Starvation for an essential amino acid induces apoptosis and oxidative stress in yeast

Experimental Cell Research 300 (2004) 345 – 353 www.elsevier.com/locate/yexcr

Starvation for an essential amino acid induces apoptosis and oxidative stress in yeast Herfried Eislera, Kai-Uwe Frfhlichb, Erich Heidenreicha,* a

Division of Molecular Genetics, Institute of Cancer Research, Medical University of Vienna, A-1090 Vienna, Austria b Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria Received 15 April 2004, revised version received 29 June 2004 Available online 3 September 2004

Abstract Protracted starvation of auxotrophic Saccharomyces cerevisiae strains for an essential amino acid is commonly used to allow investigation of adaptive mutation mechanisms during starvation-induced cell cycle arrest. Under these conditions, the majority of cells dies during the first 6 days. We investigated starving cells for markers of programmed cell death and for the production of reactive oxygen species (ROS). We observed that protracted starvation for lysine or histidine resulted in an increasing number of cells exhibiting DNA fragmentation and chromatin condensation, thus an apoptotic phenotype. Not only respiration-competent cells but also respiratory deficient rho0 cells were able to undergo programmed cell death. In addition the starving cells rapidly exhibited indicators of oxidative stress, independently of their respiratory competence. These results indicate that starvation for an essential amino acid results in severe cell stress, which may finally be the trigger of programmed cell death. D 2004 Elsevier Inc. All rights reserved. Keywords: Saccharomyces cerevisiae; Yeast; Apoptosis; TUNEL; Reactive oxygen species; Vacuole; Starvation; Stationary phase; Cellular stress; rho0

Introduction Removal of dispensable or damaged cells during embryonic development of metazoans and maintenance of tissue homeostasis relies on programmed cell death. Apoptosis avoids the side effects of accidental cell death (necrosis), which usually leads to rupture of the dying cell and to inflammation of the surrounding neighborhood due to released cell contents. During programmed cell death, the cell remains intact, but divides into apoptotic bodies to be properly removed by phagocytosis. Apoptotic cell death can be described as a sequence of characteristic phenotypical alterations: First, the lipid phosphatidylserine is exposed at the cell surface [1], followed by a characteristic condensation and bmarginationQ of the chro* Corresponding author. Division of Molecular Genetics, Institute of Cancer Research, Borschkegasse 8a, A-1090 Vienna, Austria. Fax: +43 1 4277 9651. E-mail address: [email protected] (E. Heidenreich). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.07.025

matin to the nuclear envelope [2], DNA fragmentation, and formation of membrane-enclosed cell fragments, the bapoptotic bodiesQ [1]. Beside its function in development, apoptosis plays a crucial role in preventing cancer, autoimmune diseases, and spreading of viral infections, by removing damaged, malfunctioning or infected cells [3]. Apoptosis in vertebrates is a complexly regulated mechanism, which can be activated by external signals (receptors activated by death signaling ligands), cellular stress, and damage. Programmed cell death is an ubiquitous process found in many eukaryotic organisms [4]. Recently, it was discovered that simple unicellular organisms like the budding yeast Saccharomyces cerevisiae [5–8] or like bacteria [9] also have the potential to undergo programmed cell death, suggesting that even unicellular organisms can exhibit altruistic behavior [10–12]. The initiation of budding yeast cell death by expression of human proapoptotic genes such as Bax, caspases, p53, or CED-4/Apaf1, and the rescue by coexpression of anti-

