scholarly journals Mitochondrial OXPHOS Biogenesis: Co-Regulation of Protein Synthesis, Import, and Assembly Pathways

2020 ◽  
Vol 21 (11) ◽  
pp. 3820 ◽  
Author(s):  
Jia Xin Tang ◽  
Kyle Thompson ◽  
Robert W. Taylor ◽  
Monika Oláhová
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Protein Import ◽  
Acyl Carrier Protein ◽  
Early Stage ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Fine Tuning ◽  
Mitochondrial Translation ◽  
Oxphos Complex

The assembly of mitochondrial oxidative phosphorylation (OXPHOS) complexes is an intricate process, which—given their dual-genetic control—requires tight co-regulation of two evolutionarily distinct gene expression machineries. Moreover, fine-tuning protein synthesis to the nascent assembly of OXPHOS complexes requires regulatory mechanisms such as translational plasticity and translational activators that can coordinate mitochondrial translation with the import of nuclear-encoded mitochondrial proteins. The intricacy of OXPHOS complex biogenesis is further evidenced by the requirement of many tightly orchestrated steps and ancillary factors. Early-stage ancillary chaperones have essential roles in coordinating OXPHOS assembly, whilst late-stage assembly factors—also known as the LYRM (leucine–tyrosine–arginine motif) proteins—together with the mitochondrial acyl carrier protein (ACP)—regulate the incorporation and activation of late-incorporating OXPHOS subunits and/or co-factors. In this review, we describe recent discoveries providing insights into the mechanisms required for optimal OXPHOS biogenesis, including the coordination of mitochondrial gene expression with the availability of nuclear-encoded factors entering via mitochondrial protein import systems.

scholarly journals Using mitoribosomal profiling to investigate human mitochondrial translation

2018 ◽  
Vol 2 ◽  
pp. 116
Author(s):  
Fei Gao ◽  
Maria Wesolowska ◽  
Reuven Agami ◽  
Koos Rooijers ◽  
Fabricio Loayza-Puch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Codon Position ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Base Pairs ◽  
Suboptimal Structure ◽  
Human Counterpart ◽  
Steady State Levels

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 mitospecific domain of Mrp7 (bL27) supports mitochondrial translation during fermentation and is required for effective adaptation to respiration

2021 ◽  
Author(s):  
Jessica M. Anderson ◽  
Jodie M. Box ◽  
Rosemary A. Stuart
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Growth Conditions ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Glucose Fermentation ◽  
Binding Partner ◽  
Oxphos Complex ◽  
Mitochondrial Signaling ◽  
Metabolic Conditions

We demonstrate here that mitoribosomal protein synthesis, responsible for the synthesis of oxidative phosphorylation (OXPHOS) subunits encoded by mitochondrial genome, occurs at high levels during glycolysis fermentation and in a manner uncoupled from OXPHOS complex assembly regulation. Furthermore, we provide evidence that the mitospecific domain of Mrp7 (bL27), a mitoribosomal component, is required to maintain mitochondrial protein synthesis during fermentation, but is not required under respiration growth conditions. Maintaining mitotranslation under high glucose fermentation conditions also involves Mam33 (p32/gC1qR homolog), a binding partner of Mrp7’s mitospecific domain, and together they confer a competitive advantage for a cell's ability to adapt to respiration-based metabolism when glucose becomes limiting. Furthermore, our findings support that the mitoribosome, and specifically the central protuberance (CP) region, may be differentially regulated and/or assembled, under the different metabolic conditions of fermentation and respiration. Based on our findings, we propose the purpose of mitotranslation is not limited to the assembly of OXPHOS complexes, but also plays a role in mitochondrial signaling critical for switching cellular metabolism from a glycolysis- to a respiratory-based state.

