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A Role for the Mitochondrial Protein Mrpl44 in Maintaining OXPHOS Capacity.

Yeo JH, Skinner JP, Bird MJ, Formosa LE, Zhang JG, Kluck RM, Belz GT, Chong MM - PLoS ONE (2015)

Bottom Line: This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts.We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome.These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia; St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.

ABSTRACT
We identified Mrpl44 in a search for mammalian proteins that contain RNase III domains. This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts. However, the precise Mrpl44 localization had been unclear. Here, we show by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localized to the matrix of the mitochondria. We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome. By manipulating its expression, we show that Mrpl44 may be important for regulating the expression of mtDNA-encoded genes. This was at the level of RNA expression and protein translation. This ultimately impacted ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

No MeSH data available.


ATP synthesis capacity and bioenergetics are compromised by Mrpl44 knockdown.(A) Rates of ATP synthesis were measured in NIH3T3 cells knocked down (ORF) for Mrpl44 or expressing a control shRNA, in the presence of specific substrate inhibitor combinations. Rates are expressed relative to the control Complex II-dependent rate (succinate + rotenone) on the day. The mean +/- SEM from six independent experiments is shown. (B) Oxygen consumption rates (OCR) measured by Seahorse XF24/3 extracellular flux analysis. The values were normalized to cell number, determined by CyQUANT. Four measurements were taken at each stage of the assay. (C) Calculation of maximum respiratory rate (MRR), spare respiratory capacity (SRC), extracellular acidification rate (ECAR) and OCR/ECAR ratio from the Seahorse analysis. The mean +/- SEM of 3 independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005).
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pone.0134326.g005: ATP synthesis capacity and bioenergetics are compromised by Mrpl44 knockdown.(A) Rates of ATP synthesis were measured in NIH3T3 cells knocked down (ORF) for Mrpl44 or expressing a control shRNA, in the presence of specific substrate inhibitor combinations. Rates are expressed relative to the control Complex II-dependent rate (succinate + rotenone) on the day. The mean +/- SEM from six independent experiments is shown. (B) Oxygen consumption rates (OCR) measured by Seahorse XF24/3 extracellular flux analysis. The values were normalized to cell number, determined by CyQUANT. Four measurements were taken at each stage of the assay. (C) Calculation of maximum respiratory rate (MRR), spare respiratory capacity (SRC), extracellular acidification rate (ECAR) and OCR/ECAR ratio from the Seahorse analysis. The mean +/- SEM of 3 independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005).

Mentions: Knockdown of Mrpl44 resulted in a reduction in Complex I- and Complex II-dependent rates of ATP synthesis (Fig 5A). This correlates with the reduced assembly of the OXPHOS Complexes I, III, IV and V. For both Mrpl44 KD and control cells, the Complex I and Complex II-dependent ATP synthesis were abolished by the appropriate inhibitors (Fig 5A), confirming that measurement of ATP synthesis was “substrate-specific”. ATP synthesis rates were unchanged with Mrpl44 overexpression (S7 Fig).


A Role for the Mitochondrial Protein Mrpl44 in Maintaining OXPHOS Capacity.

Yeo JH, Skinner JP, Bird MJ, Formosa LE, Zhang JG, Kluck RM, Belz GT, Chong MM - PLoS ONE (2015)

ATP synthesis capacity and bioenergetics are compromised by Mrpl44 knockdown.(A) Rates of ATP synthesis were measured in NIH3T3 cells knocked down (ORF) for Mrpl44 or expressing a control shRNA, in the presence of specific substrate inhibitor combinations. Rates are expressed relative to the control Complex II-dependent rate (succinate + rotenone) on the day. The mean +/- SEM from six independent experiments is shown. (B) Oxygen consumption rates (OCR) measured by Seahorse XF24/3 extracellular flux analysis. The values were normalized to cell number, determined by CyQUANT. Four measurements were taken at each stage of the assay. (C) Calculation of maximum respiratory rate (MRR), spare respiratory capacity (SRC), extracellular acidification rate (ECAR) and OCR/ECAR ratio from the Seahorse analysis. The mean +/- SEM of 3 independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519308&req=5

pone.0134326.g005: ATP synthesis capacity and bioenergetics are compromised by Mrpl44 knockdown.(A) Rates of ATP synthesis were measured in NIH3T3 cells knocked down (ORF) for Mrpl44 or expressing a control shRNA, in the presence of specific substrate inhibitor combinations. Rates are expressed relative to the control Complex II-dependent rate (succinate + rotenone) on the day. The mean +/- SEM from six independent experiments is shown. (B) Oxygen consumption rates (OCR) measured by Seahorse XF24/3 extracellular flux analysis. The values were normalized to cell number, determined by CyQUANT. Four measurements were taken at each stage of the assay. (C) Calculation of maximum respiratory rate (MRR), spare respiratory capacity (SRC), extracellular acidification rate (ECAR) and OCR/ECAR ratio from the Seahorse analysis. The mean +/- SEM of 3 independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (**p<0.005).
Mentions: Knockdown of Mrpl44 resulted in a reduction in Complex I- and Complex II-dependent rates of ATP synthesis (Fig 5A). This correlates with the reduced assembly of the OXPHOS Complexes I, III, IV and V. For both Mrpl44 KD and control cells, the Complex I and Complex II-dependent ATP synthesis were abolished by the appropriate inhibitors (Fig 5A), confirming that measurement of ATP synthesis was “substrate-specific”. ATP synthesis rates were unchanged with Mrpl44 overexpression (S7 Fig).

Bottom Line: This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts.We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome.These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia; St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia.

ABSTRACT
We identified Mrpl44 in a search for mammalian proteins that contain RNase III domains. This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts. However, the precise Mrpl44 localization had been unclear. Here, we show by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localized to the matrix of the mitochondria. We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome. By manipulating its expression, we show that Mrpl44 may be important for regulating the expression of mtDNA-encoded genes. This was at the level of RNA expression and protein translation. This ultimately impacted ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

No MeSH data available.