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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 forms multimers as part of the large subunit of the mitoribosome.(A) HEK293T cells were co-transfected with Mrpl44FLAG and Mrpl44GFP; or FLAGBak and Mrpl44GFP, as a negative control. Lysates were immunoprecipitated with αFLAG, then blotted with an αGFP antibody to determine co-IP. Results are representative of three independent experiments. (B) NIH3T3 whole cell extracts were fractionated by HPLC on a Superose 6 column. The fractions were collected and alternate fractions from 14 to 30 were run on an SDS-PAGE gel, then blotted with antibodies against Mrpl44 as well as Mrpl12 and Mrps15, known components of the mitoribosome. (C) Mitochondrial fractions were obtained from Mrpl44FLAG NIH3T3 cells, and FLAGBak NIH3T3 cells, as a negative control, and immunoprecipitated with αFLAG agarose beads. RNA was then extracted and analysed for pulldown of the mitochondrial rRNA subunits by quantitative RT-PCR. The mean +/- SEM of three independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (*p<0.05). (D) NIH3T3 whole cell extracts were immunoprecipitated with αrabbit Ig antibody or αMrpl44 antibody bound to Protein G sepharose beads then blotted for Mrpl44, Mrpl12 and Mrps15.
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pone.0134326.g002: Mrpl44 forms multimers as part of the large subunit of the mitoribosome.(A) HEK293T cells were co-transfected with Mrpl44FLAG and Mrpl44GFP; or FLAGBak and Mrpl44GFP, as a negative control. Lysates were immunoprecipitated with αFLAG, then blotted with an αGFP antibody to determine co-IP. Results are representative of three independent experiments. (B) NIH3T3 whole cell extracts were fractionated by HPLC on a Superose 6 column. The fractions were collected and alternate fractions from 14 to 30 were run on an SDS-PAGE gel, then blotted with antibodies against Mrpl44 as well as Mrpl12 and Mrps15, known components of the mitoribosome. (C) Mitochondrial fractions were obtained from Mrpl44FLAG NIH3T3 cells, and FLAGBak NIH3T3 cells, as a negative control, and immunoprecipitated with αFLAG agarose beads. RNA was then extracted and analysed for pulldown of the mitochondrial rRNA subunits by quantitative RT-PCR. The mean +/- SEM of three independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (*p<0.05). (D) NIH3T3 whole cell extracts were immunoprecipitated with αrabbit Ig antibody or αMrpl44 antibody bound to Protein G sepharose beads then blotted for Mrpl44, Mrpl12 and Mrps15.

Mentions: RNase III proteins typically form dimers that bind to and cleave dsRNA [15]. To determine whether Mrpl44 might form dimers or multimers, FLAG-tagged Mrpl44 (Mrpl44FLAG) was co-transfected with Mrpl44GFP into HEK293T cells. As a negative control, Mrpl44GFP was co-transfected with FLAGBak, another mitochondria-localized protein. Lysates were immunoprecipitated with αFLAG beads to capture Mrpl44FLAG or FLAGBak, then Western blotted to determine if Mrpl44GFP was co-immunoprecipitated. Indeed, Mrpl44GFP was found to interact with Mrpl44FLAG but not FLAGBak (Fig 2A). This suggests that Mrpl44 can exist in dimers or multimers.


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 forms multimers as part of the large subunit of the mitoribosome.(A) HEK293T cells were co-transfected with Mrpl44FLAG and Mrpl44GFP; or FLAGBak and Mrpl44GFP, as a negative control. Lysates were immunoprecipitated with αFLAG, then blotted with an αGFP antibody to determine co-IP. Results are representative of three independent experiments. (B) NIH3T3 whole cell extracts were fractionated by HPLC on a Superose 6 column. The fractions were collected and alternate fractions from 14 to 30 were run on an SDS-PAGE gel, then blotted with antibodies against Mrpl44 as well as Mrpl12 and Mrps15, known components of the mitoribosome. (C) Mitochondrial fractions were obtained from Mrpl44FLAG NIH3T3 cells, and FLAGBak NIH3T3 cells, as a negative control, and immunoprecipitated with αFLAG agarose beads. RNA was then extracted and analysed for pulldown of the mitochondrial rRNA subunits by quantitative RT-PCR. The mean +/- SEM of three independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (*p<0.05). (D) NIH3T3 whole cell extracts were immunoprecipitated with αrabbit Ig antibody or αMrpl44 antibody bound to Protein G sepharose beads then blotted for Mrpl44, Mrpl12 and Mrps15.
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pone.0134326.g002: Mrpl44 forms multimers as part of the large subunit of the mitoribosome.(A) HEK293T cells were co-transfected with Mrpl44FLAG and Mrpl44GFP; or FLAGBak and Mrpl44GFP, as a negative control. Lysates were immunoprecipitated with αFLAG, then blotted with an αGFP antibody to determine co-IP. Results are representative of three independent experiments. (B) NIH3T3 whole cell extracts were fractionated by HPLC on a Superose 6 column. The fractions were collected and alternate fractions from 14 to 30 were run on an SDS-PAGE gel, then blotted with antibodies against Mrpl44 as well as Mrpl12 and Mrps15, known components of the mitoribosome. (C) Mitochondrial fractions were obtained from Mrpl44FLAG NIH3T3 cells, and FLAGBak NIH3T3 cells, as a negative control, and immunoprecipitated with αFLAG agarose beads. RNA was then extracted and analysed for pulldown of the mitochondrial rRNA subunits by quantitative RT-PCR. The mean +/- SEM of three independent experiments is shown. Statistical analysis performed using multiple t-test with Holm-Sidak correction for multiple comparisons (*p<0.05). (D) NIH3T3 whole cell extracts were immunoprecipitated with αrabbit Ig antibody or αMrpl44 antibody bound to Protein G sepharose beads then blotted for Mrpl44, Mrpl12 and Mrps15.
Mentions: RNase III proteins typically form dimers that bind to and cleave dsRNA [15]. To determine whether Mrpl44 might form dimers or multimers, FLAG-tagged Mrpl44 (Mrpl44FLAG) was co-transfected with Mrpl44GFP into HEK293T cells. As a negative control, Mrpl44GFP was co-transfected with FLAGBak, another mitochondria-localized protein. Lysates were immunoprecipitated with αFLAG beads to capture Mrpl44FLAG or FLAGBak, then Western blotted to determine if Mrpl44GFP was co-immunoprecipitated. Indeed, Mrpl44GFP was found to interact with Mrpl44FLAG but not FLAGBak (Fig 2A). This suggests that Mrpl44 can exist in dimers or multimers.

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.