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apoptotic genes, indicates a similar mechanism [11]. In agreement to mammalian cells, apoptosis in yeast can be induced by cell–cell communication via pheromones [13] and by cellular stress like UV irradiation [14], starvation for carbon source on expired medium [10], or reactive oxygen species (ROS) [6]. ROS not only act as external triggers of apoptosis, but they also play a crucial role as mediators of apoptosis [6,15]. A possible benefit of apoptosis for unicellular organisms is described by the bapple scenarioQ [11]. When colonies of yeast cells suffer from nutrient depletion, more bhealthyQ cells can survive when weak cells die quickly, stopping consumption of the limiting reserves. Due to the fact that a population of yeast cells feeding on the same fruit mostly descends from one cell (spore), the survivors have the same progenitors and therefore the same genome as the dying cells; death can be a method of saving the own genome. Accordingly, it was found that nutrient depletion by cultivation of yeast on expired medium is a strong inducer of apoptosis [10]. Along with increased levels of apoptosis, a continuous, massive ROS burden of all starving cells was detected in stationary yeast [10], in spite of an induced synthesis of antioxidant enzymes [16]. ROS (O2 , HO , and H2O2) are highly reactive oxidative agents, causing damage to lipids, proteins, and DNA. Owing to the toxicity of these reactive molecules, cells of all aerobic organisms possess an efficient oxidant defense system [17]. The formation of ROS is linked to the mitochondrial respiratory chain, as delocalized electrons are the main source of oxygen radicals. Contrary to mammals, budding yeast is a facultative anaerobic organism, taking energy from fermentation of appropriate carbon sources to ethanol, e.g., when living in a surplus of sugar or under oxygen-free conditions. Accordingly, yeast is able to live without respiration. Respirationdeficient strains exhibit the bpetiteQ phenotype (slowed growth speed, smaller colonies on solid medium, no growth on nonfermentable medium). Respiration deficiencies can result from loss of nuclearly coded genes of the respiratory chain (pet genes) as well as from a partly (rho ) or complete (rho0) loss of the mitochondrial genome. Mitochondria are known to be crucial players in the regulation of apoptosis [18], but it is still discussed whether the remaining mitochondrial function of respiration-deficient cells (rho0 cells still possess mitochondria) is sufficient for induction of apoptosis. Ambiguous results were obtained with mammalian cells [19–22]. In yeast, acetic-acid-induced apoptosis was found to be abolished in rho0 cells, suggesting impediment of programmed cell death in respirationdeficient cells [23]. In this study, we examined the occurrence of apoptosis and the generation of reactive oxygen species during protracted starvation for one essential amino acid. We show that lysine starvation or histidine starvation of auxotrophic yeast cells induces strong bursts of ROS and a high level of apoptotic cell death.

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Materials and methods Strains, growth conditions, and starvation Experiments were performed with an isogenic set of three S. cerevisiae strains: the haploid respiration-competent EH150 and the haploid rho0 EH151 [24], as well as the diploid respiration-competent YLBD [25]. All strains are auxotrophic for lysine due to a mutated LYS2 allele (lys2DBglII) and auxotrophic for histidine due to a his3D200 allele. Cultures of each yeast strain were grown in liquid rich medium (YPD: 1% yeast extract, 2% peptone, 2% glucose) to late exponential phase. Then, the cells were washed and transferred to selective solid medium (SClysine, synthetic complete medium lacking lysine, or SChistidine, synthetic complete medium lacking histidine [26]) at a plating density of 108 cells/plate. The plates were incubated at 308C for 6 days to exert starvation stress on the cells. For further analyses, aliquots were removed before plating (day 0) or washed from the starvation plates after 1, 3, or 6 days of incubation. Eventually present prototrophic revertant colonies were removed before harvesting starved cells. Test for apoptotic markers For TUNEL and DAPI staining, 2  107 cells were fixed with 3.7% formaldehyde, digested with lyticase (Sigma) and h-glucuronidase (Roche), and applied to a polylysine-coated slide as described for immunofluorescence [5,27]. DNA ends were labeled with the TdTmediated dUTP nick end labeling (TUNEL) method using the bIn Situ Cell Death Detection Kit, PODQ (Roche) as described previously [6]. For nuclear staining, cells (on a microscope slide) were incubated with 1 Ag/ml diaminophenylindole (DAPI) in phosphate-buffered saline (PBS: 22.5 mM potassium-monohydrogen/dihydrogen-phosphate buffer, pH 7.0, 9 g/l NaCl) for 15 min, and then rinsed six times with PBS. A coverslide was mounted with a drop of Kaiser’s glycerol gelatine (Merck). Microscopy was carried out with 330–385-nm excitation and N420-nm emission wavelength. Image acquisition of the DAPI and the TUNEL-overview image was accomplished with a Zeiss (Axioskop) fluorescence microscope and a Visitron Systems (Visicam) digital camera. The detailed TUNEL images were acquired with a Nikon (Microphot FXA) fluorescence microscope and a Nikon (D100) digital camera. Test for reactive oxygen species Intracellular reactive oxygen species were detected by dihydrorhodamine (DHR)-123 staining, while dead cells were simultaneously marked by phloxine B. The vital dye phloxine B is expelled by normal (viable) cells, but accumulates in metabolically dead cells [13,28]. The

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frequencies of dead cells judged by phloxine B staining generally are within the limits of variation of the determination of frequencies of colony-forming units on YPD medium (H.E., unpublished observation). A number of 2  107 cells was incubated in appropriate liquid medium (YPD for day 0, SC-lysine for the other time points) with 0.5 Ag/ ml DHR-123 (Molecular Probes) and 400 Ag/ml phloxine B (Merck) for 2 h at 308C. After two washing steps with PBS, microscopy was performed with 450–490-nm excitation and N520-nm emission wavelength (fluoresceine filter). Image acquisition was accomplished with a Nikon (Microphot FXA) fluorescence microscope and a Nikon (D100) digital camera. Statistical analysis Statistical comparisons were done using Student’s t test. Percentages were arcsine-transformed before analysis [29].

