cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation
© Leadsham and Gourlay; licensee BioMed Central Ltd. 2010
Received: 1 September 2010
Accepted: 25 November 2010
Published: 25 November 2010
Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that co-ordinate mitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death.
We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one of the yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1.
We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast.
Mitochondria participate in a number of essential cellular functions, for example they are key players in ATP production, via the process of oxidative phosphorylation, which can produce up to 15 times more ATP from glucose than glycolysis alone. They are also central to metabolic regulation and facilitate diverse cell signaling events [1, 2]. Mitochondria are therefore essential for the maintenance, adaptability and survival of eukaryotic cells. In addition, these remarkable organelles have been conclusively shown to play a role in the regulation of programmed cell death processes (reviewed in ), and act as an important determinants of cellular senescence and ageing [4, 5]. The importance of understanding how mitochondria influence the metabolic status of cells becomes apparent when we consider that many muscular and neurodegenerative diseases have been linked with their dysfunction. The current list of disease pathologies in which mitochondrial function is thought to be a major contributing factor is extensive and includes diabetes , cancer , multiple sclerosis , Alzheimer's  and Parkinson's . Good evidence also exists that indicates a loss of mitochondrial function occurs during the progression of normal ageing . Mitochondrial dysfunction can lead to the production of reactive oxygen species (ROS), which are implicated in both ageing and apoptosis [12, 13], presumably as a result of their ability to damage macromolecules.
The control of mitochondrial biogenesis is complex, requiring coordinated transcription of a large number of nuclear and mitochondria encoded genes. In addition, there must be concerted control of the synthesis, import, and incorporation of proteins and lipids to existing mitochondria alongside appropriate replication of the mitochondrial DNA (mtDNA). In mammalian cells a number of have been indentified that act to co-ordinate tissue specific mitochondrial biogenesis in the face of altered metabolic demand and environmental change, including; nuclear respiratory factor-1 (NRF-1) and GA-binding protein (GABP or NRF-2), peroxisome proliferator-activated receptors (PPARα, PPARδ, and PPARγ), Mitochondrial transcription factors TFAM, TFB1M and TFB2M, Estrogen-related receptors (ERRα, ERRβ, and ERRγ) (for a recent review see ). The mitochondria of yeast are also adapted to respond to rapidly changing metabolic demands. For example switching between fermentative and non-fermentative carbon sources requires adjustment of mitochondrial activity and density . However despite the importance of the mitochondria to cellular function, the signaling mechanisms by which their activity is co-ordinated with environmental conditions in yeast are not well documented.
Previous studies in yeast have established links between Ras signaling and mitochondrial function, via cAMP/PKA dependent and independent routes [16, 17]. Within yeast Ras/cAMP/PKA signaling also controls cellular processes that include cell growth and proliferation and the induction of stress responses , making this pathway a good candidate to integrate environmental signaling with mitochondrial regulation. In yeast, protein kinase A (PKA) consists of a single regulatory subunit encoded by the BCY1 gene  and three catalytic subunits Tpk1p, Tpk2p and Tpk3p . PKA is activated by an increase in cAMP concentration, which is generated in the cell from adenylyl cyclase, Cyr1p . The three catalytic subunits of yeast exhibit high similarity and have been shown to have both specific and overlapping functions. For instance Tpk2p has been shown to influence iron uptake, trehalase synthesis, water homeostasis  and pseudohyphal growth . Tpk1p has been implicated in the branched chain amino acid biosynthesis pathway, mitochondrial iron homeostasis and mtDNA stability . Excessive PKA activity can prove deleterious, for instance the overexpression of TPK3 has been shown to inhibit growth . An important regulator of Cyr1p activation is the small regulatory GTPase Ras . The regulation of cAMP production from Cyr1p in response to Ras activation also requires the protein Srv2p/CAP [25, 26], which is able to bind both adenylyl cyclase and actin structures. We have shown previously that the accumulation of stable actin aggregates leads to the hyperactivation of the Ras/cAMP/PKA pathway . The result of actin aggregation induced Ras/cAMP/PKA signaling is that ROS are produced from dysfunctional mitochondria, facilitating cell death that displays hallmarks of yeast apoptosis [27–29]. The deletion of TPK3 is sufficient to prevent the production of ROS in actin aggregating strains, implicating this PKA subunit as a regulator of mitochondrial function [27, 30]. Further evidence for a role of Tpk3p in the regulation of mitochondrial function comes from a study in which cells lacking Tpk3p showed reduced respiratory activity .
Here we demonstrate that increased Tpk3p activity is sufficient to induce formation of dysfunctional mitochondria with striking morphological abnormalities that produce high levels of ROS. We provide evidence that the loss of mitochondria function and ROS production associated with elevated Tpk3p activity arises as a result of transcriptional changes that inhibit mitochondrial biogenesis, corrupt the electron transport chain and inhibit stress response mechanisms. We also show that the loss of mitochondrial function and production of ROS requires the activity of the transcriptional regulators HAP4, SOK2 and SKO1. This paper therefore establishes important links between cAMP/PKA signaling, nutritional sensing, mitochondrial biogenesis and ROS production.
