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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.


Knockdown of Mrpl44 decreases mitochondrial translation.(A) NIH3T3 cells knocked down for Mrpl44 (ORF) or expressing a control shRNA were radiolabelled with 35S-methionine for 120mins. Cytosolic translation was inhibited by the addition of emetine. Protein extracts were collected at 0, 30, 60 and 120 mins after 35S-methionine addition. The extracted mitochondrial proteins were separated on a 10–16% gradient SDS-PAGE then autoradiographed. To validate equal protein loading, a small section of the gel was stained with Coomassie Blue. One or two experiments is shown. (B) Densitometry analysis of the bands at 120 mins. The data were normalized to the control COII expression at 30 mins. The mean +/- SD of 2 different shRNA knockdowns (ORF and 3’UTR) is shown.
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pone.0134326.g004: Knockdown of Mrpl44 decreases mitochondrial translation.(A) NIH3T3 cells knocked down for Mrpl44 (ORF) or expressing a control shRNA were radiolabelled with 35S-methionine for 120mins. Cytosolic translation was inhibited by the addition of emetine. Protein extracts were collected at 0, 30, 60 and 120 mins after 35S-methionine addition. The extracted mitochondrial proteins were separated on a 10–16% gradient SDS-PAGE then autoradiographed. To validate equal protein loading, a small section of the gel was stained with Coomassie Blue. One or two experiments is shown. (B) Densitometry analysis of the bands at 120 mins. The data were normalized to the control COII expression at 30 mins. The mean +/- SD of 2 different shRNA knockdowns (ORF and 3’UTR) is shown.

Mentions: We next examine whether Mrpl44 might also regulate mitochondrial translation, rather than just affecting transcript maturation. A mitochondrial translation assay was performed. 35S-methionine was used to radiolabel mitochondrial translation products in the presence of emetine, which inhibits cytosolic translation. Knockdown of Mrpl44 resulted in a clear decrease in overall mitochondrial protein translation, with fewer proteins being labeled during the time course (Fig 4A). Further quantification revealed that the subunits ND3, ND4, ND5, ND6 and COIII were below detection limits with Mrpl44 knockdown (Fig 4B). Of the proteins detected during the time course, the rate of translation was decreased as well, with the subunits ND1, ND2, CytB and COI being labeled 30 min later, at the 60 min time point, compared to control cells (S6 Fig). These results indicate that Mrpl44 does not only affect the mtDNA at the RNA level, but also at the level of protein translation.


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)

Knockdown of Mrpl44 decreases mitochondrial translation.(A) NIH3T3 cells knocked down for Mrpl44 (ORF) or expressing a control shRNA were radiolabelled with 35S-methionine for 120mins. Cytosolic translation was inhibited by the addition of emetine. Protein extracts were collected at 0, 30, 60 and 120 mins after 35S-methionine addition. The extracted mitochondrial proteins were separated on a 10–16% gradient SDS-PAGE then autoradiographed. To validate equal protein loading, a small section of the gel was stained with Coomassie Blue. One or two experiments is shown. (B) Densitometry analysis of the bands at 120 mins. The data were normalized to the control COII expression at 30 mins. The mean +/- SD of 2 different shRNA knockdowns (ORF and 3’UTR) is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519308&req=5

pone.0134326.g004: Knockdown of Mrpl44 decreases mitochondrial translation.(A) NIH3T3 cells knocked down for Mrpl44 (ORF) or expressing a control shRNA were radiolabelled with 35S-methionine for 120mins. Cytosolic translation was inhibited by the addition of emetine. Protein extracts were collected at 0, 30, 60 and 120 mins after 35S-methionine addition. The extracted mitochondrial proteins were separated on a 10–16% gradient SDS-PAGE then autoradiographed. To validate equal protein loading, a small section of the gel was stained with Coomassie Blue. One or two experiments is shown. (B) Densitometry analysis of the bands at 120 mins. The data were normalized to the control COII expression at 30 mins. The mean +/- SD of 2 different shRNA knockdowns (ORF and 3’UTR) is shown.
Mentions: We next examine whether Mrpl44 might also regulate mitochondrial translation, rather than just affecting transcript maturation. A mitochondrial translation assay was performed. 35S-methionine was used to radiolabel mitochondrial translation products in the presence of emetine, which inhibits cytosolic translation. Knockdown of Mrpl44 resulted in a clear decrease in overall mitochondrial protein translation, with fewer proteins being labeled during the time course (Fig 4A). Further quantification revealed that the subunits ND3, ND4, ND5, ND6 and COIII were below detection limits with Mrpl44 knockdown (Fig 4B). Of the proteins detected during the time course, the rate of translation was decreased as well, with the subunits ND1, ND2, CytB and COI being labeled 30 min later, at the 60 min time point, compared to control cells (S6 Fig). These results indicate that Mrpl44 does not only affect the mtDNA at the RNA level, but also at the level of protein translation.

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.