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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.

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Comparison of mitochondrial parameters in puf3Δ, tor1Δ, and puf3Δ tor1Δ mutant strains.(A) Mitochondrial oxygen consumption during early log phase. (B) Western blot analysis of protein extracts derived from log phase cultures (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in log phase. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.
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pone-0020441-g005: Comparison of mitochondrial parameters in puf3Δ, tor1Δ, and puf3Δ tor1Δ mutant strains.(A) Mitochondrial oxygen consumption during early log phase. (B) Western blot analysis of protein extracts derived from log phase cultures (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in log phase. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.

Mentions: To probe more directly the potential involvement of Puf3p in the regulation of mitochondrial function by TORC1, we compared mitochondrial parameters in tor1Δ and puf3Δ single-mutant strains to those in a tor1Δ puf3Δ double-mutant strain. While both single-mutant strains had increased mitochondrial oxygen consumption and Cox2p and Cox4p levels compared to the isogenic wild-type strains, the double-mutant strains displayed an additive increase in these parameters (Figure 5A and 5B). In contrast to Cox2p and Cox4p, Pet100p (a Puf3p target) did not display an additive increase in the double-mutant strain (Figure 5B). Comparison of the “mitochondrial housekeeping” proteins Coq5p and porin to the loading control actin revealed only modest changes in overall mitochondrial abundance in any of the mutant strains compared to the wild-type control (Figure 5B). Finally, analysis of mitochondrial transcripts in this cadre of strains revealed complex regulation. While loss of Puf3p led to increased levels of the Puf3-target COX17 transcript, this was enhanced further by loss of Tor1p (Figure 5C, left). Similarly, loss of Tor1p resulted in increased amounts of COX12 and RIP1 transcripts, but this too was enhanced further (in an apparently additive manner) by lack of Puf3p (Figure 5C, center and right). Similar results with regard to respiration, steady-state mitochondrial marker protein levels, and target transcripts were observed in wild-type and puf3Δ strains treated with rapamycin (Figure 6). These data indicate that, while there might be some degree of crosstalk/synergism between the Puf3p and TORC1 pathways with regard to specific mitochondrial targets and effects, these pathways operate largely independently with regard to respiration and CLS.


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)

Comparison of mitochondrial parameters in puf3Δ, tor1Δ, and puf3Δ tor1Δ mutant strains.(A) Mitochondrial oxygen consumption during early log phase. (B) Western blot analysis of protein extracts derived from log phase cultures (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in log phase. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020441-g005: Comparison of mitochondrial parameters in puf3Δ, tor1Δ, and puf3Δ tor1Δ mutant strains.(A) Mitochondrial oxygen consumption during early log phase. (B) Western blot analysis of protein extracts derived from log phase cultures (representative of three biological replicates). (C) Steady-state levels of COX17, COX12, and RIP1 transcripts in log phase. The values indicate the mean +/− SD with p-values indicated as described in the legend of Figure 1.
Mentions: To probe more directly the potential involvement of Puf3p in the regulation of mitochondrial function by TORC1, we compared mitochondrial parameters in tor1Δ and puf3Δ single-mutant strains to those in a tor1Δ puf3Δ double-mutant strain. While both single-mutant strains had increased mitochondrial oxygen consumption and Cox2p and Cox4p levels compared to the isogenic wild-type strains, the double-mutant strains displayed an additive increase in these parameters (Figure 5A and 5B). In contrast to Cox2p and Cox4p, Pet100p (a Puf3p target) did not display an additive increase in the double-mutant strain (Figure 5B). Comparison of the “mitochondrial housekeeping” proteins Coq5p and porin to the loading control actin revealed only modest changes in overall mitochondrial abundance in any of the mutant strains compared to the wild-type control (Figure 5B). Finally, analysis of mitochondrial transcripts in this cadre of strains revealed complex regulation. While loss of Puf3p led to increased levels of the Puf3-target COX17 transcript, this was enhanced further by loss of Tor1p (Figure 5C, left). Similarly, loss of Tor1p resulted in increased amounts of COX12 and RIP1 transcripts, but this too was enhanced further (in an apparently additive manner) by lack of Puf3p (Figure 5C, center and right). Similar results with regard to respiration, steady-state mitochondrial marker protein levels, and target transcripts were observed in wild-type and puf3Δ strains treated with rapamycin (Figure 6). These data indicate that, while there might be some degree of crosstalk/synergism between the Puf3p and TORC1 pathways with regard to specific mitochondrial targets and effects, these pathways operate largely independently with regard to respiration and CLS.

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