Results and discussion Protracted starvation for a single essential amino acid triggers apoptosis In studies on spontaneous mutagenesis in stationaryphase cells, starvation for an essential amino acid is used to trigger cell cycle arrest. Especially starvation for lysine is an important condition of stationary-phase mutagenesis studies in yeast during selection for spontaneous revertants of the lys2DBglII allele [24,25,30–33]. It is known from the progressive decrease in viability and the progressive accumulation of respiration-deficient cells [34] that such starvation conditions are stressful to the cells. Further results indicated the occurrence of spontaneous double-strand breaks during prolonged starvation for an essential amino acid [25]. Therefore, we were interested in the involved mechanisms of cell stress and cell death and in the question whether the cells undergo apoptosis, similarly as they do upon long-term cultivation in exhausted complete medium [10]. We investigated the incidence of apoptotic phenotype of three different lysine-auxotrophic S. cerevisiae strains before and during continuous starvation for the essential amino acid lysine. Two respiration-competent (haploid and diploid) strains were tested to reveal the level of apoptosis during mutation studies. Additionally, a respiration-deficient rho0 strain (haploid) was analyzed for its capability of performing apoptosis, to investigate the significance of functional mitochondria for programmed cell death. We used the TUNEL (terminal uridine nucleotide end labeling) assay as indicator of apoptotic cell death. This method detects apoptotic DNA fragmentation by labeling of 3V-OH termini with modified nucleotides catalyzed by terminal deoxynucleotidyl-transferase [35,36]. Fragmented DNA is visualized as dark stain of the nuclei.

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Whereas exponentially growing cells showed hardly any sign of apoptotic cell death (Fig. 1, day 0), already a single day of starvation for lysine resulted in considerable numbers of TUNEL-stained cells (Fig. 1, day 1). At this time point, 34% of the diploid and 23% of the haploid respirationcompetent cells as well as 19% of the rho0 cells showed a TUNEL-positive phenotype (Table 1). After 3 days of starvation, the number of respirationcompetent cells exhibiting an apoptotic phenotype further increased to more than 40%, while about 26% of the respiration-deficient cells were TUNEL-positive. The frequency of TUNEL-stained cells of the respiration-competent strains correlates well with the frequency of phloxine Bstained, metabolically dead cells of a corresponding sample (Table 1). In the rho0 strain, on the other hand, a much higher frequency of dead cells than of apoptotic cells was observed, indicating considerable amounts of accidental cell death. Persistent starvation for 3 more days led to a further accumulation of dead cells, while a small (statistically nonsignificant) decrease of TUNEL-positive cells was evident (Table 1). As discussed in the next section, a progressive degradation of cellular components and decay of DNA in cells already dead for up to 5 days could explain a weaker or lacking staining of some fraction of cells. In summary, our data support that lysine starvation is a strong inducer of programmed cell death, in respirationcompetent as well as in rho0 cells. A similar increase in TUNEL-positive cells was observed during protracted starvation of strain EH150 for histidine (see online Supplementary Fig. 1 and Supplementary Table 1). Mitochondrial function seems to play an important role in regulation and promotion of programmed cell death [15]. However, controversial results have so far been reported concerning the question if rho0 cells are able to undergo apoptosis [19–23]. Our data at hand document that the remaining mitochondrial function of rho0 cells is sufficient to support apoptosis. Time-dependent changes of the apoptotic phenotype Apoptotically dying cells undergo different changes in phenotype. One of the first signs of apoptosis is the externalization of phosphatidylserine [8]. The following characteristic is a TUNEL-positive nuclear ring structure resulting from DNA fragmentation and simultaneous chromatin condensation. As evident from Fig. 1 and Table 1, up to half of the cells died apoptotically during 6 days of starvation-induced cell cycle arrest. While the overall occurrence of the apoptotic phenotype was monitored, we noticed a distinct morphological change of the predominant TUNEL phenotype. The staining pattern of the TUNEL-positive cells after 1-day starvation as well as of the rare occurrences of TUNELpositive cells during proliferation (day 0) indicated condensed fragmented chromatin, often ring- or sickle-shaped

