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Vacuolar functions determine the mode of cell death
Biochimica et Biophysica Acta 1793 (2009) 540–545
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r
Vacuolar functions determine the mode of cell death Alexandra Schauer, Heide Knauer, Christoph Ruckenstuhl, Heike Fussi, Michael Durchschlag, Ulrike Potocnik, Kai-Uwe Fröhlich ⁎ Institute of Molecular Biosciences, University of Graz, Humboldtstraβe 50, 8010 Graz, Austria
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Article history: Received 9 September 2008 Received in revised form 13 November 2008 Accepted 17 November 2008 Available online 27 November 2008 Keywords: Vacuole Necrosis Apoptosis Acetic acid pH regulation
a b s t r a c t The yeast vacuole plays a crucial role in cell homeostasis including pH regulation and degradation of proteins and organelles. Class C VPS genes code for proteins essential for vacuolar and endosomal vesicle fusion, their deletion results in the absence of a detectable vacuole. We found that single gene deletions of class C VPS genes result in a drastically enhanced sensitivity to treatment with acetic acid whereas sensitivity towards H2O2 remains largely unaffected. Interestingly acetic acid treatment known as an established inducer of yeast apoptosis leads to necrosis in class C VPS deletion strains. Their intracellular pH drops from 6.7 to 5.5 after acetic acid treatment, while in wild type the pH drops to just 6.3. When the intracellular pH in wild type is lowered below pH 5.5 using a higher concentration of acetic acid, the survival rate is similarly low as in the class C VPS mutants, however, the death phenotype is predominantly apoptotic. Hence, the vacuole not only prevents acetic acid induced cell death by buffering the cytosolic pH, but it also has a proapoptotic function. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In the past few years, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis is the best studied form of programmed cell death, phenotypically characterised by cytological marker events. Typical apoptotic markers have been discovered in yeast like externalization of phosphatidylserine to the outer leaflet of the plasma membrane, DNA fragmentation and chromatin condensation [1]. Homologues and orthologues of key players in metazoan apoptosis have been discovered in Saccharomyces cerevisiae including caspase orthologue metacaspase Yca1p [2], apoptosis inducing factor Aif1p [3], Endonuclease G [4], Nma111p, a member of the HtrA2/Omi protein family [5], and Bir1p, an inhibitor of apoptosis (IAP) protein [6]. In contrast to the organised apoptotic death, necrosis has initially been described as an uncontrolled cell death where cells lose control of their ionic balance, imbibe water, and lyse. But necrosis appears to be more than an accident. Whereas the morphology of necrosis is variable some marker events are found consistently like ATP depletion or loss of membrane pumps as well as production of reactive oxygen species (ROS) and cytoplasmic Ca2+ increase [7,8]. Furthermore, in mammals necrosis can be prevented by inhibition of certain enzymes or processes. Knockout of the cyclophilin D gene confers resistance to necrosis induced by ROS or Ca2+ overload in fibroblasts or hepatocytes [9–11]. On the other hand stressed baby mouse kidney epithelial cells
⁎ Corresponding author. Fax: +43 316 380 9898. E-mail address: [email protected] (K.-U. Fröhlich). 0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.11.006
knocked out in the proapoptotic proteins Bax and Bak and additionally blocked in autophagy die displaying a necrotic phenotype [12]. So there are increasing evidences that necrosis also can be a well orchestrated form of programmed cell death. In both cases (apoptosis and necrosis) the lysosomal compartment plays an important role. For example, low amounts of lysosomotropic toxins can trigger apoptosis and high doses can initiate necrosis via lysosomal membrane permeabilization [13]. The yeast S. cerevisiae contains a single large lysosome termed the vacuole. It plays an important role in pH- and ion-homeostasis, and is used as a storage compartment for ions. Another important function of the vacuole, especially during nutrient limitation, is the bulk degradation of proteins and even whole organelles by means of autophagy. Key components of the sophisticated transport pathway into the vacuole are the class C VPS (vacuolar protein sorting) proteins (Vps16p, Vps33p, Pep3p and Pep5p) which are part of the HOPS and CORVET complex. The HOPS complex (for homotypic fusion and vacuole protein sorting) additionally contains Vps41p and Vam6p and interacts with Vam3p, a vacuolar SNARE (soluble N-ethylmaleimide-sensitive-factor attachment receptor) and also activates GTPase Ypt7p/Rab which in turn initiates docking [14,15]. The HOPS complex ensures the accurate and efficient fusion of the autophagosome with the vacuole and couples the function of SNAREs and Rab GTPases [15–18]. At the endosome this function is executed by the CORVET (class C core vacuole/endosome tethering) complex which is a homologue of the HOPS complex containing the above described four class C VPS proteins and additionally Vps3p and Vps8p. The CORVET complex interacts with Rab5 homologue VPS21p [19].
