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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.


Mrpl44 is predominantly localized to the mitochondria.(A) Shown are NIH3T3 fibroblasts, CommaD P24 mammary epithelial cells and N2A neuronal cells expressing an Mrpl44GFP fusion. The cells were co-stained with MitoTrackerRed along with DAPI and analyzed by wide-field microscopy (400X magnification). (B) NIH3T3 cells were sub-fractionated into the following fractions: cytosol and heavy membranes (C+HM), nuclear (N), cytosol (C), mitochondrial outer membrane and intermembrane space (IO) and mitoplast (MP). Whole cell extract (WCE) is also shown. Fractions were blotted with antibodies against Mrpl44 as well as Grp75, a known mitochondrial protein, Drosha, a nuclear protein, and β-actin, a cytoskeletal protein.
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pone.0134326.g001: Mrpl44 is predominantly localized to the mitochondria.(A) Shown are NIH3T3 fibroblasts, CommaD P24 mammary epithelial cells and N2A neuronal cells expressing an Mrpl44GFP fusion. The cells were co-stained with MitoTrackerRed along with DAPI and analyzed by wide-field microscopy (400X magnification). (B) NIH3T3 cells were sub-fractionated into the following fractions: cytosol and heavy membranes (C+HM), nuclear (N), cytosol (C), mitochondrial outer membrane and intermembrane space (IO) and mitoplast (MP). Whole cell extract (WCE) is also shown. Fractions were blotted with antibodies against Mrpl44 as well as Grp75, a known mitochondrial protein, Drosha, a nuclear protein, and β-actin, a cytoskeletal protein.

Mentions: To better define the cellular localization of Mrpl44, we generated an Mrpl44GFP fusion construct and transduced it into the cell lines NIH3T3, CommaD P24 and N2A cells. Mrpl44GFP co-localized with MitoTrackerRED in all cell lines examined (Fig 1A). We also examined for co-localization with PD1 in the endoplasmic reticulum and phalloidin, which binds filamentous actin, but found no co-localization (data not shown). This indicates that Mrpl44GFP is specifically localized to the mitochondria. In contrast, untagged GFP showed no specific localization (S2 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)

Mrpl44 is predominantly localized to the mitochondria.(A) Shown are NIH3T3 fibroblasts, CommaD P24 mammary epithelial cells and N2A neuronal cells expressing an Mrpl44GFP fusion. The cells were co-stained with MitoTrackerRed along with DAPI and analyzed by wide-field microscopy (400X magnification). (B) NIH3T3 cells were sub-fractionated into the following fractions: cytosol and heavy membranes (C+HM), nuclear (N), cytosol (C), mitochondrial outer membrane and intermembrane space (IO) and mitoplast (MP). Whole cell extract (WCE) is also shown. Fractions were blotted with antibodies against Mrpl44 as well as Grp75, a known mitochondrial protein, Drosha, a nuclear protein, and β-actin, a cytoskeletal protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519308&req=5

pone.0134326.g001: Mrpl44 is predominantly localized to the mitochondria.(A) Shown are NIH3T3 fibroblasts, CommaD P24 mammary epithelial cells and N2A neuronal cells expressing an Mrpl44GFP fusion. The cells were co-stained with MitoTrackerRed along with DAPI and analyzed by wide-field microscopy (400X magnification). (B) NIH3T3 cells were sub-fractionated into the following fractions: cytosol and heavy membranes (C+HM), nuclear (N), cytosol (C), mitochondrial outer membrane and intermembrane space (IO) and mitoplast (MP). Whole cell extract (WCE) is also shown. Fractions were blotted with antibodies against Mrpl44 as well as Grp75, a known mitochondrial protein, Drosha, a nuclear protein, and β-actin, a cytoskeletal protein.
Mentions: To better define the cellular localization of Mrpl44, we generated an Mrpl44GFP fusion construct and transduced it into the cell lines NIH3T3, CommaD P24 and N2A cells. Mrpl44GFP co-localized with MitoTrackerRED in all cell lines examined (Fig 1A). We also examined for co-localization with PD1 in the endoplasmic reticulum and phalloidin, which binds filamentous actin, but found no co-localization (data not shown). This indicates that Mrpl44GFP is specifically localized to the mitochondria. In contrast, untagged GFP showed no specific localization (S2 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.