Isolation and incubation conditions to study heart mitochondrial protein synthesis

1990 ◽  
Vol 258 (3) ◽  
pp. E492-E502 ◽  
Author(s):  
E. E. McKee ◽  
B. L. Grier ◽  
G. S. Thompson ◽  
J. D. McCourt
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Adenine Nucleotide ◽  
Isolated Heart ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Specific Radioactivity ◽  
Companion Paper ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

scholarly journals Using mitoribosomal profiling to investigate human mitochondrial translation

2017 ◽  
Vol 2 ◽  
pp. 116 ◽  
Author(s):  
Fei Gao ◽  
Maria Wesolowska ◽  
Reuven Agami ◽  
Koos Rooijers ◽  
Fabricio Loayza-Puch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Codon Position ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Base Pairs ◽  
Suboptimal Structure ◽  
Human Counterpart ◽  
Steady State Levels

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.

scholarly journals Mitochondrial gene expression in Saccharomyces cerevisiae. Proteolysis of nascent chains in isolated yeast mitochondria optimized for protein synthesis

1991 ◽  
Vol 274 (1) ◽  
pp. 199-205 ◽  
Author(s):  
C L Black-Schaefer ◽  
J D McCourt ◽  
R O Poyton ◽  
E E McKee
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Gene Products ◽  
Yeast Mitochondria ◽  
Mitochondrial Translation ◽  
True Rate ◽  
Isolated Mitochondria ◽  
Enzyme Complexes

We demonstrate here that mitochondrial translation products synthesized by isolated yeast mitochondria are subject to rapid proteolysis. The loss of label from mitochondrial peptides synthesized in vitro comes from two distinct pools of peptides: one that is rapidly degraded (t1/2 of minutes) and one that is much more resistant to proteolysis (t1/2 of hours). As the length of the incubation period increases, the percentage of labelled peptides in the rapidly-turning-over pool decreases and cannot be detected after 60 min of incubation. This proteolysis is inhibited by chloramphenicol and is dependent on the presence of ATP. The loss of label during the chase occurs from fully completed translation products. The proteolysis observed here markedly affects measurements of rates of mitochondrial protein synthesis in isolated yeast mitochondria. In earlier work, in which proteolysis was not considered, mitochondrial translation was thought to stop after 20-30 min of incubation. In the present study, by taking proteolysis into account, we demonstrate that the rate of translation in isolated mitochondria is actually constant for nearly 60 min and then decreases to near zero by 80 min of incorporation. These findings have allowed us to devise a procedure for measuring the ‘true’ rate of translation in isolated mitochondria. In addition, they suggest that mitochondrial translation products which normally assemble with nuclear-encoded gene products into multimeric enzyme complexes are unstable without their nuclear-encoded counterparts.

Coupling of mitochondrial metabolism and protein synthesis in heart mitochondria

1990 ◽  
Vol 258 (3) ◽  
pp. E503-E510 ◽  
Author(s):  
E. E. McKee ◽  
B. L. Grier ◽  
G. S. Thompson ◽  
A. C. Leung ◽  
J. D. McCourt
Keyword(s):  
Protein Synthesis ◽  
Adenine Nucleotide ◽  
Isolated Heart ◽  
Mitochondrial Gene ◽  
Energy Charge ◽  
Mitochondrial Protein ◽  
Mitochondrial Metabolism ◽  
Heart Mitochondria ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. In this paper the potential coupling of mitochondrial metabolism to mitochondrial protein synthesis was investigated and the concentration of nucleotides and substrates for optimal translation in isolated rat heart mitochondria was determined. It was demonstrated that optimal isolated heart mitochondrial protein synthesis required the presence of an oxidizable substrate. Of the substrates tested, glutamate (20 mM) supported translation best followed by malate, succinate, and alpha-ketoglutarate, whereas pyruvate supported synthesis poorly. Unlike other recent mammalian mitochondrial systems, the presence of an oxidizable substrate was required for translation even in the presence of medium ATP and an exogenous energy-generating system. Mitochondrial translation also required the presence of adenine nucleotide that could be added as ADP or ATP; however, ATP added above 0.5 mM became progressively inhibitory. As a result, synthesis was supported significantly better by ATP synthesized by the system from added ADP, than by ATP added directly to the system. However, if the phosphorylation of ADP was prevented by limiting the phosphate concentration, ADP itself strongly inhibited mitochondrial protein synthesis. This inhibition appeared to be closely related to the energy charge of the system rather than to absolute levels of ADP, indicating for the first time that mitochondrial translation, like its cytoplasmic counterpart is regulated by energy charge. Last, this system did not require the inhibition of guanine nucleotide or exogenous energy-generating systems.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 674
Author(s):  
Francesco Capriglia ◽  
Francesca Rizzo ◽  
Giuseppe Petrosillo ◽  
Veronica Morea ◽  
Giulia d’Amati ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Molecular Mechanisms ◽  
Mitochondrial Protein ◽  
Trna Synthetase ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Mitochondrial Functions ◽  
Carboxy Terminal