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In view of the pivotal role of the lysosome in mammalian cell death, it seemed worthwhile to investigate the role of the vacuole in yeast cell death scenarios. Intracellular acidification induced with nigericin (K+/H+ antiporter) has been reported to cause cell death in yeast [20]. To link intracellular acidification, mode of cell death and the vacuole, appropriate candidate genes like the class C VPS genes whose single deletion results in the absence of a visible vacuole were tested. Class C VPS deletion strains show an enhanced sensitivity to treatment with acetic acid pH = 3 whereas to hydrogen peroxide (H2O2) treatment they react almost like wild type. We also report that acetic acid induced apoptosis requires an intact vacuolar fusion machinery. 2. Materials and methods 2.1. Yeast strains and growth conditions The single gene deletion strains Δvps16, Δvps33, Δpep3, and Δpep5, respectively of S. cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) were purchased from EUROSCARF (Frankfurt, Germany). All strains were grown on SC medium containing 0.17% yeast nitrogen base without amino acids and ammonium sulphate (Difco), 0.5% (NH4)2SO4 and 30 mg/l of all amino acids (except 80 mg/l histidine and 200 mg/l leucine), 30 mg/l adenine, and 320 mg/l uracil with 2% glucose (SCD).
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3. Results 3.1. Vacuolar class C VPS deletion strains show a reduced life span resulting from necrotic cell death Among the more than 50 genes required for vacuolar protein sorting in S. cerevisiae, only four VPS genes belong to the class C group, of which single gene deletions lack a readily identifiable vacuole [23]. The vacuolar class C VPS genes, VPS16, VPS33, PEP3, and PEP5, code for the components shared by the HOPS and the CORVETcomplex implicated in vacuolar and endoplasmic fusion. For yeast, chronological ageing is defined as the survival of cells in stationary cultures and is used as a model system for ageing of post-mitotic cells and tissues. To determine the impact of an absent vacuole during chronological ageing appropriate experiments were performed. We found that the class C VPS deletion strains show a decreased chronological life span compared to wild type (Fig.1A). The viability of Δpep3 and Δpep5 strains decreased to 20–40% at day 2 and finally no survival could be observed at day 3 whereas wild type shows 50% survival at day 3. This is accompanied by an enhanced accumulation of ROS in the mutant strains (Fig. 1B). Growth medium (of wild type and of class C VPS deletion strains) acidifies during cultivation of yeast as shown by Fabrizio et al. [24], detectable from day 1 on. The deletion strains show large amounts of propidium iodide (PI) positive cells indicating necrotic cell death (Fig. 1C). The decreased chronological life span is shared with other VPS deletion strains (e.g. vps19, vps34, data not shown).
2.2. Survival assay and chronological ageing For experiments testing oxygen stress or acid tolerance, experiments were performed as described [2]. At a cell count of 3.5 × 106, strains were incubated in 10 ml SC medium with indicated concentrations of acetic acid or 0.7 mM hydrogen peroxide for 3 h. Two different acetic acid solutions were used: acetic acid pH = 4.05 (10 M acetic acid, 3.4 M K-acetate stock) or acetic acid pH = 3 (10 M acetic acid, 0.09 M K-acetate stock). Addition of 100 mM acetic acid pH = 4.05 decreases the pH of the growth medium from pH 6 to 4.5, and addition of 100 mM acetic acid pH = 3 to 3. The percentage of survival was determined relative to an untreated control. Chronological ageing experiments were performed as described [21]. 2.3. Cell death marker assays AnnexinV/PI costaining was performed as described [1]. For quantifications using flow cytometry 30,000 cells were evaluated with BD FACSAria and analyzed with BD FACSDiva software. For dihydroethidium (DHE) staining, 1 × 106 cells were harvested by centrifugation, resuspended in 250 μl of 2.5 μg/ml DHE in PBS, and incubated in the dark for 10 min. Relative fluorescence units (RFU) were determined by using a fluorescence reader (Tecan, GeniusPRO), or stained cells were counted by using flow cytometry. For propidium iodide (PI) staining, 1 × 106 cells were harvested by centrifugation, resuspended in 250 μl 2.5 μg/ml PI in PBS, and incubated in the dark for 10 min. Stained cells were counted by using flow cytometry. For TUNEL staining, 1 × 107 cells were harvested by centrifugation and staining was performed as described [4]. 10,000 cells of each sample were evaluated by flow cytometry. 2.4. Yeast intracellular pH measurement Intracellular pH of yeast cells was assessed by FACS analysis of cells stained with the pH-dependent fluorescent dye SNARF-4F (Invitrogen, Austria), essentially following the method described by Valli et al. [22]. In order to ensure sufficient activation of the dye the incubation time was increased to 30 min.
