Limits...
Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p.

Chatenay-Lapointe M, Shadel GS - PLoS ONE (2011)

Bottom Line: Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility.Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling.Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

Show MeSH

Related in: MedlinePlus

Analysis of puf3Δ Yeast Strains.Analysis of wild-type (wt) and an isogenic puf3Δ strains are shown. (A) Mitochondrial oxygen consumption assayed at 12 hours (log phase), 24 hours (early stationary phase), and 48 hours post-inoculation (1 day into stationary phase). The bars represent the mean ± SD with p-values as indicated (*<0.05, **<0.01, ***<0.001). Statistical analysis was performed with Prism 5 software, using a Student's t-test (unpaired, two-tailed). (B) Western blot of indicated proteins in log-phase growth (representative of three biological replicates). (C) Autoradiogram of separated mtDNA-encoded proteins labeled with 35S-methionine and 35S -cysteine at logarithmic growth. A section of the same gel stained with coomassie blue to demonstrate loading is shown underneath. (D) mtDNA copy number.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105058&req=5

pone-0020441-g001: Analysis of puf3Δ Yeast Strains.Analysis of wild-type (wt) and an isogenic puf3Δ strains are shown. (A) Mitochondrial oxygen consumption assayed at 12 hours (log phase), 24 hours (early stationary phase), and 48 hours post-inoculation (1 day into stationary phase). The bars represent the mean ± SD with p-values as indicated (*<0.05, **<0.01, ***<0.001). Statistical analysis was performed with Prism 5 software, using a Student's t-test (unpaired, two-tailed). (B) Western blot of indicated proteins in log-phase growth (representative of three biological replicates). (C) Autoradiogram of separated mtDNA-encoded proteins labeled with 35S-methionine and 35S -cysteine at logarithmic growth. A section of the same gel stained with coomassie blue to demonstrate loading is shown underneath. (D) mtDNA copy number.

Mentions: We analyzed multiple mitochondrial parameters in wild-type (DBY2006) and isogenic puf3Δ yeast strains grown in synthetic dextrose medium. Lack of Puf3p increased mitochondrial oxygen consumption during logarithmic growth and at early stationary phase time points, however, this was not sustained (and even decreased slightly) later in stationary phase (Figure 1A). The increase in mitochondrial respiration observed in log-phase growth was accompanied by a corresponding robust increases in the steady-state levels of multiple mitochondrial proteins, including Cox2p and Cox4p, mtDNA-encoded and nucleus-encoded OXPHOS subunits, respectively (Figure 1B) and Pet100p, a nucleus-encoded protein with a Puf3 element in its 3′ UTR. We observed no significant increases in “mitochondrial housekeeping” markers Coq5p and porin compared to the actin loading control, suggesting that overall mitochondrial biogenesis was not increased in puf3Δ cells (Figure 1B). There was also no increase in mtDNA per cell in puf3Δ strains, which is consistent with mitochondrial biogenesis not being altered (Figure 1D). Finally, there was an apparently global up-regulation of mitochondrial translation in puf3Δ strains compared to the wild-type control during log-phase growth (Figure 1C). The up-regulation of mitochondrial translation and OXPHOS subunits per se (i.e. without a corresponding increase in overall mitochondrial biogenesis) is consistent with Puf3p being a negative regulator of the subset of nucleus-encoded genes involved in mitochondrial translation and OXPHOS assembly [23], but not overall organelle biogenesis.


Repression of mitochondrial translation, respiration and a metabolic cycle-regulated gene, SLF1, by the yeast Pumilio-family protein Puf3p.

Chatenay-Lapointe M, Shadel GS - PLoS ONE (2011)