Yeast strains, plasmids, Media and Growth Conditions
Yeast strains used in this study
mata his3Δ1 leu2Δ met15Δ ura3Δ
mata his3Δ1 leu2Δ met15Δ ura3Δ Δpde2::KanMx
mata his3Δ1 leu2Δ met15Δ ura3Δ Δpde2::HIS3 Δtpk3::KanMx
mata his3Δ1 leu2Δ met15Δ ura3Δ Δpde2::HIS3 Δtpk1::KanMx
mata his3Δ1 leu2Δ met15Δ ura3Δ Δpde2::HIS3 Δtpk2::KanMx
mata his3Δ1 leu2Δ met15Δ ura3Δ Δpde2::HIS3 Δtpk1::KanMx Δtpk3::LEU2
mata his3Δ1 leu2Δ met15Δ ura3Δ Δpde2::HIS3 Δtpk2::KanMx Δtpk3::LEU2
mata his3Δ1 leu2Δ met15Δ ura3Δ Δpde2::HIS3 Δtpk2::KanMx Δtpk3::LEU2
mata his3Δ1 leu2Δ met15Δ ura3Δ Δcox4::KanMx
RNA Isolation and Affymatrix Microarray Procedure
Strains CGY502 (Δpde2) and CGY217 (Δpde2Δtpk3) were grown for 24 hours in YPD (1% yeast extract, 2% Bacto-peptone, 2% glucose) supplemented with 4 mM cAMP at 30°C in a rotary air incubator. Cells from triplicate 20 ml cultures were harvested by centrifugation and resuspended in a small volume of media. The suspension was dispensed dropwise into liquid nitrogen. Triplicate cell pellets were maintained on dry ice and sent to the COGEME Transcriptome facility at the University of Manchester http://cogeme.ex.ac.uk/index.html for RNA isolation and microarray analysis. A yeast2 Affymetrix array was used in these experiments and statistical analysis on a gene by gene basis carried out using Limma software. Quality Control analysis was carried out using dchip software. Raw and normailsed data from the microarray has been deposited in the Gene Expression Omnibus (GEO) database, accession number GSE25541.
High Resolution Respirometry
Intact cell respiration was determined at 30°C using an Oxygraph-2 k system (Oroboros, Innsbruck, Austria) equipped with two chambers. Data was analysed using DatLab software. Yeast cells (2 ml) at a concentration of 3.5 × 106/ml, in minimal media without glucose, were added to each chamber. All assays were conducted in biological triplicate. The chambers were closed and Routine respiration was recorded. LEAK respiration was determined by the addition of 150 μM TET (Sigma), an ATP synthase inhibitor. Uncoupled respiration was then determined by the addition of the ionophore FCCP (12 μM) (Sigma). The addition of 2 μM Antimycin A (Sigma) accounted for non-mitochondrial oxygen consumption.
Cell number was accurately determined using a haemocytometer and total protein extracted using as optimized protein extraction for method suitable for quantitative proteomics . Samples were separated by SDS PAGE and transferred to PVDF membranes before probing with primary antibodies at the final concentrations, anti-Cox4 (mitoscience 1: 1000), anti-Cox2 (mitoscience 1:1000), anti-Por1 (Molecular Probes, Invitrogen 1:500) and anti-actin (1:2000).
Rhodamine-phalloidin and DAPI staining were performed as previously described for F-actin . Cells were viewed with an Olympus IX-81 fluorescence microscope with a 150 W Xenon/mercury lamp and an Olympus 150× Plan NeoFluor oil-immersion objective. GFP/RFP Co-localisation studies were performed using an Optosplit II Image Splitter (Cairn Scientific). Images were captured using a Hammamatsu ORCA AG digital camera using Olympus Cell R software.
Analysis of mitochondria
Assessment of reactive oxygen species content and mitochondrial membrane potential were carried out as previously described.
GFP labeled mitochondria were visualized using the plasmids pVTU100U-mtGFP and pYX122-mtGFP .
Analysis of cell death in yeast colonies
Between 10 and 20 cells were plated onto 9 cm YPAD agar plates containing 10 μM Phloxine B (Sigma). Colonies were grown for five days before being visualized using a Leica M2FLIII microscope and documented with a Leica DC300F colour camera. To visualise a cross section through the colony, a glass coverslip was inserted directly, bisecting the colony, and removed from the agar. Dissected colonies were visualized and documented immediately. All images were taken using the same illumination conditions and exposure times.
Iodine staining of yeast cells as an assay for glycogen content was carried out as previously described (Care et al., 2004).
cAMP/PKA regulation of respiratory function
The addition of cAMP to cells lacking both PDE2 and the TPK1 subunit genes led to a reduction in respiration similar to that observed in the Δpde2 single mutant. However while the level of routine respiration differed between strains (Figure 1A), triple mutant Δpde2Δtpk2Δtpk3, Δpde2Δtpk1Δtpk3 or Δpde2Δtpk1Δtpk2 strains did not exhibit the collapse of respiratory activity observed Δpde2 or Δpde2Δ tpk1 cells when cAMP levels were elevated (Figure 1A). Similarly Δpde2Δ tpk2 or Δpde2Δtpk3 double mutant cells were resistant to the effects of elevated cAMP. Collectively these data suggest that the collapse of respiration observed in Δpde2 cells when grown in the presence of exogenous cAMP requires the presence of both TPK2 and TPK3, but occurs independently of TPK1.
We then examined the accumulation of ROS in response to cAMP elevation within cell populations expressing Tpk1p, Tpk2p or Tpk3p as their sole source of PKA activity (Figure 1B). We found that the elevation of cAMP in strains expressing TPK1 only resulted in no difference in the number of cells producing high ROS levels within a population (Figure 1B). In contrast, cells expressing TPK2 or TPK3 only did show an increase in the number of ROS producing cells when cAMP levels were increased (Figure 1B). These data are in accordance with the findings in Figure 1A, that both TPK2 and TPK3 play a role in the regulation of respiratory activity in response to cAMP elevation. However it should be noted that cells expressing TPK3 only displayed the most significant increase in the number of ROS producing cells (Figure 1B), indicating that this PKA subunit may play a more fundamental role in the regulation of radical production.