3.2. Class C VPS deletion strains show a drastically enhanced cell death with a predominantly necrotic phenotype after treatment with acetic acid pH = 3 Next, we investigated the reaction of class C VPS deletion strains towards classical inducers of apoptosis like acetic acid and H2O2 [25,26]. In survival assays H2O2 treated Δpep3 and Δpep5 deletion strains show no difference compared to wild type (Fig. 2A), with low ROS accumulation within cells (Fig. 2B) and low amounts of necrotic cells (Fig. 2C). PI/AnnexinV costaining reveals that H2O2 induces apoptosis in class C VPS null mutants (see Supplementary Fig. 1). Acetic acid pH = 3 treatment results in massive death of the class C VPS deletion strains, only about 5% of the cells survive (Fig. 2A), an increased ROS accumulation (about 100% DHE positive cells) and high amounts of necrotic cells (about 100% PI positive cells) in the class C VPS knock outs (Fig. 2B and C). In contrast, wild type treated with acetic acid pH = 3 shows 10.5% necrotic cells. Deletion strains in other vacuolar components like some subunits of the vacuolar ATPase (vma6, vma8 and vma13) or adaptor proteins for high-fidelity vesicle docking and fusion like vps19 are less sensitive towards acetic acid pH = 3 (see Supplementary Fig. 2), indicating a specific effect of class C VPS deletion strains. 3.3. pH influences survival after acetic acid treatment of class C VPS deletion strains To evaluate the role of pH on cell death in yeast various acetic acid treatments were used, lowering the pH of the growth medium to 4.5 or 3. Wild type cells showed no difference in survival after treatment with acetic acid pH = 4.05 or pH = 3 (Fig. 2A). In contrast, class C VPS deletion strains showed a far better survival when treated with acetic acid pH = 4.05 than with pH = 3 (Fig. 2A), and a lower portion of DHE and PI positive cells (Fig. 2B and C). Δvps33, Δpep3, and Δpep5 strains nearly reach wild type survival after treatment with acetic acid pH = 4.05. Class C VPS deletion strains treated with 100 mM acetic acid pH = 4.05 show DHE staining in less than 40% of the cells, while treatment with acetic acid pH = 3 results in almost 100% DHE positive cells (Fig. 2B). A similar effect could be observed in the PI stainings, indicative for necrosis (Fig. 2C).
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C VPS mutants a similar drop of intracellular pH could be detected (Fig. 3D). 3.4. In contrast to class C VPS deletion strains, low intracellular pH leads to apoptosis in wild type To get comparable survival rates between wild type and deletion strains, wild type cells were treated with higher concentrations of acetic acid (225 mM acetic acid pH = 4.05 or 150 mM acetic acid
Fig. 1. Deletion of class C VPS genes decreases chronological life span and increases ROS production. (A) Chronological life-span curves (i.e., cfu of 500 plated cells) of Δvps16 (♦), Δvps33 ( ), Δpep3 (▲) Δpep5 ( ), and of wild type (⁎). Data represent means of three independent experiments. (B) Quantification (fluorescence reader) of ROS accumulation using DHE-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments. (C) Quantification (FACS analysis) of necrotic cells using PI-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments.
To find out whether the different pH treatments have an influence on the mode of cell death, PI/AnnexinV costaining was performed. Seventy-eight percent of wild type cells treated with 100 mM acetic acid exhibit apoptotic markers, regardless of pH (Fig. 3B). In the class C VPS deletion strains acetic acid pH = 4.05 causes lower amounts of necrotic cells (about 20%) in contrast to treatment with acetic acid pH = 3 which causes a predominantly necrotic death (Fig. 3B and C). Measurement of the intracellular pH value of acetic acid treated cells showed that unstressed wild type and Δpep3 cells have an intracellular pH value of 6.7 (Fig. 3D) which is mostly unaffected by addition of 100 mM acetic acid pH = 4.05 for both strains. With 100 mM acetic acid pH = 3, the intracellular pH drops to 6.3 in wild type cells but to 5.5 in Δpep3. In the other class
Fig. 2. Acetic acid pH = 3 decreases survival of class C VPS deletion strains and increases ROS and necrosis. (A) Survival of Δvps16, Δvps33, Δpep3, Δpep5 and wild type. Survival was determined after cell death induction with 0.7 mM H2O2, 100 mM acetic acid (pH = 3) or 100 mM acetic acid (pH = 4.05). Data represent means of three independent experiments. (B) Quantification (FACS analysis) of ROS accumulation using DHE-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments. (C) Quantification (FACS analysis) of necrotic cells using PI-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments.
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pH = 3), resulting in similar survival rates and intracellular pH drop as in mutant strains with 100 mM acetic acid pH = 3 (Fig. 3A and D). But while more than 80% of the wild type cells die, the amount of necrotic cells just increases to about 20% of the whole cellular population. In contrast, in the class C VPS knock outs almost 100% of the cells die with a necrotic phenotype after 100 mM acetic acid pH = 3 treatment (Fig. 3A and C). Apoptotic cells can become leaky during further cultivation, resulting in PI positives. To distinguish this secondary necrosis from primary accidental or regulated necrosis kinetic assays were performed. Over a period of 3 h the effects of 50 mM or 100 mM acetic acid pH = 3 or 225 mM acetic acid pH = 4.05 on survival and modes of death of wild type and Δpep3 strains were observed (Fig. 4). In wild type about 3% of the cells die with a necrotic phenotype after all three treatments. In contrast, in the Δpep3 strain necrosis gradually enhances over time with all acetic acid treatments (Fig. 4B), no TUNEL positives are observed over the time course (Fig. 4C). This indicates that apoptosis is suppressed by the lack of the vacuole and primary necrosis is the only way to go for class C VPS deletion strains. In some cases, the sum of TUNEL-positive (apoptotic) and PIpositive (necrotic) cells is lower than that of dead cells (e.g., wild type with 225 mM acetic acid pH = 4.05 in Fig. 4). This is probably due to a delayed effect of the experimental stress on survival even after plating on the cell count plates, and in differences in the kinetics of apoptotic marker development. As our wild type studies show, treatment with acetic acid concentrations up to 150 mM at low intracellular pH levels do not necessarily lead to necrosis, a functional vacuole directs the mode of death under acetic conditions towards apoptosis. Of course, this capacity can be overloaded, resulting in necrosis at very high concentrations or long exposure [25]. 