Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan 11;11(1):e0146511.
doi: 10.1371/journal.pone.0146511. eCollection 2016.

Reduced Glucose Sensation Can Increase the Fitness of Saccharomyces cerevisiae Lacking Mitochondrial DNA

Emel Akdoğan  1 , Mehmet Tardu  2 , Görkem Garipler  1 , Gülkız Baytek  1 , İ Halil Kavakli  1   2 , Cory D Dunn  1
Affiliations

Reduced Glucose Sensation Can Increase the Fitness of Saccharomyces cerevisiae Lacking Mitochondrial DNA

Emel Akdoğan et al. PLoS One. .

Abstract

Damage to the mitochondrial genome (mtDNA) can lead to diseases for which there are no clearly effective treatments. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. We found that restricting glucose or otherwise reducing the activity of the protein kinase A (PKA) pathway can lead to improved proliferation of Saccharomyces cerevisiae cells lacking mtDNA and that the transcriptional response to mtDNA loss is reduced in cells with diminished PKA activity. We have excluded many pathways and proteins from being individually responsible for the benefits provided to cells lacking mtDNA by PKA inhibition, and we found that robust import of mitochondrial polytopic membrane proteins may be required in order for cells without mtDNA to receive the full benefits of PKA reduction. Finally, we have discovered that the transcription of genes involved in arginine biosynthesis and aromatic amino acid catabolism is altered after mtDNA damage. Our results highlight the potential importance of nutrient detection and availability on the outcome of mitochondrial dysfunction.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Decreased PKA activity can increase proliferation of cells lacking mtDNA.
(A) Overexpression of cAMP phosphodiesterase Pde2p increases the fitness of cells lacking mtDNA. Strain BY4743 (WT) was transformed with empty, high-copy vector pRS426 or plasmid b89 (2μ-PDE2). Strains were tested for their response to mtDNA loss by incubation in selective medium lacking or containing 25 μg/ml EtBr, with subsequent incubation on solid SC-Ura medium for 2 d. (B) Lack of Tpk3p increases the fitness of cells lacking mtDNA. Strains BY4742 (WT), CDD884 (tpk1Δ), CDD885 (tpk2Δ), CDD886 (tpk3Δ), CDD908 (tpk1Δ tpk3Δ), CDD922 (tpk2Δ tpk3Δ), and CDD923 (tpk1Δ tpk2Δ) were tested for their response to mtDNA deletion with incubation on solid YEPD medium for 2 d. (C) Cells deleted of Gpa2p or Gpr1p exhibit increased fitness after mtDNA deletion. Strains BY4742 (WT), CDD886 (tpk3Δ), CDD849 (gpa2Δ), and CDD850 (gpr1Δ) were treated as in (B).
Fig 2
Fig 2. Glucose inhibits proliferation of cells deleted of mtDNA.
(A) Decreasing glucose concentration leads to increased proliferation of cells lacking mtDNA. Strain BY4742 (WT) was cultured in YEPD medium containing 2%, 0.5%, or 0.2% glucose and tested for the response to mtDNA deletion. Cells were incubated for 3 d. (B) Proliferation of ρ0 cells by Gpa2p or Gpr1p deletion is not improved further upon lowering the glucose concentration. Strains BY4742 (WT), CDD849 (gpa2Δ), and CDD850 (gpr1Δ) were treated as in (A), yet incubated on solid medium for 2 d.
Fig 3
Fig 3. Overexpression of Pde2p can diminish the transcriptional response to mtDNA deletion.
(A) IDR target genes activated by mtDNA loss are attenuated upon Pde2p overexpression. Wild-type strain BY4743 transformed with vector pRS426, plasmid b89 (pRS426-PDE2), or plasmid M489 (pRS426-TIP41) was treated with EtBr for 24 hr to force mtDNA loss. Gene expression levels were determined by next-generation sequencing and normalized to BY4743 ρ+ cells harboring vector pRS426. Genes selected for analysis were activated more than three-fold in ρ0 cells expressing vector pRS426 over ρ+ cells expressing the same plasmid, were statistically significant upon comparison of these two conditions (q < 0.05), and were listed as IDR targets in [127] or [128]. (B) Genes activated by the PDR pathway in ρ0 cells are reduced in expression by Pde2p overexpression. Analysis was performed as in (A), except genes selected for analysis were identified as PDR pathway targets in [129] or [130]. (C) Genes activated by the RTG signaling pathway in cells lacking a mitochondrial genome are decreased in expression by