Tpk3p activity is sufficient to induce respiratory collapse and ROS production
Typical respirometry profiles for wild type and Δpde2 cells cultured in the presence of 4 mM cAMP are presented (Figure 2A). Significant differences were apparent between the respirometry profiles obtained for wild type and Δpde2 strains when grown in the presence of exogenous cAMP. While wild type cells typically demonstrated a routine respiration of around 6 pmols/s/106 cells under these conditions, cells lacking PDE2 showed a significant reduction, typically less than 2 pmols/s/106 cells (Figure 2A). This suggests that high levels of cAMP signaling suppress the respiratory activity of yeast mitochondria. To investigate this further we examined the respirometry profiles of wild type, Δpde2, Δpde2Δtpk3 and Δpde2Δtpk3 + TPK3 grown in the presence or absence of cAMP (Figure 2B and summarised in Additional File 2). When cultured in the absence of cAMP both wild type and the double mutant (Δpde2Δtpk3) displayed similar routine respiration rates and maximal ETS levels upon FCCP addition. Interestingly the single mutant, Δpde2, showed a 20% increase in routine respiration and a maximal ETS that appeared higher than wild type when grown in the absence of additional cAMP (Figure 2B). Overexpression of TPK3 from a plasmid in Δpde2Δtpk3 resulted in a significant reduction in routine respiration, and notably cells failed to significantly increase respiration rate upon FCCP addition. In all strains the LEAK level of respiration appeared to be constant (Figure 2B).
The addition of 4 mM cAMP did appear to have a small effect on respiratory activity in wild type cells using this highly sensitive assay. We observed a 17% reduction in routine respiration when compared to wild type cells cultured without cAMP. A similar reduction was observed in the LEAK level of respiration, but the maximal ETS level remained unchanged. When cells lacking PDE2 were grown in the presence of exogenous cAMP we consistently observed a dramatic fall in routine respiration and maximal ETS rate (Figure 2C). Confirmation that Tpkp3 was responsible was obtained as respiratory activity was largely restored in Δpde2Δtpk3 cells grown under the same conditions (Figure 2C). Further evidence was obtained by the re-expression of TPK3 in Δpde2Δtpk3 cells which resulted in the loss of respiration in the presence of exogenous cAMP. In addition, the addition of FCCP did not elevate respiration in either Δpde2 Δtpk3 or Δpde2 Δtpk3+TPK3 cells, pointing to a loss of proton gradient across the inner mitochondrial membrane, further supporting the notion that elevated Tpkp3 activity leads to a loss of mitochondrial membrane potential. The data provided may also suggest that elevated levels of Tpk3p activity leads to reduction in the levels of complete and functional electron transport complexes.
Effects of TPK3 on ROS production and mitochondrial morphology
In order to investigate the effects of Tpk3p on mitochondrial morphology we made use of a plasmid that allows the expression of a mitochondrial targeted RFP protein (a kind gift from J. Shaw University of Utah) (Figure 3B). While a reticular network was seen in wild-type cells, mitochondria in Δpde2 mutant cells appeared reduced in number, were fragmented and visibly enlarged when grown in the presence of exogenous cAMP (Figure 3B). The abnormal mitochondria observed could be restored to a wild type appearance in cells lacking both PDE2 and TPK3 (Figure 3B). We also noted that in the Δpde2 mutant only a few enlarged structures of mtDNA could be seen when compared to the numerous structures visible in wild type (data not shown). In line with the improvement in mitochondrial morphology, the number of mt nucleoids was increased in Δpde2Δtpk3 cells (data not shown). In addition fragmentation of genomic DNA (gDNA) was apparent in Δpde2 cells, a phenotype indicative of apoptosis. In contrast nuclear DNA fragmentation did not appear in Δpde2Δtpk3 cells (data not shown), consistent with our previous reports that Tpk3p induced mitochondrial dysfunction can trigger an apoptotic form of cell death [27, 30]. In line with the ROS accumulation result (Figure 3A), the re-expression of TPK3 in Δpde2Δtpk3 was sufficient to restore the highly fragmented mitochondrial appearance observed in Δpde2 cells.
To confirm that the kinase activity of Tpk3p is responsible for the respiratory and morphological defects observed in Dpde2 cells when cAMP levels are elevated we generated a kinase dead version of the enzyme. Deminoff et al (2006) demonstrated that the mutagenesis of residues K336 and H338 to alanine in the yeast PKA, Tpk1p produced a protein lacking kinase activity. As the catalytic domain of TPK1 and TPK3 are highly conserved, we mutagenised the equivalent K337 and H339 residues of Tpk3p to alanine. In line with these mutations rendering Tpk3p inactive, the expression of TPK3 KD in Dpde2Dtpk3 cells did not lead to the collapse of respiration (Figure 3C) or to the manifestation of a highly fragmented mitochondrial network (Figure 3D). These data further suggest that elevated kinase activity of Tpk3p is sufficient to induce respiratory collapse and ROS production in yeast mitochondria.