4. Discussion In the 1950s the free radical theory of ageing was postulated by Harman [27]. In accordance to this theory we could demonstrate that class C VPS null mutants exhibit a drastically reduced survival in chronological ageing and accumulate high amounts of ROS which predominantly leads to necrosis. ROS cause irreversible damage mainly to mitochondria, resulting in a further increase of the ROS level [28]. Degradation of damaged structures is mediated via autophagy, requiring an efficient vacuole fusion machinery. Defects in autophagy in class C VPS knock outs could enhance the vicious circle of accumulation of damaged mitochondria and oxidised proteins, resulting in enhanced production of ROS and even more oxidative damage. Furthermore the acidification of the media during cultivation [24] could result in a drop of the intracellular pH in the class C VPS null mutants leading to enhanced ROS accumulation within the cells. While chronological ageing of wild type yeast predominantly results in apoptotic death, and can be delayed by antiapoptotic treatment (disruption of yeast caspase YCA1 [21]), mutants with Fig. 3. A functional vacuole is needed for acetic acid induced apoptosis. (A) Survival of wild type and Δpep3. Survival was determined after cell death induction with 100 mM or 225 mM acetic acid (pH = 4.05), or 100 mM or 150 mM acetic acid (pH = 3). Data represent means of three independent experiments. (B) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using AnnexinV/ PI costaining of spheroplasts in wild type and Δpep3. Note that digestion of the cell wall may result in membrane damage and PI staining. For precise determination of necrosis, see panel C. Data represent means of three independent experiments. (C) Quantification (FACS analysis) of necrotic cells using PI-staining of wild type and Δpep3. Data represent means of three independent experiments. (D) Intracellular pH determined by staining with the pH-dependent fluorescent dye SNARF-4F of wild type and Δpep3 after induction with acetic acid (pH = 4.05) or acetic acid (pH = 3), the intracellular pH of Δpep5, Δvps16 and Δvps33 is displayed after induction with 100 mM acetic acid (pH = 3). The broken line indicates the mean of intracellular pH in unstressed wild type and class C VPS deletion strains. Data represent means of three independent experiments.
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treatment: as an acidifying agent resulting in necrotic death, which can be prevented by vacuolar activities, and as a proapoptotic signal molecule, an effect which can not be prevented by, but instead requires a functional vacuole. As a result, acetic acid at pH = 3 or 4.05 has similar effects on wild type, a slight intracellular drop in pH has no influence on survival or the mode of death. Loss of vacuolar function in the class C VPS knock out strains had no effect with 100 mM acetic acid pH = 4.05 but results in intracellular acidification and massive necrotic death with acetic acid pH = 3. To achieve similar drop in the intracellular pH in wild type higher concentrations of acetic acid (225 mM acetic acid pH = 4.05 or 150 mM acetic acid pH = 3) were required. However, while the death rate is similar to class C VPS deletion strains, death is predominantly apoptotic in wild type. These data indicate that a functional vacuole is not only needed for intracellular pH regulation but also for executing apoptosis during low pH stress. In human cells a decrease in intracellular pH also acts as an initial trigger for a cascade of events resulting in apoptosis [29] in accordance to our results with yeast. Secretory pathway and pH regulation within the cell are crucial factors of cell death regulation and maintenance of cell integrity. In conclusion our results fit the hypothesis of Golstein and Kroemer [8]: programmed cell death developed from a necrotic mechanism and acquired additional, alternative terminal phenotypes (apoptotic, autophagic). In such a scenario, mechanisms to suppress the development of the original necrotic phenotype are required to allow the implementation of the alternatives like apoptosis or autophagy. The vacuole has a central role in the execution of autophagy, and in case of acetic acid stress also for preventing necrosis. Our findings suggest a role of the vacuole/lysosomes in a switch from necrotic to apoptotic death phenotype, giving it control over all responses to acetic stress and making it the central organelle in the regulation of cell death pathways under acetic conditions. Acknowledgements We are grateful to the Fonds zur Förderung der wissenschaftlichen Forschung (Austria) for grant S-9303-B05 to A.S., H.K. and K.-U.F. and Doctoral College scholarship for M.D., and to the European Commission, project TransDeath for A.S., C.R. and K.-U.F. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbamcr.2008.11.006. References
Fig. 4. Acetic acid leads to gradual increase of necrosis in Δpep3 strain. Wild type (dashed line, filled symbols) and Δpep3 (continuous line, empty symbols) were treated with 225 mM acetic acid (pH = 4.05; △, ▲), or 100 mM (□, ■) or 50 mM (○, ●) acetic acid (pH = 3). Data represent means of three independent experiments. (A) Survival, (B) necrotic cells (FACS analysis of PI-staining), and (C) apoptotic cells (FACS analysis of TUNEL-staining, with untreated control displayed as diamonds (◊, ♦)) were determined in time series.
