumorigenesis in the colon. Thus, mitochondrial genome instability and ROS enhance intestinal tumorigenesis and Tfam(+/-) mice are a relevant model to address the role of mtDNA instability in disease states in which mitochondrial dysfunction is implicated, such as cancer, neurodegeneration, and aging.
American Journal Of Pathology 11/2011; 180(1):24-31. · 4.89 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation.
Free radical biology & medicine 02/2011; 50(10):1234-41. · 5.42 Impact Factor
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ABSTRACT: Mammalian mitochondrial mRNAs are basically leaderless, having few or no untranslated nucleotides prior to the 5'-start codon. We demonstrate here that mammalian mitochondrial 55 S ribosomes preferentially form initiation complexes at a 5'-terminal AUG codon over an internal AUG. The preferential use of the 5'-start codon is also seen on mitochondrial 28 S small subunits, which suggests that mitochondrial translation initiation on leaderless mRNAs does not require the large ribosomal subunit. The selection of the 5'-AUG is dependent on the presence of fMet-tRNA and is enhanced by the presence of the mitochondrial initiation factor IF2(mt). In prokaryotes, IF3 is believed to antagonize initiation on leaderless mRNAs. However, IF3(mt) stimulates initiation complex formation on leaderless mRNAs when tested with 55 S ribosomes. The addition of even a few nucleotides 5' to the AUG codon significantly reduces the efficiency of initiation, highlighting the importance of the leaderless or nearly leaderless nature of mitochondrial mRNAs. In addition, very few initiation complexes could form on a hybrid mRNA construct consisting of tRNA(Met) attached at the 5'-end of a mitochondrial protein-coding sequence. This observation demonstrates that post-transcriptional processing must occur prior to translation in mammalian mitochondria.
Journal of Biological Chemistry 09/2010; 285(36):28379-86. · 4.77 Impact Factor
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ABSTRACT: Mammalian mitochondria synthesize a set of thirteen proteins that are essential for energy generation via oxidative phosphorylation. The genes for all of the factors required for synthesis of the mitochondrially encoded proteins are located in the nuclear genome. A number of disease-causing mutations have been identified in these genes. In this manuscript, we have elucidated the mechanisms of translational failure for two disease states characterized by lethal mutations in mitochondrial elongation factor Ts (EF-Ts(mt)) and elongation factor Tu (EF-Tu(mt)). EF-Tu(mt) delivers the aminoacyl-tRNA (aa-tRNA) to the ribosome during the elongation phase of protein synthesis. EF-Ts(mt) regenerates EF-Tu(mt):GTP from EF-Tu(mt):GDP. A mutation of EF-Ts(mt) (R325W) leads to a two-fold reduction in its ability to stimulate the activity of EF-Tu(mt) in poly(U)-directed polypeptide chain elongation. This loss of activity is caused by a significant reduction in the ability of EF-Ts(mt) R325W to bind EF-Tu(mt), leading to a defect in nucleotide exchange. A mutation of Arg336 to Gln in EF-Tu(mt) causes infantile encephalopathy caused by defects in mitochondrial translation. EF-Tu(mt) R336Q is as active as the wild-type protein in polymerization using Escherichia coli 70S ribosomes and E. coli [(14)C]Phe-tRNA but is inactive in polymerization with mitochondrial [(14)C]Phe-tRNA and mitochondrial 55S ribosomes. The R336Q mutation causes a two-fold decrease in ternary complex formation with E. coli aa-tRNA but completely inactivates EF-Tu(mt) for binding to mitochondrial aa-tRNA. Clearly the R336Q mutation in EF-Tu(mt) has a far more drastic effect on its interaction with mitochondrial aa-tRNAs than bacterial aa-tRNAs.
Biochimica et Biophysica Acta 05/2010; 1802(7-8):692-8. · 4.66 Impact Factor
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ABSTRACT: Two mammalian mitochondrial initiation factors have been identified. Initiation factor 2 (IF2(mt)) selects the initiator tRNA (fMet-tRNA) and promotes its binding to the ribosome. Initiation factor 3 (IF3(mt)) promotes the dissociation of the 55S mitochondrial ribosome into subunits and may play additional, less-well-understood, roles in initiation complex formation. Native bovine IF2(mt) was purified from liver a number of years ago. The yield of this factor is very low making biochemical studies difficult. The cDNA for bovine IF2(mt) was expressed in Escherichia coli under the control of the T7 polymerase promoter in a vector that provides a His(6)-tag at the C-terminus of the expressed protein. This factor was expressed in E. coli and purified by chromatography on Ni-NTA resins. The expressed protein has a number of degradation products in partially purified preparations and this factor is then further purified by high-performance liquid chromatography or gravity chromatography on anion exchange resins. IF3(mt) has never been purified from any mammalian system. However, the cDNA for this protein can be identified in the expressed sequence tag (EST) libraries. The portion of the sequence encoding the region of human IF3(mt) predicted to be present in the mitochondrially imported form of this factor was cloned and expressed in E. coli using a vector that provides a C-terminal His(6)-tag. The tagged factor is partially purified on Ni-NTA resins. However, a major proteolytic fragment arising from a defined cleavage of this protein is present in these preparations. This contaminant can be removed by a single step of high-performance liquid chromatography on a cation exchange resin. Alternatively, the mature form of IF3(mt) can be purified by two sequential passes through a gravity S-Sepharose column.
Methods in Enzymology 02/2007; 430:59-78. · 2.04 Impact Factor
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ABSTRACT: Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2mt) and mitochondrial initiation factor 3 (IF3mt). This observation indicates that IF2mt and IF3mt, while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3mt dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2mt. The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.
Biochemical and Biophysical Research Communications.