cAMP/Tpk3p Signalling regulates cell death in yeast colonies
Microarray analysis of changes in gene expression mediated by Tpk3p activity
Selected genes downregulated by elevated cAMP/TPK3 signalling
PKA catalytic subunit
PKA catalytic subunit
Mitochondrial external NADH dehydrogenase, catalyzes the oxidation of cytosolic NADH;
Mitochondrial external NADH dehydrogenase, catalyzes the oxidation of cytosolic NADH;
Iron-sulfur protein subunit of succinate dehydrogenase Complex II
Catalyzes the second step in ubiquinone (coenzyme Q) biosynthesis
Rieske Iron Sulphur Protein of Complex III
Cytochrome c1, catalytic subunit of complex III
Cytochrome c1 heme lyase, involved in maturation of CYT1
Cytochrome c, isoform 1;transfers electrons from Complex III to Complex IV
Subunit IV of cytochrome c oxidase (Complex IV)
Heme A:farnesyltransferase, required for cytochrome c oxidase activity;
Copper-binding protein of the mitochondrial inner membrane, required for cytochrome c oxidase
Regulates mitochondrial expression of subunits 6 (Atp6p) and 8 (Atp8p) of Complex V
Subunit k of the mitochondrial F1F0 ATP synthase
Cytosolic superoxide dismutase
Mitochondrial superoxide dismutase; protects cells against oxygen toxicity
Cytosolic catalase T, has a role in protection from oxidative damage by hydrogen peroxide
Selected genes uppregulated by elevated cAMP/TPK3 signalling
Responds to DNA damage or replication arrest, transcription is induced by DNA damage
Thiazole synthase, required for thiamine biosynthesis and for mitochondrial genome stability
Glucose transporter, expressed in the presence of glucose, repressed when glucose is limited
Supressor of SUC2 expressed under high glucose levels
OMM protein, linksArp2/3 complex with the mitochore promotes degradation of mRNAs for select nuclear-encoded mitochondrial proteins
IMM ADP/ATP translocator, exchanges cytosolic ADP for mitochondrially synthesized ATP; roles in maintenance of viability and in respiration
A known feature of PKA activity is the ability to suppress the activation of biosynthesis of the storage carbohydrates glycogen and trehalose. This effect was noted in our array data. Several genes whose products are involved in glycogen and trehalose biosynthesis were downregulated in a Tpk3p dependent manner (Additional File 5). As a further control to verify the validity of our microarray data we investigated whether elevated Tpkp3 activity resulted in the suppression of glycogen accumulation as cells enter the stationary phase of growth. Wild type, Δpde2 and Δpde2 Δtpk3 cells were grown for 48 h and stained for glycogen accumulation as described in materials and methods. Wild type cells start to accumulate glycogen at this point of growth (Figure 5C), however we found that cells lacking PDE2 fail to accumulate glycogen. This failure to accumulate glycogen could be reversed by the additional deletion of TPK3 (Figure 5C).
The microarray data also suggested that the elevation of cAMP levels leds to a Tpk3p dependent reduction in COX4 transcript levels, a core component of Cytochrome c Oxidase. We tested whether this observation could be re-capitulated at the protein level by conducting a western blot using wild type, Δpde2 and Δpde2Δtpk3 strains were grown to diauxic shift in the absence or presence of 4 mM cAMP. In all strains grown without the addition of exogenous cAMP there appeared to be similar levels of Cox4p (Figure 5D). However upon addition of cAMP Cox4p was barely detectable in Δpde2 cells and a partial restoration was observed in Δpde2Δtpk3 cells. As Cox4p is essential for complex IV stability, these data suggested that high levels of cAMP and Tpk3p activity may lead to a loss or respiration as a result of the loss of Cytochrome c oxidase from the mitochondria. In line with this the level of Cox2p, which is encoded by the mitochondrial genome, was also greatly reduced upon cAMP elevation in Δpde2 cells (Figure 5D).
cAMP/PKA signaling regulates mitochondrial function and cell death via transcription factor activity
HAP4 activity leads to the cAMP/PKA dependent biogenesis of dysfunctional mitochondria prone to ROS production
The activity of cAMP/PKA signalling pathways appears to play an important role in the regulation of cell death in eukaryotes. For example it has been shown in lymphoid cells that elevated cAMP/PKA levels cause transcriptional changes that promote mitochondrial dependent apoptosis  andcan induce apoptosis via the up-regulation of Smac/DIABLO in cultured HeLa cells . Our previous studies suggest that in yeast excessive PKA activity can also trigger, albeit via distinct signalling mechanisms to those observed to date in mammalian systems, mitochondria dependent apoptosis [27, 28, 30]. In yeast cAMP/PKA stimulated apoptosis involves the production of ROS from mitochondria. The mitochondrial state that leads to ROS production in actin aggregating cells requires PKA activity, and in particular the function of one of the three PKA subunits found in S. cerevisiae, Tpk3p [27, 30]. Our data presented here supports a role for PKA signalling in regulating the activity of mitochondria via transcriptional control. In support of this our microarray analysis of the effects of cAMP elevation revealed a Tpk3p dependent suppression of a significant number of genes involved in mitochondrial function. Of particular note were genes whose products contribute to the electron transport system. The Tpk3p dependent downregulation of genes required for respiration also resulted in a significant reduction in respiratory capacity. Both the regulation of transcription and respiration could be restored by deletion of TPK3, implicating this single enzyme as an important regulator of mitochondrial biogenesis. Another possibility that must be considered is that Tpk3p localises to the mitochondria and regulates function directly via phosphorylation of protein targets. PKA activity within the mitochondria has been widely reported in mammalian systems and is known to modulate the activity of the respiratory chain . Interestingly a fully functional carbon dioxide sensing cAMP/PKA signalling system has recently been identified within human HeLa and COS-1 cell lines . In yeast, the mitochondrial histone-like protein Abf2 has been shown to be phosphorylated by PKA, suggesting that activity of cAMP dependent phosphorylation also occurs within mitochondria of lower eukaryotes . Phosphorylation of Abf2p by PKA was shown to inhibit its ability to interact with DNA, and the loss of ABF2 resulted in mtDNA instability . However fluorescence microscopy studies suggest that Tpk1p, Tpk2p and Tpk3p do not co-localise with the mitochondria (our unpublished results). In addition to this, the published mitochondrial proteome from S. cerevisae, despite identifying a number of protein kinases within the organelle, did not reveal the presence of PKA enzymes . Further studies are required to fully address whether, as is the case in mammalian cells, cAMP/PKA signalling occurs within yeast mitochondria.
