Journal: Cellular and Molecular Life Sciences
Article Title: GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation
doi: 10.1007/s00018-023-05014-0
Figure Lengend Snippet: Defects in mitoribosomal assembly and mitochondrial translation caused by GTPBP8 depletion can be rescued by GTPBP8 expression. A The representative mitoribosome profiles in GTPBP8-depleted and GTPBP8-myc-expressed cells. The mt-SSU, mt-LSU, and monosome fractions are highlighted by orange, green, and purple, respectively. B Quantified gradient distribution of indicated mitoribosomal proteins upon GTPBP8-silencing and GTPBP8-myc-expression. Lines represent the mean of three independent experiments. The mt-SSU, mt-LSU, and monosome fractions are highlighted by orange, green, and purple, respectively. C Rescue of the indicated protein levels in the mt-LSU (fractions 9 and 10), mt-SSU (fractions 6 and 7) and monosome fractions (fractions 12 and 13). Quantification of mitoribosomal protein abundance was shown on the right. D Rescue of mitochondrial translation defect caused by GTPBP8 depletion. Top: Metabolic labeling of mitochondrial translation products with [ 35 S]-methionine/cysteine for 30 min in control, GTPBP8-depleted or rescued cells. Bottom: immunoblotting for the steady state level of GTPBP8. E Quantification of 35 S intensity for mitochondrially translated proteins relative to the corresponding intensity in Coomassie staining. Error bars represent the mean ± SD of three independent repetitions. *P ≤ 0.05, * *P ≤ 0.01, and ** *P ≤ 0.001, Student’s t-test . F Steady state levels of MT-CO1 and mitoribosomal proteins in control, GTPBP8 depleted, and GTPBP8-myc rescued cells. TOM40 and GAPDH were used as loading controls. G Quantification of protein levels in F . *P ≤ 0.05, * *P ≤ 0.01, and ** *P ≤ 0.001, Student’s t -test. H Rescue of the oxygen consumption rate (OCR) by expressing GTPBP8-myc in GTPBP8-depleted cells, measured using a Seahorse XF96 e Analyzer. The OCR was determined by adding mitochondrial function inhibitors sequentially at the indicated times. The OCR was normalized to the total protein amount used for the assay. I Quantification of the basal, ATP linked and maximal respiration capacities from the OCR measurement in H . J Rescue of cellular ATP level by expressing GTPBP8-myc in GTPBP8- depleted cells. Error bars represent the mean ± SD of three independent experiments. *P ≤ 0.05, * *P ≤ 0.01 and ** *P ≤ 0.001, Student’s t -test
Article Snippet: The following primary antibodies were used: mouse anti-ATP5A (1:1000, 14748, Abcam), rabbit anti-GFP (1:4000, 50430-2-AP, Proteintech), mouse anti-MT-CO1 (1:2000, 14705, Abcam), rabbit anti-COXIV (1:1000, MA5-15078, ThermoFisher Scientific), rabbit anti-CytB (1:3000, 55090-1-AP, Proteintech), mouse anti-CytC (1:10,000, 05-479, Millipore), goat anti-GAPDH (1:1000, sc-20357, Santa Cruz Biotechnology), rabbit anti-GTPBP5 (1:2000, 20133-1-AP, Proteintech), rabbit anti-GTPBP7 (1:4000, 13742-1-AP, Proteintech), rabbit anti-GTPBP8 (1:500, HPA034831, Sigma), rabbit anti-GTPBP10 (1:500, NBP1-85055, Novus Biologicals), mouse anti-HSP60 (1:20,000, SMC-110, StressMarq Biosciences), rabbit anti-uL1m[MRPL1] (1:2000, 16254-1-AP, Proteintech), rabbit anti-uL2m[MRPL2] (1:2000, 16492-1-AP, Proteintech), rabbit anti-uL11m[MRPL11] (1:6000, 15543-1-AP, Proteintech), rabbit anti-bL12m[MRPL12] (1:4000, 14795-1-AP, Proteintech), rabbit anti-uL16m[MRPL16] (1:500, HPA054133, Sigma), rabbit anti-bL21m[MRPL21] (1:3000, PA5-31939, ThermoFisher Scientific), rabbit anti-uL24m[MRPL24] (1:2000, 16224-1-AP, Proteintech), rabbit anti-bL27m[MRPL27] (1:1000, 14765-1-AP, Proteintech), rabbit anti-mS27[MRPS27] (1:1000, 17280-1-AP, Proteintech), rabbit anti-mL65[MRPS30] (1:1000, 18441-1-AP, Proteintech), rabbit anti-mS35[MRPS35] (1:10,000, 16457-1-AP, Proteintech), rabbit anti-bL36m[MRPL36] (1:1000, ab126517, Abcam), rabbit anti-mL40[MRPL40] (1:1000, HPA006181, Sigma), rabbit anti-mL45[MRPL45] (1:4000, 15682-1-AP, Proteintech), rabbit anti-mL46[MRPL46] (1:2000, 16611-1-AP, Proteintech), rabbit anti-bL31m[MRPL55] (1:1000, 17679-1-AP, Proteintech), rabbit anti-mL64[MRPL59] (1:500, 16260-1-AP, Proteintech), mouse anti-myc (1:1000, sc-40, Santa Cruz Biotechnology), mouse anti-NDUFA9 (1:1000, 20312-1-AP, Proteintech), mouse anti-TOM20 (1:1000, sc-17764, Santa Cruz Biotechnology), rabbit anti-TOM40 (1:1000, 18409-1-AP, Proteintech), rabbit anti-TRMT61B (1:500, 26009-1-AP, Proteintech), mouse anti-SDHA (1:1000, 14715, Abcam).
