human embryonic kidney cells  (ATCC)

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: (A ) Schematic of the mitochondrial oxidative phosphorylation system, including the four enzymes comprising the electron transport chain (complexes I to IV) and the F1Fo-ATP synthase. The ADP/ATP translocator ANT and the phosphate carrier PiC are also depicted. The red lines indicate the site of action of specific inhibitors AA, antimycin A; ATR, atractyloside; BA, bongkrekic acid; Citreo, citreoviridin; KCN, potassium cyanide; Oligo, oligomycin; Rot, rotenone. ( B ) Effect of treatments for 80 min (50 min before the addition of 35S-methionine and through the 30 min pulse time) with the indicated OxPhos inhibitors on the incorporation of 35S-methionine into de novo synthesized mitochondrial polypeptides in whole HEK293T cells. ( C ) To follow the time-course effect of oligomycin on mitochondrial protein synthesis, cells were incubated with 2 μM oligomycin for 50 or 10 min before the addition of 35S-methionine and through the 30 min pulse time, or 10 min into the pulse and only through the last 20 min of labelling (see timeline scheme). In (B) and (C), cells were incubated in the presence of emetine to inhibit cytoplasmic protein synthesis. The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and exposed to X-ray film, after which the signal was developed by autoradiography. Polypeptides synthesized by mitochondrial ribosomes are indicated on the left side. Immunoblotting against ACTIN was used as a loading control. The graphs represent the quantification of protein synthesis (PS) signal by densitometry, normalized to ACTIN, across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3), One-way Anova with Dunnett multiple comparisons. ****: p<0.0001. ( D ) Time-course and dose-response assays on the inhibitory effect of oligomycin on ATPase activity. ( E-F ) Mitochondrial membrane potential (DYm) in HEK293T WT cells, treated or not with indicated OxPhos inhibitors. The assay measures the accumulation of tetramethylrhodamine methyl ester (TMRM) into mitochondria using flow cytometry. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 4-5)., One-way ANOVA with Dunnett multiple comparisons. ****: p<0.0001; ***: p<0.001, **: p<0.01, *: p<0.05.

Article Snippet: Human HEK293T embryonic kidney cells (CRL-3216), osteosarcoma 143B (CRL-8303), glioblastoma U-87 MG (HTB-14 ) , and neonatal fibroblasts (CCD-1064Sk) were obtained from the American Type Culture Collection (ATCC).

Techniques: Phospho-proteomics, Synthesized, Incubation, SDS Page, Membrane, Autoradiography, Western Blot, Control, Activity Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: ( A ) Effect of the indicated OxPhos inhibitors on ATPase activity. ( B ) Mitochondrial membrane potential (ΔΨm) in HEK293T WT cells, treated or not with indicated OxPhos inhibitors. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. ( C and E ) Effect of treatments for 80 min (50 min before the addition of 35 S-methionine and through the 30 min pulse time) with the indicated compounds on the incorporation of 35 S-methionine into de novo synthesized mitochondrial polypeptides in whole HEK293T cells, in the presence of emetine to inhibit cytoplasmic protein synthesis. Polypeptides synthesized by mitochondrial ribosomes are indicated on the left side. Immunoblotting against ACTIN was used as a loading control. The graphs represent the quantification by densitometry of the protein synthesis (PS) signal normalized by ACTIN across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3). One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, ****: p<0.0001. ( D ) Mitochondrial ATP levels assessed by the ATP bioluminescence assay (free Mg 2+ -ATP) or LC/MS (total ATP) in mitochondria isolated from HEK293T WT cells treated with the indicated OxPhos inhibitors. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3-9), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Article Snippet: Human HEK293T embryonic kidney cells (CRL-3216), osteosarcoma 143B (CRL-8303), glioblastoma U-87 MG (HTB-14 ) , and neonatal fibroblasts (CCD-1064Sk) were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Membrane, Synthesized, Western Blot, Control, ATP Bioluminescent Assay, Liquid Chromatography with Mass Spectroscopy, Isolation

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: ( A-B ) Metagene plots of ribosome-protected fragments (RPFs) mapped to the mitochondrial genome in HEK293T cells treated with DMSO (upper panels, blue tracks) or oligomycin (lower panels, red tracks). Read densities are expressed as reads per million (RPM), scaled to codon positions. Gene positions are shown below the plots, with darker shading marking the first of overlapping open reading frames. ORFs encoded on the heavy (sense) and light (anti-sense) strands are indicated by the direction of the boxed arrows; the single light-strand ORF ( ND6 ) is shown in dark grey. (A) RPFs of 28-35 nt, and (B) RPFs of 20-24 nt are displayed. ( C ) Codon-specific ribosome occupancy profiles under DMSO and oligomycin conditions. Relative enrichment is plotted for each codon (color-coded by amino acid).