Further evidence that cAMP/Tpk3 signalling regulates mitochondrial biogenesis at the level of transcription comes from our finding that respiratory function, ROS production and cell death can be manipulated by rescued to varying degrees by the transcription factors SOK2, SKO1 and HAP4, which were predicted to play a role via bioinformatic analysis of our microarray data. Both SOK2 and SKO1 are transcription factors that have been previously implicated in cAMP signalling systems, and act as suppressor elements within stress signalling pathways. SOK2 is known to be regulated by cAMP/PKA activity  and to play an important role in the regulation of cell death in colonies in response to ammonia signalling . SKO1 is a repressor that mediates HOG pathway-dependent regulation by binding to cAMP response elements (CRE) sites in target promoters . It is also known to play a regulatory role within cAMP/PKA stress signalling and can be phosphorylated by both HOG1 and PKA at independent sites . It is likely that some of the toxicity associated with elevated cAMP/PKA activity occurs as a result of increased stress response suppression via the activity of SOK2 and SKO1. In line with this, the deletion of either SOK2 or SKO1 led to a partial restoration of respiratory activity and reduction in ROS production in Δpde2 cells grown in the presence of cAMP.
More significantly, the deletion of HAP4, a known mitochondrial biogenesis factor , prevented ROS production and cell death in Δpde2 cells grown in the presence of cAMP. This suggests that the activity of HAP4 present within cells experiencing high cAMP/PKA signalling is responsible for the production of ROS. The overexpression of HAP4 led to the partial restoration of mitochondrial biogenesis in Δpde2 cells grown under conditions of cAMP elevation. However the additional mitochondria produced were not competent to respire and continued to produce high levels of ROS. We found that the overexpression of HAP4 in wild type cells also gave rise to mitochondria with reduced respiratory capacity, which were prone to ROS production. These data suggest that as yet unidentified factors aside from HAP4 are required to ensure the biogenesis of fully functional mitochondria. As the loss of TPK3 led to the restoration of fully functional mitochondria in Δpde2 cells grown under conditions of cAMP elevation it is likely that PKA function regulates mitochondrial activity via more than one effector to co-ordinate biogenesis in response to environmental change and metabolic demand. The search for further downstream targets of Tpk3p that are involved in mitochondrial regulation and biogenesis is currently underway. Elevation of Tpk3p activity also led to striking morphological defects, with mitochondria appearing fewer in number and as larger swollen structures. These mitochondria also exhibited reduced membrane potential and a reduction in respiratory chain components. Very similar features were reported to occur in mutants lacking Mdm38p and were attributed to the role that this protein plays in K+/H+ exchange  raising the possibility that elevated Tpk3p induces a loss of homeostasis within the organelle.
Our observations suggest that elevated Tpkp3 activity induces a novel mitochondrial state that results in non-respiring mitochondria prone to the production of ROS. A key question arising from this research is therefore, why do mitochondria damaged by elevated Tpk3p activity produce ROS? Interestingly a previous study has proposed that constitutive activation of the Ras pathway, via expression of the Ras val19 allele, leads to a state 4 non-phosphorylating respiration that is prone to ROS production . This study also suggested that the respiratory activity and mitochondrial membrane potential of Ras val19 cells becomes elevated, offering a plausible explanation as to the origin of ROS . However in actin aggregating cells, which also exhibit constitutive Ras activation, and in Δpde2 cells grown in the presence of 4 mM cAMP, there exists little or no mitochondrial membrane potential . This was confirmed by treatment of cells with FCCP, a proton ionophore, that allows protons to flow back through the inner mitochondrial membrane into the matrix. This releases the inhibition caused by the build up of a proton gradient and allows the electron transport system to operate at its maximal rate (ETS). In cells with functional mitochondria, FCCP treatment normally results in an increase in oxygen consumption of around 30-40% (Figure 3A). However cells lacking PDE2 failed to respond to FCCP when grown in the presence of exogenous cAMP. This supports our suggestion that elevated Tpk3p activity results in a loss of mitochondrial membrane potential. We would argue that the constitutive activation of Ras/cAMP/PKA signalling can lead to altered mitochondrial function prone to ROS function that does not occur as a result of state 4 respiration.
In summary, our study has revealed that the loss of regulation of cAMP/PKA signalling leads to cell death that requires TPK3 dependent transcriptional remodelling that suppresses stress response signalling and promotes mitochondrial dysfunction. Tpk3p appears to facilitate cell death via action on several downstream targets, notably HAP4, which induce the formation of dysfunctional mitochondria which fail to respire and produce high levels of ROS. The loss of respiratory capacity within mitochondria has been associated with a wide range of human disorders including stroke, cardioencephalomyopathy, hepatic failure and Leigh's syndrome [56, 57] and is also associated with aging and aging-related degenerative diseases such as Alzheimer's . We hypothesise that the increased production of respiratory incompetent mitochondria that are prone to ROS production may contribute to the pathology of a variety of age related disease states. Further research in this area utilising yeast as a model eukaryote may well provide insights of important medical relevance.
electron transport system
Protein Kinase A
Reactive Oxygen Species
Thanks to Dr. Tobias von der Harr and Dr. G.U.Innes for critical reading of this manuscript and helpful comments. This work was sponsored by a Medical Research Council (MRC) career development fellowship to CWG (ref No. 78573).