The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

scholarly journals Mitogenome Analysis of Four Lamiinae Species (Coleoptera: Cerambycidae) and Gene Expression Responses by Monochamus alternatus When Infected with the Parasitic Nematode, Bursaphelenchus mucronatus

Insects ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 453
Author(s):  
Zi-Yi Zhang ◽  
Jia-Yin Guan ◽  
Yu-Rou Cao ◽  
Xin-Yi Dai ◽  
Kenneth B. Storey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phylogenetic Trees ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Monochamus Alternatus ◽  
Genome Database ◽  
Protein Coding ◽  
Nd5 Gene

We determined the mitochondrial gene sequence of Monochamus alternatus and three other mitogenomes of Lamiinae (Insect: Coleoptera: Cerambycidae) belonging to three genera (Aulaconotus, Apriona and Paraglenea) to enrich the mitochondrial genome database of Lamiinae and further explore the phylogenetic relationships within the subfamily. Phylogenetic trees of the Lamiinae were built using the Bayesian inference (BI) and maximum likelihood (ML) methods and the monophyly of Monochamus, Anoplophora, and Batocera genera was supported. Anoplophora chinensis, An. glabripennis and Aristobia reticulator were closely related, suggesting they may also be potential vectors for the transmission of the pine wood pathogenic nematode (Bursaphelenchus xylophilus) in addition to M. alternatus, a well-known vector of pine wilt disease. There is a special symbiotic relationship between M. alternatus and Bursaphelenchus xylophilus. As the native sympatric sibling species of B. xylophilus, B. mucronatus also has a specific relationship that is often overlooked. The analysis of mitochondrial gene expression aimed to explore the effect of B. mucronatus on the energy metabolism of the respiratory chain of M. alternatus adults. Using RT-qPCR, we determined and analyzed the expression of eight mitochondrial protein-coding genes (COI, COII, COIII, ND1, ND4, ND5, ATP6, and Cty b) between M. alternatus infected by B. mucronatus and M. alternatus without the nematode. Expression of all the eight mitochondrial genes were up-regulated, particularly the ND4 and ND5 gene, which were up-regulated by 4–5-fold (p < 0.01). Since longicorn beetles have immune responses to nematodes, we believe that their relationship should not be viewed as symbiotic, but classed as parasitic.

A genetic link between an mRNA-specific translational activator and the translation system in yeast mitochondria.

Genetics ◽  
1990 ◽  
Vol 125 (3) ◽  
pp. 495-503 ◽  
Author(s):  
P Haffter ◽  
T W McMullin ◽  
T D Fox
Keyword(s):  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Open Reading Frame ◽  
Null Alleles ◽  
Translation System ◽  
Amino Acid Residues ◽  
Mitochondrial Translation ◽  
Reading Frame ◽  
Large Deletions ◽  
Translational Activator

Abstract Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the products of at least three nuclear genes, PET122, PET494 and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of the gene product were found to be suppressed by unlinked nuclear mutations. These unlinked suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise wild-type strain and reduces translation of all mitochondrial gene products in cells grown at high temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123. The sequence of a DNA fragment carrying PET123 contains one major open reading frame encoding a basic protein of 318 amino acids. Inactivation of the chromosomal copy of PET123 by interruption of this open reading frame causes cells to become rho- (sustain large deletions in their mtDNA). This phenotype is characteristic for null alleles of genes whose products are essential for general mitochondrial protein synthesis. Thus our data strongly suggest that the PET123 protein is a component of the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activator PET122.

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