reduced chronological life span have been found to exhibit a necrotic death phenotype [4], indicating that the cause is not acceleration of the wild type program, but an additional mechanism. In contrast, presence of a vacuole appears not to be important for H2O2 mediated apoptotic cell death, which we demonstrate in our stress experiments. H2O2 treatment leads to apoptosis in wild type as well as in class C VPS deletion strains (see Supplementary Fig. 1). Acetic acid induces apoptosis with its typical markers like chromatin condensation, DNA fragmentation, and phosphatidylserine externalization [25]. Our data indicate a dual role of acetic acid
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r
Vacuolar functions determine the mode of cell death Alexandra Schauer, Heide Knauer, Christoph Ruckenstuhl, Heike Fussi, Michael Durchschlag, Ulrike Potocnik, Kai-Uwe Fröhlich ⁎ Institute of Molecular Biosciences, University of Graz, Humboldtstraβe 50, 8010 Graz, Austria
a r t i c l e
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Article history: Received 9 September 2008 Received in revised form 13 November 2008 Accepted 17 November 2008 Available online 27 November 2008 Keywords: Vacuole Necrosis Apoptosis Acetic acid pH regulation
a b s t r a c t The yeast vacuole plays a crucial role in cell homeostasis including pH regulation and degradation of proteins and organelles. Class C VPS genes code for proteins essential for vacuolar and endosomal vesicle fusion, their deletion results in the absence of a detectable vacuole. We found that single gene deletions of class C VPS genes result in a drastically enhanced sensitivity to treatment with acetic acid whereas sensitivity towards H2O2 remains largely unaffected. Interestingly acetic acid treatment known as an established inducer of yeast apoptosis leads to necrosis in class C VPS deletion strains. Their intracellular pH drops from 6.7 to 5.5 after acetic acid treatment, while in wild type the pH drops to just 6.3. When the intracellular pH in wild type is lowered below pH 5.5 using a higher concentration of acetic acid, the survival rate is similarly low as in the class C VPS mutants, however, the death phenotype is predominantly apoptotic. Hence, the vacuole not only prevents acetic acid induced cell death by buffering the cytosolic pH, but it also has a proapoptotic function. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In the past few years, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis is the best studied form of programmed cell death, phenotypically characterised by cytological marker events. Typical apoptotic markers have been discovered in yeast like externalization of phosphatidylserine to the outer leaflet of the plasma membrane, DNA fragmentation and chromatin condensation [1]. Homologues and orthologues of key players in metazoan apoptosis have been discovered in Saccharomyces cerevisiae including caspase orthologue metacaspase Yca1p [2], apoptosis inducing factor Aif1p [3], Endonuclease G [4], Nma111p, a member of the HtrA2/Omi protein family [5], and Bir1p, an inhibitor of apoptosis (IAP) protein [6]. In contrast to the organised apoptotic death, necrosis has initially been described as an uncontrolled cell death where cells lose control of their ionic balance, imbibe water, and lyse. But necrosis appears to be more than an accident. Whereas the morphology of necrosis is variable some marker events are found consistently like ATP depletion or loss of membrane pumps as well as production of reactive oxygen species (ROS) and cytoplasmic Ca2+ increase [7,8]. Furthermore, in mammals necrosis can be prevented by inhibition of certain enzymes or processes. Knockout of the cyclophilin D gene confers resistance to necrosis induced by ROS or Ca2+ overload in fibroblasts or hepatocytes [9–11]. On the other hand stressed baby mouse kidney epithelial cells
⁎ Corresponding author. Fax: +43 316 380 9898. E-mail address: [email protected] (K.-U. Fröhlich). 0167-4889/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.11.006
knocked out in the proapoptotic proteins Bax and Bak and additionally blocked in autophagy die displaying a necrotic phenotype [12]. So there are increasing evidences that necrosis also can be a well orchestrated form of programmed cell death. In both cases (apoptosis and necrosis) the lysosomal compartment plays an important role. For example, low amounts of lysosomotropic toxins can trigger apoptosis and high doses can initiate necrosis via lysosomal membrane permeabilization [13]. The yeast S. cerevisiae contains a single large lysosome termed the vacuole. It plays an important role in pH- and ion-homeostasis, and is used as a storage compartment for ions. Another important function of the vacuole, especially during nutrient limitation, is the bulk degradation of proteins and even whole organelles by means of autophagy. Key components of the sophisticated transport pathway into the vacuole are the class C VPS (vacuolar protein sorting) proteins (Vps16p, Vps33p, Pep3p and Pep5p) which are part of the HOPS and CORVET complex. The HOPS complex (for homotypic fusion and vacuole protein sorting) additionally contains Vps41p and Vam6p and interacts with Vam3p, a vacuolar SNARE (soluble N-ethylmaleimide-sensitive-factor attachment receptor) and also activates GTPase Ypt7p/Rab which in turn initiates docking [14,15]. The HOPS complex ensures the accurate and efficient fusion of the autophagosome with the vacuole and couples the function of SNAREs and Rab GTPases [15–18]. At the endosome this function is executed by the CORVET (class C core vacuole/endosome tethering) complex which is a homologue of the HOPS complex containing the above described four class C VPS proteins and additionally Vps3p and Vps8p. The CORVET complex interacts with Rab5 homologue VPS21p [19].