- Butow RA, Avadhani NG: Mitochondrial Signaling: The Retrograde Response. Molecular Cell. 2004, 14 (1): 1-15. 10.1016/S1097-2765(04)00179-0.View ArticlePubMedGoogle Scholar
- McBride HM, Neuspiel M, Wasiak S: Mitochondria: More Than Just a Powerhouse. Current Biology. 2006, 16 (14): R551-R560. 10.1016/j.cub.2006.06.054.View ArticlePubMedGoogle Scholar
- Wang C, Youle RJ: The Role of Mitochondria in Apoptosis. Annual review of genetics. 2009Google Scholar
- Passos JF, Simillion C, Hallinan J, Wipat A, von Zglinicki T: Cellular senescence: unravelling complexity. Age Dordrecht, Netherlands. 2009Google Scholar
- Chan SL, Wei Z, Chigurupathi S, Tu W: Compromised respiratory adaptation and thermoregulation in aging and age-related diseases. Ageing research reviews. 2009Google Scholar
- Turner N, Heilbronn LK: Is mitochondrial dysfunction a cause of insulin resistance?. Trends in endocrinology and metabolism: TEM. 2008, 19 (9): 324-330. 10.1016/j.tem.2008.08.001.View ArticlePubMedGoogle Scholar
- Gogvadze V, Orrenius S, Zhivotovsky B: Mitochondria as targets for chemotherapy. Apoptosis. 2009, 14 (4): 624-640. 10.1007/s10495-009-0323-0.View ArticlePubMedGoogle Scholar
- Mahad D, Lassmann H, Turnbull D: Review: Mitochondria and disease progression in multiple sclerosis. Neuropathology and applied neurobiology. 2008, 34 (6): 577-589. 10.1111/j.1365-2990.2008.00987.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Gibson GE, Karuppagounder SS, Shi Q: Oxidant-induced changes in mitochondria and calcium dynamics in the pathophysiology of Alzheimer's disease. Annals of the New York Academy of Sciences. 2008, 1147: 221-232. 10.1196/annals.1427.038.PubMed CentralView ArticlePubMedGoogle Scholar
- Henchcliffe C, Beal MF: Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis. Nature clinical practice. 2008, 4 (11): 600-609. 10.1038/ncpneuro0924.View ArticlePubMedGoogle Scholar
- Boveris A, Navarro A: Brain mitochondrial dysfunction in aging. IUBMB life. 2008, 60 (5): 308-314. 10.1002/iub.46.View ArticlePubMedGoogle Scholar
- Ishii N: Role of oxidative stress from mitochondria on aging and cancer. Cornea. 2007, 26 (9 Suppl 1): S3-9. 10.1097/ICO.0b013e31812f6745.View ArticlePubMedGoogle Scholar
- Orrenius S: Reactive oxygen species in mitochondria-mediated cell death. Drug metabolism reviews. 2007, 39 (2-3): 443-455. 10.1080/03602530701468516.View ArticlePubMedGoogle Scholar
- Hock MB, Kralli A: Transcriptional control of mitochondrial biogenesis and function. Annual review of physiology. 2009, 71: 177-203. 10.1146/annurev.physiol.010908.163119.View ArticlePubMedGoogle Scholar
- Ohlmeier S, Kastaniotis AJ, Hiltunen JK, Bergmann U: The Yeast Mitochondrial Proteome, a Study of Fermentative and Respiratory Growth. J Biol Chem. 2004, 279 (6): 3956-3979. 10.1074/jbc.M310160200.View ArticlePubMedGoogle Scholar
- Hlavata L, Aguilaniu H, Pichova A, Nystrom T: The oncogenic RAS2 val19 mutation locks respiration, independently of PKA, in a mode prone to generate ROS. The EMBO journal. 2003, 22 (13): 3337-3345. 10.1093/emboj/cdg314.PubMed CentralView ArticlePubMedGoogle Scholar
- Hlavata L, Nachin L, Jezek P, Nystrom T: Elevated Ras/protein kinase A activity in Saccharomyces cerevisiae reduces proliferation rate and lifespan by two different reactive oxygen species-dependent routes. Aging cell. 2008, 7 (2): 148-157. 10.1111/j.1474-9726.2007.00361.x.View ArticlePubMedGoogle Scholar
- Reinders A, Burckert N, Boller T, Wiemken A, De Virgilio C: Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase. Genes & development. 1998, 12 (18): 2943-2955.View ArticleGoogle Scholar
- Toda T, Cameron S, Sass P, Zoller M, Scott J, McCullen B, Hurwitz M, Krebs E, Wigler M: Cloning and characterisation of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Molecular and cellular biology. 1987, 7: 1371-1377.PubMed CentralView ArticlePubMedGoogle Scholar
- Toda T, Cameron S, Sass P, Zoller M, Wigler M: Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell. 1987, 50: 277-287. 10.1016/0092-8674(87)90223-6.View ArticlePubMedGoogle Scholar
- Toda T, Uno I, Ishikawa T, Powers S, Kataoka T, Broek D, Cameron S, Broach J, Matsumoto K, Wigler M: In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985, 40 (1): 27-36. 10.1016/0092-8674(85)90305-8.View ArticlePubMedGoogle Scholar
- Robertson LS, Causton HC, Young RA, Fink GR: The yeast A kinases differentially regulate iron uptake and respiratory function. Proceedings of the National Academy of Sciences of the United States of America. 2000, 97 (11): 5984-5988. 10.1073/pnas.100113397.PubMed CentralView ArticlePubMedGoogle Scholar