Analysis of puf3Δ Yeast Strains.Analysis of wild-type (wt) and an isogenic puf3Δ strains are shown. (A) Mitochondrial oxygen consumption assayed at 12 hours (log phase), 24 hours (early stationary phase), and 48 hours post-inoculation (1 day into stationary phase). The bars represent the mean ± SD with p-values as indicated (*<0.05, **<0.01, ***<0.001). Statistical analysis was performed with Prism 5 software, using a Student's t-test (unpaired, two-tailed). (B) Western blot of indicated proteins in log-phase growth (representative of three biological replicates). (C) Autoradiogram of separated mtDNA-encoded proteins labeled with 35S-methionine and 35S -cysteine at logarithmic growth. A section of the same gel stained with coomassie blue to demonstrate loading is shown underneath. (D) mtDNA copy number.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105058&req=5

pone-0020441-g001: Analysis of puf3Δ Yeast Strains.Analysis of wild-type (wt) and an isogenic puf3Δ strains are shown. (A) Mitochondrial oxygen consumption assayed at 12 hours (log phase), 24 hours (early stationary phase), and 48 hours post-inoculation (1 day into stationary phase). The bars represent the mean ± SD with p-values as indicated (*<0.05, **<0.01, ***<0.001). Statistical analysis was performed with Prism 5 software, using a Student's t-test (unpaired, two-tailed). (B) Western blot of indicated proteins in log-phase growth (representative of three biological replicates). (C) Autoradiogram of separated mtDNA-encoded proteins labeled with 35S-methionine and 35S -cysteine at logarithmic growth. A section of the same gel stained with coomassie blue to demonstrate loading is shown underneath. (D) mtDNA copy number.
Mentions: We analyzed multiple mitochondrial parameters in wild-type (DBY2006) and isogenic puf3Δ yeast strains grown in synthetic dextrose medium. Lack of Puf3p increased mitochondrial oxygen consumption during logarithmic growth and at early stationary phase time points, however, this was not sustained (and even decreased slightly) later in stationary phase (Figure 1A). The increase in mitochondrial respiration observed in log-phase growth was accompanied by a corresponding robust increases in the steady-state levels of multiple mitochondrial proteins, including Cox2p and Cox4p, mtDNA-encoded and nucleus-encoded OXPHOS subunits, respectively (Figure 1B) and Pet100p, a nucleus-encoded protein with a Puf3 element in its 3′ UTR. We observed no significant increases in “mitochondrial housekeeping” markers Coq5p and porin compared to the actin loading control, suggesting that overall mitochondrial biogenesis was not increased in puf3Δ cells (Figure 1B). There was also no increase in mtDNA per cell in puf3Δ strains, which is consistent with mitochondrial biogenesis not being altered (Figure 1D). Finally, there was an apparently global up-regulation of mitochondrial translation in puf3Δ strains compared to the wild-type control during log-phase growth (Figure 1C). The up-regulation of mitochondrial translation and OXPHOS subunits per se (i.e. without a corresponding increase in overall mitochondrial biogenesis) is consistent with Puf3p being a negative regulator of the subset of nucleus-encoded genes involved in mitochondrial translation and OXPHOS assembly [23], but not overall organelle biogenesis.

Bottom Line: Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility.Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling.Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Synthesis and assembly of the mitochondrial oxidative phosphorylation (OXPHOS) system requires genes located both in the nuclear and mitochondrial genomes, but how gene expression is coordinated between these two compartments is not fully understood. One level of control is through regulated expression mitochondrial ribosomal proteins and other factors required for mitochondrial translation and OXPHOS assembly, which are all products of nuclear genes that are subsequently imported into mitochondria. Interestingly, this cadre of genes in budding yeast has in common a 3'-UTR element that is bound by the Pumilio family protein, Puf3p, and is coordinately regulated under many conditions, including during the yeast metabolic cycle. Multiple functions have been assigned to Puf3p, including promoting mRNA degradation, localizing nucleus-encoded mitochondrial transcripts to the outer mitochondrial membrane, and facilitating mitochondria-cytoskeletal interactions and motility. Here we show that Puf3p has a general repressive effect on mitochondrial OXPHOS abundance, translation, and respiration that does not involve changes in overall mitochondrial biogenesis and largely independent of TORC1-mitochondrial signaling. We also identified the cytoplasmic translation factor Slf1p as yeast metabolic cycle-regulated gene that is repressed by Puf3p at the post-transcriptional level and promotes respiration and extension of yeast chronological life span when over-expressed. Altogether, these results should facilitate future studies on which of the many functions of Puf3p is most relevant for regulating mitochondrial gene expression and the role of nuclear-mitochondrial communication in aging and longevity.

Show MeSH
Related in: MedlinePlus