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In view of the pivotal role of the lysosome in mammalian cell death, it seemed worthwhile to investigate the role of the vacuole in yeast cell death scenarios. Intracellular acidification induced with nigericin (K+/H+ antiporter) has been reported to cause cell death in yeast [20]. To link intracellular acidification, mode of cell death and the vacuole, appropriate candidate genes like the class C VPS genes whose single deletion results in the absence of a visible vacuole were tested. Class C VPS deletion strains show an enhanced sensitivity to treatment with acetic acid pH = 3 whereas to hydrogen peroxide (H2O2) treatment they react almost like wild type. We also report that acetic acid induced apoptosis requires an intact vacuolar fusion machinery. 2. Materials and methods 2.1. Yeast strains and growth conditions The single gene deletion strains Δvps16, Δvps33, Δpep3, and Δpep5, respectively of S. cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) were purchased from EUROSCARF (Frankfurt, Germany). All strains were grown on SC medium containing 0.17% yeast nitrogen base without amino acids and ammonium sulphate (Difco), 0.5% (NH4)2SO4 and 30 mg/l of all amino acids (except 80 mg/l histidine and 200 mg/l leucine), 30 mg/l adenine, and 320 mg/l uracil with 2% glucose (SCD).
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3. Results 3.1. Vacuolar class C VPS deletion strains show a reduced life span resulting from necrotic cell death Among the more than 50 genes required for vacuolar protein sorting in S. cerevisiae, only four VPS genes belong to the class C group, of which single gene deletions lack a readily identifiable vacuole [23]. The vacuolar class C VPS genes, VPS16, VPS33, PEP3, and PEP5, code for the components shared by the HOPS and the CORVETcomplex implicated in vacuolar and endoplasmic fusion. For yeast, chronological ageing is defined as the survival of cells in stationary cultures and is used as a model system for ageing of post-mitotic cells and tissues. To determine the impact of an absent vacuole during chronological ageing appropriate experiments were performed. We found that the class C VPS deletion strains show a decreased chronological life span compared to wild type (Fig.1A). The viability of Δpep3 and Δpep5 strains decreased to 20–40% at day 2 and finally no survival could be observed at day 3 whereas wild type shows 50% survival at day 3. This is accompanied by an enhanced accumulation of ROS in the mutant strains (Fig. 1B). Growth medium (of wild type and of class C VPS deletion strains) acidifies during cultivation of yeast as shown by Fabrizio et al. [24], detectable from day 1 on. The deletion strains show large amounts of propidium iodide (PI) positive cells indicating necrotic cell death (Fig. 1C). The decreased chronological life span is shared with other VPS deletion strains (e.g. vps19, vps34, data not shown).
2.2. Survival assay and chronological ageing For experiments testing oxygen stress or acid tolerance, experiments were performed as described [2]. At a cell count of 3.5 × 106, strains were incubated in 10 ml SC medium with indicated concentrations of acetic acid or 0.7 mM hydrogen peroxide for 3 h. Two different acetic acid solutions were used: acetic acid pH = 4.05 (10 M acetic acid, 3.4 M K-acetate stock) or acetic acid pH = 3 (10 M acetic acid, 0.09 M K-acetate stock). Addition of 100 mM acetic acid pH = 4.05 decreases the pH of the growth medium from pH 6 to 4.5, and addition of 100 mM acetic acid pH = 3 to 3. The percentage of survival was determined relative to an untreated control. Chronological ageing experiments were performed as described [21]. 2.3. Cell death marker assays AnnexinV/PI costaining was performed as described [1]. For quantifications using flow cytometry 30,000 cells were evaluated with BD FACSAria and analyzed with BD FACSDiva software. For dihydroethidium (DHE) staining, 1 × 106 cells were harvested by centrifugation, resuspended in 250 μl of 2.5 μg/ml DHE in PBS, and incubated in the dark for 10 min. Relative fluorescence units (RFU) were determined by using a fluorescence reader (Tecan, GeniusPRO), or stained cells were counted by using flow cytometry. For propidium iodide (PI) staining, 1 × 106 cells were harvested by centrifugation, resuspended in 250 μl 2.5 μg/ml PI in PBS, and incubated in the dark for 10 min. Stained cells were counted by using flow cytometry. For TUNEL staining, 1 × 107 cells were harvested by centrifugation and staining was performed as described [4]. 10,000 cells of each sample were evaluated by flow cytometry. 2.4. Yeast intracellular pH measurement Intracellular pH of yeast cells was assessed by FACS analysis of cells stained with the pH-dependent fluorescent dye SNARF-4F (Invitrogen, Austria), essentially following the method described by Valli et al. [22]. In order to ensure sufficient activation of the dye the incubation time was increased to 30 min.
3.2. Class C VPS deletion strains show a drastically enhanced cell death with a predominantly necrotic phenotype after treatment with acetic acid pH = 3 Next, we investigated the reaction of class C VPS deletion strains towards classical inducers of apoptosis like acetic acid and H2O2 [25,26]. In survival assays H2O2 treated Δpep3 and Δpep5 deletion strains show no difference compared to wild type (Fig. 2A), with low ROS accumulation within cells (Fig. 2B) and low amounts of necrotic cells (Fig. 2C). PI/AnnexinV costaining reveals that H2O2 induces apoptosis in class C VPS null mutants (see Supplementary Fig. 1). Acetic acid pH = 3 treatment results in massive death of the class C VPS deletion strains, only about 5% of the cells survive (Fig. 2A), an increased ROS accumulation (about 100% DHE positive cells) and high amounts of necrotic cells (about 100% PI positive cells) in the class C VPS knock outs (Fig. 2B and C). In contrast, wild type treated with acetic acid pH = 3 shows 10.5% necrotic cells. Deletion strains in other vacuolar components like some subunits of the vacuolar ATPase (vma6, vma8 and vma13) or adaptor proteins for high-fidelity vesicle docking and fusion like vps19 are less sensitive towards acetic acid pH = 3 (see Supplementary Fig. 2), indicating a specific effect of class C VPS deletion strains. 3.3. pH influences survival after acetic acid treatment of class C VPS deletion strains To evaluate the role of pH on cell death in yeast various acetic acid treatments were used, lowering the pH of the growth medium to 4.5 or 3. Wild type cells showed no difference in survival after treatment with acetic acid pH = 4.05 or pH = 3 (Fig. 2A). In contrast, class C VPS deletion strains showed a far better survival when treated with acetic acid pH = 4.05 than with pH = 3 (Fig. 2A), and a lower portion of DHE and PI positive cells (Fig. 2B and C). Δvps33, Δpep3, and Δpep5 strains nearly reach wild type survival after treatment with acetic acid pH = 4.05. Class C VPS deletion strains treated with 100 mM acetic acid pH = 4.05 show DHE staining in less than 40% of the cells, while treatment with acetic acid pH = 3 results in almost 100% DHE positive cells (Fig. 2B). A similar effect could be observed in the PI stainings, indicative for necrosis (Fig. 2C).