- Robertson LS, Fink GR: The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proceedings of the National Academy of Sciences of the United States of America. 1998, 95 (23): 13783-13787. 10.1073/pnas.95.23.13783.PubMed CentralView ArticlePubMedGoogle Scholar
- Akada R, Yamamoto J, Yamashita I: Screening and identification of yeast sequences that cause growth inhibition when overexpressed. Mol Gen Genet. 1997, 254: 267-274. 10.1007/s004380050415.View ArticlePubMedGoogle Scholar
- Gerst JE, Ferguson K, Vojtek A, Wigler M, Field J: CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex. Molecular and cellular biology. 1991, 11 (3): 1248-1257.PubMed CentralView ArticlePubMedGoogle Scholar
- Mintzer KA, Field J: Interactions between adenylyl cyclase, CAP and RAS from Saccharomyces cerevisiae. Cellular signalling. 1994, 6 (6): 681-694. 10.1016/0898-6568(94)90050-7.View ArticlePubMedGoogle Scholar
- Gourlay CW, Ayscough KR: Actin-Induced Hyperactivation of the Ras Signalling Pathway Leads to Apoptosis in Saccharomyces cerevisiae. Molecular and cellular biology. 2006, 26 (17): 6487-6501. 10.1128/MCB.00117-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Gourlay CW, Carpp LN, Timpson P, Winder SJ, Ayscough KR: A role for the actin cytoskeleton in cell death and aging in yeast. J Cell Biol. 2004, 164 (6): 803-809. 10.1083/jcb.200310148.PubMed CentralView ArticlePubMedGoogle Scholar
- Gourlay CW, Ayscough KR: Identification of an upstream regulatory pathway controlling actin-mediated apoptosis in yeast. J Cell Sci. 2005, 118: 2119-2132. 10.1242/jcs.02337.View ArticlePubMedGoogle Scholar
- Leadsham JE, Miller K, Ayscough KR, Colombo S, Martegani E, Sudbery P, Gourlay CW: Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast. J Cell Sci. 2009, 122: 706-715. 10.1242/jcs.042424.PubMed CentralView ArticlePubMedGoogle Scholar
- Chevtzoff C, Vallortigara J, Averet N, Rigoulet M, Devin A: The yeast cAMP protein kinase Tpk3p is involved in the regulation of mitochondrial enzymatic content during growth. Biochem Biophys Acta. 2005, 1706: 117-125. 10.1016/j.bbabio.2004.10.001.PubMedGoogle Scholar
- Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH: A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic acids research. 2002, 30 (6): e23-10.1093/nar/30.6.e23.PubMed CentralView ArticlePubMedGoogle Scholar
- Mazon MJ, Behrens MM, Morgado E, Portillo F: Low activity of the yeast cAMP-dependent protein kinase catalytic subunit Tpk3 is due to the poor expression of the TPK3 gene. European journal of biochemistry/FEBS. 1993, 213 (1): 501-506. 10.1111/j.1432-1033.1993.tb17787.x.View ArticlePubMedGoogle Scholar
- Lascaris R, Bussemaker HJ, Boorsma A, Piper M, van der Spek H, Grivell L, Blom J: Hap4p overexpression in glucose-grown Saccharomyces cerevisiae induces cells to enter a novel metabolic state. Genome biology. 2003, 4 (1): R3-10.1186/gb-2002-4-1-r3.PubMed CentralView ArticlePubMedGoogle Scholar
- von der Haar T: Optimized protein extraction for quantitative proteomics of yeasts. PloS one. 2007, 2 (10): e1078-10.1371/journal.pone.0001078.PubMed CentralView ArticlePubMedGoogle Scholar
- Gourlay CW, Carpp LN, Timpson P, Winder SJ, Ayscough KR: A role for the actin cytoskeleton in cell death and aging in yeast. J Cell Biol. 2004, 164 (6): 803-809. 10.1083/jcb.200310148.PubMed CentralView ArticlePubMedGoogle Scholar
- Westermann B, Neupert W: Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae. Yeast. 2000, 16 (15): 1421-1427. 10.1002/1097-0061(200011)16:15<1421::AID-YEA624>3.0.CO;2-U.View ArticlePubMedGoogle Scholar
- Wilson RB, Renault G, Jacquet M, Tatchell K: The pde2 gene of Saccharomyces cerevisiae is allelic to rcal and encodes a phosphodiesterase which protects the cell from extracellullar cAMP. FEBS letters. 1993, 325 (3): 191-195. 10.1016/0014-5793(93)81071-7.View ArticlePubMedGoogle Scholar
- Nagley P, Hall RM, Ooi BG: Amino acid substitutions in mitochondrial ATPase subunit 9 of saccharomyces cerevisiae leading to oligomycin or venturicidin resistance. FEBS letters. 1986, 195 (1-2): 159-163. 10.1016/0014-5793(86)80152-1.View ArticlePubMedGoogle Scholar
- Cain K, Griffiths DE: Studies of energy-linked reactions. Biochem J. 1977, 162: 575-580.PubMed CentralView ArticlePubMedGoogle Scholar