Article Snippet: Human HEK293T embryonic kidney cells (CRL-3216), osteosarcoma 143B (CRL-8303), glioblastoma U-87 MG (HTB-14 ) , and neonatal fibroblasts (CCD-1064Sk) were obtained from the American Type Culture Collection (ATCC).

Techniques:

Journal: bioRxiv

Article Title: Matrix nucleotide homeostasis couples energetic state to mitochondrial translation

doi: 10.64898/2026.06.04.730174

Figure Lengend Snippet: ( A ) Total mitochondrial GTP levels assessed by LC/MS in mitochondria isolated from HEK293T WT cells, expressing or not Saccharomyces cerevisiae GGC1, treated for 80 min with the indicated OxPhos inhibitors or pre-treated with ATR for 16h. This analysis was performed in parallel to the ATP measurements presented in . Dots represent individual values, and the columns are the mean ± SD (error bars, n = 3-9), One-way ANOVA with Dunnett multiple comparisons. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. ( B ) Schematic illustrating the strategy to selectively increase matrix GTP independently of adenine nucleotide exchange. HEK293T cells expressing empty vector, wild-type yeast mitochondrial GTP/GDP carrier (Ggc1), or an empty vector (EV) were treated with oligomycin (Oligo), oligomycin plus FCCP, or oligomycin + FCCP + bongkrekic acid (BA). FCCP was used to normalize membrane potential (ΔΨm), and BA to inhibit ANT-mediated adenine nucleotide exchange. Mitochondrial translation was assessed by 35 S-methionine incorporation in the presence of emetine. ( C ) Representative autoradiograph of 35 S-methionine incorporation into de novo synthesized mitochondrial polypeptides in HEK293T cells expressing vector or Ggc1, treated as indicated. Cytosolic translation was inhibited with emetine. ACTIN immunoblot serves as loading control. The graph shows the densitometric analysis of total 35 S-methionine incorporation normalized to ACTIN from n = X independent experiments. Data are mean ± SD. One-way ANOVA with Tukey (or Dunnett) multiple comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001. ( D ) Quantification by densitometry of the protein synthesis (PS) signal in panel (C), normalized by ACTIN across three independent experiments. Dots represent individual values, and the columns are the mean ± SD (error bars, n = 2-3). One-way ANOVA with Dunnett multiple comparisons. ****: p<0.0001. ( E ) Model of mitochondrial translational control by matrix nucleotide phosphorylation potential. Inhibition of the F₁F₀-ATP synthase by oligomycin blocks proton re-entry into the mitochondrial matrix, causing membrane hyperpolarization (ΔΨ↑). The elevated ΔΨm impairs adenine nucleotide exchange across the inner mitochondrial membrane, limiting ATP import and retention in the matrix. Reduced matrix ATP compromises nucleoside diphosphate kinase (NDPK)-mediated regeneration of GTP from GDP, leading to depletion of the mitochondrial GTP pool. Because mitochondrial translation initiation and elongation require GTP-dependent factors (mtIF2, mtEF-Tu, mtEF-G), reduced GTP availability is expected to limit mitochondrial translation by restricting the activity of GTP-dependent translation factors.

Article Snippet: Human HEK293T embryonic kidney cells (CRL-3216), osteosarcoma 143B (CRL-8303), glioblastoma U-87 MG (HTB-14 ) , and neonatal fibroblasts (CCD-1064Sk) were obtained from the American Type Culture Collection (ATCC).

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Expressing, Plasmid Preparation, Membrane, Autoradiography, Synthesized, Western Blot, Control, Phospho-proteomics, Inhibition, Activity Assay