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C VPS mutants a similar drop of intracellular pH could be detected (Fig. 3D). 3.4. In contrast to class C VPS deletion strains, low intracellular pH leads to apoptosis in wild type To get comparable survival rates between wild type and deletion strains, wild type cells were treated with higher concentrations of acetic acid (225 mM acetic acid pH = 4.05 or 150 mM acetic acid
Fig. 1. Deletion of class C VPS genes decreases chronological life span and increases ROS production. (A) Chronological life-span curves (i.e., cfu of 500 plated cells) of Δvps16 (♦), Δvps33 ( ), Δpep3 (▲) Δpep5 ( ), and of wild type (⁎). Data represent means of three independent experiments. (B) Quantification (fluorescence reader) of ROS accumulation using DHE-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments. (C) Quantification (FACS analysis) of necrotic cells using PI-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments.
To find out whether the different pH treatments have an influence on the mode of cell death, PI/AnnexinV costaining was performed. Seventy-eight percent of wild type cells treated with 100 mM acetic acid exhibit apoptotic markers, regardless of pH (Fig. 3B). In the class C VPS deletion strains acetic acid pH = 4.05 causes lower amounts of necrotic cells (about 20%) in contrast to treatment with acetic acid pH = 3 which causes a predominantly necrotic death (Fig. 3B and C). Measurement of the intracellular pH value of acetic acid treated cells showed that unstressed wild type and Δpep3 cells have an intracellular pH value of 6.7 (Fig. 3D) which is mostly unaffected by addition of 100 mM acetic acid pH = 4.05 for both strains. With 100 mM acetic acid pH = 3, the intracellular pH drops to 6.3 in wild type cells but to 5.5 in Δpep3. In the other class
Fig. 2. Acetic acid pH = 3 decreases survival of class C VPS deletion strains and increases ROS and necrosis. (A) Survival of Δvps16, Δvps33, Δpep3, Δpep5 and wild type. Survival was determined after cell death induction with 0.7 mM H2O2, 100 mM acetic acid (pH = 3) or 100 mM acetic acid (pH = 4.05). Data represent means of three independent experiments. (B) Quantification (FACS analysis) of ROS accumulation using DHE-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments. (C) Quantification (FACS analysis) of necrotic cells using PI-staining of class C VPS deletion strains and wild type. Data represent means of three independent experiments.
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pH = 3), resulting in similar survival rates and intracellular pH drop as in mutant strains with 100 mM acetic acid pH = 3 (Fig. 3A and D). But while more than 80% of the wild type cells die, the amount of necrotic cells just increases to about 20% of the whole cellular population. In contrast, in the class C VPS knock outs almost 100% of the cells die with a necrotic phenotype after 100 mM acetic acid pH = 3 treatment (Fig. 3A and C). Apoptotic cells can become leaky during further cultivation, resulting in PI positives. To distinguish this secondary necrosis from primary accidental or regulated necrosis kinetic assays were performed. Over a period of 3 h the effects of 50 mM or 100 mM acetic acid pH = 3 or 225 mM acetic acid pH = 4.05 on survival and modes of death of wild type and Δpep3 strains were observed (Fig. 4). In wild type about 3% of the cells die with a necrotic phenotype after all three treatments. In contrast, in the Δpep3 strain necrosis gradually enhances over time with all acetic acid treatments (Fig. 4B), no TUNEL positives are observed over the time course (Fig. 4C). This indicates that apoptosis is suppressed by the lack of the vacuole and primary necrosis is the only way to go for class C VPS deletion strains. In some cases, the sum of TUNEL-positive (apoptotic) and PIpositive (necrotic) cells is lower than that of dead cells (e.g., wild type with 225 mM acetic acid pH = 4.05 in Fig. 4). This is probably due to a delayed effect of the experimental stress on survival even after plating on the cell count plates, and in differences in the kinetics of apoptotic marker development. As our wild type studies show, treatment with acetic acid concentrations up to 150 mM at low intracellular pH levels do not necessarily lead to necrosis, a functional vacuole directs the mode of death under acetic conditions towards apoptosis. Of course, this capacity can be overloaded, resulting in necrosis at very high concentrations or long exposure [25]. 4. Discussion In the 1950s the free radical theory of ageing was postulated by Harman [27]. In accordance to this theory we could demonstrate that class C VPS null mutants exhibit a drastically reduced survival in chronological ageing and accumulate high amounts of ROS which predominantly leads to necrosis. ROS cause irreversible damage mainly to mitochondria, resulting in a further increase of the ROS level [28]. Degradation of damaged structures is mediated via autophagy, requiring an efficient vacuole fusion machinery. Defects in autophagy in class C VPS knock outs could enhance the vicious circle of accumulation of damaged mitochondria and oxidised proteins, resulting in enhanced production of ROS and even more oxidative damage. Furthermore the acidification of the media during cultivation [24] could result in a drop of the intracellular pH in the class C VPS null mutants leading to enhanced ROS accumulation within the cells. While chronological ageing of wild type yeast predominantly results in apoptotic death, and can be delayed by antiapoptotic treatment (disruption of yeast caspase YCA1 [21]), mutants with Fig. 3. A functional vacuole is needed for acetic acid induced apoptosis. (A) Survival of wild type and Δpep3. Survival was determined after cell death induction with 100 mM or 225 mM acetic acid (pH = 4.05), or 100 mM or 150 mM acetic acid (pH = 3). Data represent means of three independent experiments. (B) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using AnnexinV/ PI costaining of spheroplasts in wild type and Δpep3. Note that digestion of the cell wall may result in membrane damage and PI staining. For precise determination of necrosis, see panel C. Data represent means of three independent experiments. (C) Quantification (FACS analysis) of necrotic cells using PI-staining of wild type and Δpep3. Data represent means of three independent experiments. (D) Intracellular pH determined by staining with the pH-dependent fluorescent dye SNARF-4F of wild type and Δpep3 after induction with acetic acid (pH = 4.05) or acetic acid (pH = 3), the intracellular pH of Δpep5, Δvps16 and Δvps33 is displayed after induction with 100 mM acetic acid (pH = 3). The broken line indicates the mean of intracellular pH in unstressed wild type and class C VPS deletion strains. Data represent means of three independent experiments.
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treatment: as an acidifying agent resulting in necrotic death, which can be prevented by vacuolar activities, and as a proapoptotic signal molecule, an effect which can not be prevented by, but instead requires a functional vacuole. As a result, acetic acid at pH = 3 or 4.05 has similar effects on wild type, a slight intracellular drop in pH has no influence on survival or the mode of death. Loss of vacuolar function in the class C VPS knock out strains had no effect with 100 mM acetic acid pH = 4.05 but results in intracellular acidification and massive necrotic death with acetic acid pH = 3. To achieve similar drop in the intracellular pH in wild type higher concentrations of acetic acid (225 mM acetic acid pH = 4.05 or 150 mM acetic acid pH = 3) were required. However, while the death rate is similar to class C VPS deletion strains, death is predominantly apoptotic in wild type. These data indicate that a functional vacuole is not only needed for intracellular pH regulation but also for executing apoptosis during low pH stress. In human cells a decrease in intracellular pH also acts as an initial trigger for a cascade of events resulting in apoptosis [29] in accordance to our results with yeast. Secretory pathway and pH regulation within the cell are crucial factors of cell death regulation and maintenance of cell integrity. In conclusion our results fit the hypothesis of Golstein and Kroemer [8]: programmed cell death developed from a necrotic mechanism and acquired additional, alternative terminal phenotypes (apoptotic, autophagic). In such a scenario, mechanisms to suppress the development of the original necrotic phenotype are required to allow the implementation of the alternatives like apoptosis or autophagy. The vacuole has a central role in the execution of autophagy, and in case of acetic acid stress also for preventing necrosis. Our findings suggest a role of the vacuole/lysosomes in a switch from necrotic to apoptotic death phenotype, giving it control over all responses to acetic stress and making it the central organelle in the regulation of cell death pathways under acetic conditions. Acknowledgements We are grateful to the Fonds zur Förderung der wissenschaftlichen Forschung (Austria) for grant S-9303-B05 to A.S., H.K. and K.-U.F. and Doctoral College scholarship for M.D., and to the European Commission, project TransDeath for A.S., C.R. and K.-U.F. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbamcr.2008.11.006. References
Fig. 4. Acetic acid leads to gradual increase of necrosis in Δpep3 strain. Wild type (dashed line, filled symbols) and Δpep3 (continuous line, empty symbols) were treated with 225 mM acetic acid (pH = 4.05; △, ▲), or 100 mM (□, ■) or 50 mM (○, ●) acetic acid (pH = 3). Data represent means of three independent experiments. (A) Survival, (B) necrotic cells (FACS analysis of PI-staining), and (C) apoptotic cells (FACS analysis of TUNEL-staining, with untreated control displayed as diamonds (◊, ♦)) were determined in time series.
reduced chronological life span have been found to exhibit a necrotic death phenotype [4], indicating that the cause is not acceleration of the wild type program, but an additional mechanism. In contrast, presence of a vacuole appears not to be important for H2O2 mediated apoptotic cell death, which we demonstrate in our stress experiments. H2O2 treatment leads to apoptosis in wild type as well as in class C VPS deletion strains (see Supplementary Fig. 1). Acetic acid induces apoptosis with its typical markers like chromatin condensation, DNA fragmentation, and phosphatidylserine externalization [25]. Our data indicate a dual role of acetic acid
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