- Vachova L, Devaux F, Kucerova H, Ricicova M, Jacq C, Palkova Z: Sok2p transcription factor is involved in adaptive program relevant for long term survival of Saccharomyces cerevisiae colonies. The Journal of biological chemistry. 2004, 279 (36): 37973-37981. 10.1074/jbc.M404594200.View ArticlePubMedGoogle Scholar
- Vachova L, Palkova Z: Physiological regulation of yeast cell death in multicellular colonies is triggered by ammonia. The Journal of cell biology. 2005, 169 (5): 711-717. 10.1083/jcb.200410064.PubMed CentralView ArticlePubMedGoogle Scholar
- Garcia-Rodriguez LJ, Gay AC, Pon LA: Puf3p, a Pumilio family RNA binding protein, localizes to mitochondria and regulates mitochondrial biogenesis and motility in budding yeast. The Journal of cell biology. 2007, 176 (2): 197-207. 10.1083/jcb.200606054.PubMed CentralView ArticlePubMedGoogle Scholar
- Forsburg SL, Guarente L: Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes & development. 1989, 3 (8): 1166-1178.View ArticleGoogle Scholar
- Zhang L, Zambon AC, Vranizan K, Pothula K, Conklin BR, Insel PA: Gene expression signatures of cAMP/protein kinase A (PKA)-promoted, mitochondrial-dependent apoptosis. Comparative analysis of wild-type and cAMP-deathless S49 lymphoma cells. The Journal of biological chemistry. 2008, 283 (7): 4304-4313. 10.1074/jbc.M708673200.View ArticlePubMedGoogle Scholar
- Martinez-Velazquez M, Melendez-Zajgla J, Maldonado V: Apoptosis induced by cAMP requires Smac/DIABLO transcriptional upregulation. Cellular signalling. 2007, 19 (6): 1212-1220. 10.1016/j.cellsig.2007.01.001.View ArticlePubMedGoogle Scholar
- Pagliarini DJ, Dixon JE: Mitochondrial modulation: reversible phosphorylation takes center stage?. Trends in biochemical sciences. 2006, 31 (1): 26-34. 10.1016/j.tibs.2005.11.005.View ArticlePubMedGoogle Scholar
- Acin-Perez R, Salazar E, Kamenetsky M, Buck J, Levin LR, Manfredi G: Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation. Cell metabolism. 2009, 9 (3): 265-276. 10.1016/j.cmet.2009.01.012.PubMed CentralView ArticlePubMedGoogle Scholar
- Cho JH, Lee YK, Chae CB: The modulation of the biological activities of mitochondrial histone Abf2p by yeast PKA and its possible role in the regulation of mitochondrial DNA content during glucose repression. Biochimica et biophysica acta. 2001, 1522 (3): 175-186.View ArticlePubMedGoogle Scholar
- Sickmann A, Reinders J, Wagner Y, Joppich C, Zahedi R, Meyer HE, Schonfisch B, Perschil I, Chacinska A, Guiard B: The proteome of Saccharomyces cerevisiae mitochondria. Proceedings of the National Academy of Sciences of the United States of America. 2003, 100 (23): 13207-13212. 10.1073/pnas.2135385100.PubMed CentralView ArticlePubMedGoogle Scholar
- Ward MP, Gimeno CJ, Fink GR, Garrett S: SOK2 may regulate cyclic AMP-dependent protein kinase-stimulated growth and pseudohyphal development by repressing transcription. Molecular and cellular biology. 1995, 15 (12): 6854-6863.PubMed CentralView ArticlePubMedGoogle Scholar
- Rep M, Proft M, Remize F, Tamas M, Serrano R, Thevelein JM, Hohmann S: The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage. Molecular microbiology. 2001, 40 (5): 1067-1083. 10.1046/j.1365-2958.2001.02384.x.View ArticlePubMedGoogle Scholar
- Pascual-Ahuir A, Posas F, Serrano R, Proft M: Multiple levels of control regulate the yeast cAMP-response element-binding protein repressor Sko1p in response to stress. The Journal of biological chemistry. 2001, 276 (40): 37373-37378. 10.1074/jbc.M105755200.View ArticlePubMedGoogle Scholar
- Lascaris R, Piwowarski J, van der Spek H, Teixeira de Mattos J, Grivell L, Blom J: Overexpression of HAP4 in glucose-derepressed yeast cells reveals respiratory control of glucose-regulated genes. Microbiology (Reading, England). 2004, 150 (Pt 4): 929-934.View ArticleGoogle Scholar
- Nowikovsky K, Reipert S, Devenish RJ, Schweyen RJ: Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy. Cell death and differentiation. 2007, 14 (9): 1647-1656. 10.1038/sj.cdd.4402167.View ArticlePubMedGoogle Scholar
- Barrientos A, Barros MH, Valnot I, Rotig A, Rustin P, Tzagoloff A: Cytochrome oxidase in health and disease. Gene. 2002, 286 (1): 53-63. 10.1016/S0378-1119(01)00803-4.View ArticlePubMedGoogle Scholar
- Shoubridge EA: Cytochrome c oxidase deficiency. American journal of medical genetics. 2001, 106 (1): 46-52. 10.1002/ajmg.1378.View ArticlePubMedGoogle Scholar
- Sullivan PG, Brown MR: Mitochondrial aging and dysfunction in Alzheimer's disease. Progress in neuro-psychopharmacology & biological psychiatry. 2005, 29 (3): 407-410.View ArticleGoogle Scholar
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