Pde2p overproduction. Analysis was performed as in (A), with RTG pathway targets provided by [41]. (D) Arginine biosynthesis genes are upregulated upon mtDNA loss, but this response is reduced upon PKA inhibition. Analysis was performed as in (A), with arginine biosynthesis genes reported by [124]. (E) Two genes involved in aromatic amino acid breakdown and reduced in expression following mtDNA loss are recovered in expression when Pde2p is overexpressed. Analysis was performed as in (A). ARO9 and ARO10 were selected after a more than three-fold (q < 0.05) reduction in expression when comparing ρ0 cells to ρ+ cells containing vector pRS426. Quantitative expression data can be found in S1 Table. Data are visualized using [131].
Fig 4
Fig 4. Cells lacking the glucose-sensing proteins Gpa2p or Gpr1p do not increase the localization of a mitochondria-targeted fluorescent protein to mitochondria.
(A) Strain BY4742 (WT) carrying pHS12 and either harboring or lacking mtDNA was examined by fluorescence microscopy to demonstrate the mitochondrial location of Cox4p(1–21)-GFP in ρ+ cells and its cytosolic location ρ0 cells. Mitochondria are visible in >99% of the cells within a ρ+ culture [16] (B) Strains BY4742 (WT), CDD849 (gpa2Δ), CDD850 (gpr1Δ), and CDD496 (vma2Δ) carrying pHS12 and deleted of mtDNA were examined by fluorescence microscopy, with representative images displayed. Scale bar, 5μm. (C) These cultures were scored, blind to genotype, for localization of Cox4p(1–21)-GFP to mitochondria (n > 200 cells).
Fig 5
Fig 5. Several transcription factors driving stress resistance following PKA inhibition are not individually responsible for the benefits provided by Pde2p overexpression to cells lacking mtDNA.
(A) Cells lacking Gis1p and mtDNA are increased in proliferation upon Pde2p overexpression. Strains BY4742 (WT) and CDD801 (gis1Δ) were treated as in Fig 1A. (B) Cells lacking both Msn2p and Msn4p exhibit increased fitness following mtDNA loss upon Pde2p overexpression. Strains BY4741 (WT) and CDD838 (msn2Δ msn4Δ) were treated as in Fig 1A. (C) The Rim15 kinase is not required in order for Pde2p overexpression to benefit ρ0 cells. Strains CDD463 (WT) and CDD841 (rim15Δ) were treated as in Fig 1A. (D) A potential reduction of Hsf1p function does not prevent increased ρ0 cell fitness upon overexpression of Pde2p. Strains BY4741 (WT) and CDD910 (hsf1-DAmP) were treated as in Fig 1A.
Fig 6
Fig 6. Chaperones Hsp12p and Hsp26p are not required for increased proliferation of cells lacking mtDNA upon Pde2p overexpression.
(A) HSP12 and HSP26 transcripts are overexpressed upon PKA inhibition in ρ0 cells. HSP12 and HSP26 transcript abundance within ρ0 cells upon overexpression of Pde2p, overproduction of Tip41p, or upon maintenance of empty vector pRS426 were normalized to gene expression in ρ+ cells carrying an empty vector. (B) Neither Hsp12p nor Hsp26p are individually responsible for the benefits provided by Pde2p overexpression to cells deleted of mtDNA. Strains CDD463 (WT), CDD542 (hsp12Δ), CDD534 (hsp26Δ) were treated as in Fig 1A.
Fig 7
Fig 7. Several cellular processes and signaling pathways controlled by PKA activity are not individually responsible for the outcome of PKA inhibition for cells deleted of mtDNA.
(A) Repression of transcriptional targets of Dot6p and Tod6p is not the sole mechanism by which high-copy Pde2p benefits ρ0 cells. Strains CDD289 (WT) and CDD567 (dot6Δ tod6Δ) were treated as in Fig 1A. (B) Repression of Maf1p targets is not required in order for Pde2p overexpression to benefit cells lacking mtDNA. Strains BY4741 (WT) and CDD928 (maf1Δ) were treated as in Fig 1A. (C) Activity of the Snf1 kinase is not required in order for Pde2p overproduction to increase proliferation of cells deprived of a mitochondrial genome. Strains CDD463 (WT) and CDD604 (snf1Δ) were treated as in Fig 1A. (D) Deletion of Sds23p and Sds24p does not prevent overexpression of Pde2p from increasing the division rate of cells lacking mtDNA. Strains BY4741 (WT) and CDD921 (sds23Δ sds24Δ) were treated as in Fig 1A. (E) Cells lacking P-body component Pat1p exhibit increased fitness after mtDNA loss. Strains BY4742 (WT) and CDD879 (pat1Δ) were treated as in Fig 1B, except ρ+ cells were incubated on solid YEPD medium for 1 d, while ρ0 cells were incubated for 2 d. (F) PKA inhibition by Pde2p overexpression is unlikely to significantly affect V1VO-ATPase assembly. Strains BY4742 (WT) and CDD496 (vma2Δ) were treated as in Fig 1A. ρ+ cultures were additionally plated to SC-Ura medium buffered to pH 7.3 using 100 mM HEPES-KOH, pH 7.5 and incubated for 3 d.