Techniques: Expressing, Labeling, Western Blot, Staining
![Loss of GTPBP8 impairs mitochondrial translation. A Steady state levels of oxidative phosphorylation complex proteins encoded by mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) after depletion of GTPBP8. The right panel shows quantification of the relative MT-CO1 and MT-CytB protein abundance normalized to TOM40. B The mRNA expression levels of MT-CO1 and MT-CytB normalized to 18S rRNA. C Steady-state levels of mtDNA content detected by quantitative PCR. The total mtDNA levels were normalized to β-globin. D Metabolic labeling of mitochondrial translation products with [ 35 S]-methionine/cysteine for the indicated time in U2OS cells. E The heatmap depicts the quantified 35 S intensity in D for mitochondrially translated proteins relative to the corresponding intensity in Coomassie staining. The color bar represents the normalized 35 S intensity value. F Quantification of 35 S intensity for mitochondrially translated 13 proteins after depletion of GTPBP8. The values were normalized to the corresponding intensity in Coomassie staining. Error bars represent the mean ± SD of three independent experiments. ** P ≤ 0.01 and *** P ≤ 0.001, Student’s t -test. Metabolic labeling of mitochondrial translation products with [ 35 S]-methionine/cysteine was performed for 30 min in U2OS cells after depletion of GTPBP8. The cells were treated with control and GTPBP8 siRNA for 5 days. G Metabolic labeling of mitochondrially translated proteins with [ 35 S]-methionine/cysteine for 30 min with or without 6 h of chasing in U2OS cells. In A – C , error bars represent the mean ± SD of three independent experiments. Student’s t -test. H The heatmap depicts the quantified 35 S intensity in panel G for mitochondrially translated proteins relative to the corresponding intensity in Coomassie staining. The color bar represents the normalized 35 S intensity value](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4211/pmc10654211/pmc10654211__18_2023_5014_Fig3_HTML.jpg)
![Defects in mitoribosomal assembly and mitochondrial translation caused by GTPBP8 depletion can be rescued by GTPBP8 expression. A The representative mitoribosome profiles in GTPBP8-depleted and GTPBP8-myc-expressed cells. The mt-SSU, mt-LSU, and monosome fractions are highlighted by orange, green, and purple, respectively. B Quantified gradient distribution of indicated mitoribosomal proteins upon GTPBP8-silencing and GTPBP8-myc-expression. Lines represent the mean of three independent experiments. The mt-SSU, mt-LSU, and monosome fractions are highlighted by orange, green, and purple, respectively. C Rescue of the indicated protein levels in the mt-LSU (fractions 9 and 10), mt-SSU (fractions 6 and 7) and monosome fractions (fractions 12 and 13). Quantification of mitoribosomal protein abundance was shown on the right. D Rescue of mitochondrial translation defect caused by GTPBP8 depletion. Top: Metabolic labeling of mitochondrial translation products with [ 35 S]-methionine/cysteine for 30 min in control, GTPBP8-depleted or rescued cells. Bottom: immunoblotting for the steady state level of GTPBP8. E Quantification of 35 S intensity for mitochondrially translated proteins relative to the corresponding intensity in Coomassie staining. Error bars represent the mean ± SD of three independent repetitions. *P ≤ 0.05, * *P ≤ 0.01, and ** *P ≤ 0.001, Student’s t-test . F Steady state levels of MT-CO1 and mitoribosomal proteins in control, GTPBP8 depleted, and GTPBP8-myc rescued cells. TOM40 and GAPDH were used as loading controls. G Quantification of protein levels in F . *P ≤ 0.05, * *P ≤ 0.01, and ** *P ≤ 0.001, Student’s t -test. H Rescue of the oxygen consumption rate (OCR) by expressing GTPBP8-myc in GTPBP8-depleted cells, measured using a Seahorse XF96 e Analyzer. The OCR was determined by adding mitochondrial function inhibitors sequentially at the indicated times. The OCR was normalized to the total protein amount used for the assay. I Quantification of the basal, ATP linked and maximal respiration capacities from the OCR measurement in H . J Rescue of cellular ATP level by expressing GTPBP8-myc in GTPBP8- depleted cells. Error bars represent the mean ± SD of three independent experiments. *P ≤ 0.05, * *P ≤ 0.01 and ** *P ≤ 0.001, Student’s t -test](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4211/pmc10654211/pmc10654211__18_2023_5014_Fig7_HTML.jpg)