scholarly journals Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

2022 ◽  
Author(s):  
Yusuke Kimura ◽  
Hironori Saito ◽  
Tatsuya Osaki ◽  
Yasuhiro Ikegami ◽  
Taisei Wakigawa ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Click Reaction ◽  
Mitochondrial Protein ◽  
Cell Types ◽  
Growth Conditions ◽  
Cellular Heterogeneity ◽  
Metabolic Labeling ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

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.

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 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.

Insulin stimulates mitochondrial protein synthesis and respiration in isolated perfused rat heart.

1990 ◽  
Vol 259 (3) ◽  
pp. E413
Author(s):  
E E McKee ◽  
B L Grier
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Control Level ◽  
Respiratory Control ◽  
Mitochondrial Proteins ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Perfused Hearts

The rates of synthesis of mitochondrial proteins by both the cytoplasmic and mitochondrial protein synthetic systems, as well as parameters of respiration, were measured and compared in mitochondria isolated from fresh, control perfused, and insulin-perfused rat hearts. The respiratory control ratio (RCR) in mitochondria from fresh hearts was 8.1 +/- 0.4 and decreased to 6.0 +/- 0.2 (P less than 0.001 vs. fresh) in mitochondria from control perfused hearts and to 6.7 +/- 0.2 (P less than 0.005 vs. fresh and P less than 0.02 vs. control perfused) for mitochondria from hearts perfused in the presence of insulin. A positive correlation between the RCR and the rate of mitochondrial translation was demonstrated in mitochondria from fresh hearts. In mitochondria isolated from control perfused hearts, the rate of protein synthesis decreased to 84 +/- 3% of the fresh rate after 30 min of perfusion and fell further to 64 +/- 3% after 3 h of perfusion. The inclusion of insulin in the perfusion buffer stimulated mitochondrial protein synthesis 1.2-fold by 1 h (P less than 0.005) and 1.34-fold by 3 h of perfusion (P less than 0.001). The addition of insulin to 1-h control perfused hearts shifted the rate of mitochondrial protein synthesis from the control level to the insulin-perfused level within 30 min of additional perfusion, whereas 1 h was required to shift the RCR values of these mitochondria from control levels to insulin-perfused levels. Thus, whereas RCR was a useful predictor of mitochondrial translation rates, it did not account for the effects of insulin on mitochondrial translation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Fidelity of translation initiation is required for coordinated respiratory complex assembly

2019 ◽  
Vol 5 (12) ◽  
pp. eaay2118 ◽  
Author(s):  
Danielle L. Rudler ◽  
Laetitia A. Hughes ◽  
Kara L. Perks ◽  
Tara R. Richman ◽  
Irina Kuznetsova ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mitochondrial Protein ◽  
Molecular Machines ◽  
Ribosome Profiling ◽  
Untranslated Regions ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Initiation Factors ◽  
Mitochondrial Ribosomes

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle–specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.

scholarly journals Differences in the products of mitochondrial protein synthesis in vivo in human and mouse cells and their potential use as markers for the mitochondrial genome in human–mouse cell hybrids

1974 ◽  
Vol 144 (1) ◽  
pp. 161-164 ◽  
Author(s):  
Alec Jeffreys ◽  
Ian Craig
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Detailed Comparison ◽  
Cell Types ◽  
Mouse Cell ◽  
Human Cells ◽  
Mitochondrial Protein Synthesis ◽  
Cell Hybrids ◽  
Different Cell Types

The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [35S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human–mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.

Glutamyl-tRNAGln amidotransferase is essential for mammalian mitochondrial translation in vivo

2014 ◽  
Vol 460 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Lucía Echevarría ◽  
Paula Clemente ◽  
Rosana Hernández-Sierra ◽  
María Esther Gallardo ◽  
Miguel A. Fernández-Moreno ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Synthesis ◽  
Indirect Pathway ◽  
Mitochondrial Translation

We have demonstrated that in mitochondria of mammalian cells the aminoacylation of tRNAGln is produced by an indirect pathway involving the enzyme glutamyl-tRNAGln amidotransferase. Misaminoacylated Glu-tRNAGln is rejected from the ribosomes maintaining the fidelity of the mitochondrial protein synthesis.

scholarly journals The lability of the products of mitochondrial protein synthesis in Saccharomyces cerevisiae. A novel method for protein half-life determination

1978 ◽  
Vol 170 (3) ◽  
pp. 569-576 ◽  
Author(s):  
G Y Bakalkin ◽  
S L Kalnov ◽  
A V Galkin ◽  
A S Zubatov ◽  
V N Luzikov
Keyword(s):  
Protein Synthesis ◽  
Half Life ◽  
Mitochondrial Protein ◽  
Double Labelling ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Novel Method ◽  
The Difference ◽  
First Time

A method for the determination of the half-life of mitochondrial translation products in yeast in vivo is proposed. The method uses inhibitors of cytoplasmic and mitochondrial protein synthesis and is based on double-labelling pulse-chase techniques, the second label being used to estimate ‘post-incorporation’ during the ‘chase’. For the first time the difference between post-incroporation and the widely known recycling of the label is considered. These studies show that, in the turnover of mitochondrial translation products, the problem is of post-incorporation into mitochondria (especially from the cell sap) is predominant. The results obtained with this procedure indicate that the half-life of the products of mitochondrial protein synthesis in yeast at the late-exponential phase is about 60 min. The results suggest that mitochondrial transplantation products are subject to proteolysis to acid-soluble forms.

scholarly journals Mitochondrial translation occurs preferentially in the peri-nuclear mitochondrial network of cultured human cells.

2021 ◽  
Author(s):  
Christin Albus ◽  
Rolando Berlinguer-Palmini ◽  
Caroline Hewison ◽  
Fiona McFarlane ◽  
Elisabeta-Ana Savu ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mitochondrial Protein ◽  
Cell Types ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Network ◽  
Mitochondrial Translation ◽  
Mitochondrial Rrna ◽  
Mrna Transcripts ◽  
Cultured Human Cells ◽  
Substantial Heterogeneity

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

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