Fig 8
Fig 8. Overexpression of Pde2p can rescue the petite-negative phenotype of several mutants defective for mitochondrial function.
(A) High-copy Pde2p can allow mutants deficient in activity of the i-AAA protease to remain viable following mtDNA loss. Strains BY4741 (WT), CDD13 (mgr1Δ), and CDD15 (mgr3Δ) were treated as in Fig 1A, with additional incubation of ρ0 cells to 4 d in order to demonstrate suppression of the petite-negative phenotype. (B) PKA inhibition by Pde2p overexpression can rescue the petite-negative phenotype of mutants deficient in mitochondrial protein import and assembly. Strains BY4741 (WT), CDD11 (mgr2Δ), and CDD17 (phb1Δ) were treated as in (A). (C) Overexpression of Pde2p allows cells lacking mtDNA to proliferate in the absence of F1-ATPase activity. Strains CDD463 (WT) and CDD215 (atp2Δ) were treated as in Fig 1A, with further incubation of ρ0 cells to 6 d.
Fig 9
Fig 9. The outer membrane protein receptor Tom70 is required for cells lacking mtDNA to receive the proliferation boost associated with Pde2p overexpression.
(A) Overexpression of Tip41p, but not Pde2p, allows viability of cells lacking both Tom70p and mtDNA. Strains BY4741 (WT) and CDD897 (tom70Δ) were transformed with empty pRS426 vector, 2μ-PDE2 plasmid b89, or 2μ-TIP41 plasmid M489 and treated as in Fig 8C. (B) Phosphorylation of S174 on Tom70p does not determine the outcome of mtDNA damage. Strain CDD913 (tom70Δ) was transformed with empty vector pRS314, plasmid b110 (pRS314-TOM70), plasmid b111 (pRS314-tom70-S174A), or plasmid b112 (pRS314-tom70-S174E). Resulting genotypes are shown. Strains were tested for their response to mtDNA deletion as in Fig 1A except cells were incubated on solid SC-Trp medium for 2 d. (C) Tom70p is not upregulated in ρ0 cells overexpressing Pde2p. Whole cell extracts from strains CDD926 (TOM70-GFP) and CDD927 (TOM70) either overexpressing Pde2p from plasmid b89 or harboring an empty pRS426 vector and either containing or lacking mtDNA were analyzed by immunoblotting using antibodies recognizing GFP or hexokinase.
Fig 10
Fig 10. Pde2p overexpression does not benefit cells lacking mtDNA by Aac2p upregulation or through Mir1p upregulation.
(A) Aac2p is not upregulated in ρ0 cells overproducing Pde2p. Whole cell extracts from aac2Δ strain CDD859 expressing FLAG-tagged Aac2p from plasmid b84 and either containing or deleted of mtDNA and either harboring 2μ-PDE2 plasmid b89 or empty vector pRS426 were analyzed using antibodies raised against the FLAG epitope tag or recognizing hexokinase. (B) Deletion of Mir1p provides an increase in proliferation rate to cells lacking mtDNA. Strains CDD862 (WT) and CDD863 (mir1Δ) were treated as in Fig 1B, except ρ+ cells were incubated for 1 d and ρ0 cells were incubated for 3 d.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Kispal G, Sipos K, Lange H, Fekete Z, Bedekovics T, Janaky T, et al. Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria. EMBO J. 2005. ed. 2005;24: 589–598. 10.1038/sj.emboj.7600541 - DOI - PMC - PubMed
    1. Lill R, Hoffmann B, Molik S, Pierik AJ, Rietzschel N, Stehling O, et al. The role of mitochondria in cellular iron–sulfur protein biogenesis and iron metabolism. BBA-Molecular Cell Research. Elsevier B.V; 2012;: 1–18. 10.1016/j.bbamcr.2012.05.009 - DOI - PubMed
    1. Gray MW. The Pre-Endosymbiont Hypothesis: A New Perspective on the Origin and Evolution of Mitochondria. Cold Spring Harbor Perspectives in Biology. 2014;6: a016097–a016097. 10.1101/cshperspect.a016097 - DOI - PMC - PubMed
    1. Voet D, Voet JG. Biochemistry, 4th Edition 4 ed. Wiley Global Education; 2010.
    1. Schaefer AM, McFarland R, Blakely EL, He L, Whittaker RG, Taylor RW, et al. Prevalence of mitochondrial DNA disease in adults. Ann Neurol. 2007 ed. 2008;63: 35–39. 10.1002/ana.21217 - DOI - PubMed

Publication types

MeSH terms

Substances