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Fig. 1. Starvation for the essential amino acid lysine induces apoptosis. TUNEL assays of three S. cerevisiae strains (rows A and B: haploid and diploid respiration-competent cells, respectively; row C: rho0 cells) were performed to investigate the incidence of apoptotic phenotype during continuous lysine starvation of the cells. The experiments were performed three times with similar outcome. This figure shows one representative set of results. Scale bar = 10 Am.

along the nuclear envelope (Fig. 2). Upon protracted lysine starvation (days 3 and 6), the frequency of this phenotype decreased in favor of staining of the whole nucleus or nuclear fragments. Whereas the cytoplasm was basically unstained during proliferation or after 1 day of starvation, during later stages (days 3 and 6), the TUNEL signal frequently effused from the nucleus to the cytoplasm resulting in staining of the whole cell with or without accentuation of the nucleus (Fig. 2). A DAPI stain of day 6 cells revealed fuzzy and prolate chromatin characteristic for apoptotic cells along with and in contrast to well-defined compact nuclei of nonapoptotic cells (Fig. 2, right column). Due to the inevitable fixation procedure it is not possible to follow the fate of individual apoptotic cells. However, our

quantification of TUNEL-positive cells (Table 1) supports that day 0 and day 1 represent fresh apoptoses, while day 3 consists of a mixture of fresh and old apoptoses, and day 6 mainly comprises old apoptoses. Therefore, we assume that the morphological changes observed from day 0 to day 6 bona fide represent a chronology of apoptotic progression in yeast. The phenotype of our TUNEL-positive cells after amino acid starvation is similar to the phenotype of mammalian apoptosis [36]. The TUNEL-stained chromatin rings we detected after 1 to 3 days of lysine starvation are known as an early effect of apoptosis, appearing shortly after the apoptotic trigger [8,36]. As a later step, the nucleus is fragmented [36], as we found it often happening at day 3. A similar phenotype is described for DAPI-stained sta-

Table 1 Fraction of TUNEL-stained and phloxine B-stained cells in the course of lysine starvation Duration of starvation (days)

TUNEL-positive cells (%)

Phloxine-B-stained cells (%) rho0

Respiration-competent Haploid 0 1 3 6

1.1 22.9 44.2 36.7

F F F F

Diploid 0.7 8.9** 7.5*** 6.7***

0.9 33.6 42.0 36.9

F F F F

Haploid 0.5 4.3*** 4.2*** 13.5**

0.9 19.1 26.1 23.1

rho0

Respiration-competent

F F F F

0.2 2.6*** 6.3*** 5.1***

1.1 19.2 48.0 56.5

F F F F

Diploid 0.4 2.9*** 2.6*** 3.8***

0.6 37.3 45.2 66.7

F F F F

0.4 2.9*** 1.4*** 2.7***

0.4 20.6 58.5 61.5

F F F F

0.2 5.4*** 3.1*** 7.2***

All values represent means F standard deviation, determined by assessing 600 cells of three experimental series. All day 1, 3, and 6 values are significantly different from their respective day 0 values (**P V 0.01, ***P V 0.001).

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Fig. 2. Changes in the apoptotic phenotype during lysine starvation. All three strains (rows A and B: haploid and diploid respiration-competent cells, respectively; row C: rho0 cells) exhibit a similar time-dependent change of the apoptotic phenotype. During exponential growth (day 0, rare cases) and after 1 day of starvation, TUNEL-stained ring structures (chromatin concentrated at the nuclear envelope) and stained nuclei are visible. After 3 days, typically also the cytoplasm surrounding a very dark nucleus is stained. After 6 days of starvation, many cells are stained throughout. A DAPI stain of day 6 cells reveals chromatin rings and degraded nuclei in all three strains. Scale bar = 5 Am.

tionary-phase yeast cdc48 mutants after several days of incubation [8]. However, the terminal stages of apoptosis are different in yeast and in vertebrates. Whereas vertebrate cells break into apoptotic vesicles to be phagocytosed by macrophages or neighboring cells [37], aged apoptotic yeast cells persist in the culture [10]. Interestingly, long-time-starved yeast cultures enriched in apoptotic cells release low-molecularweight substances, which are able to improve the survival of aged cells [10]. Therefore, it appears to be an altruistic strategy of late apoptotic cells to assist their neighboring cells by a catabolic breakdown of polymers like proteins and DNA to soluble monomers able to pass the cell wall. Such a progressive degeneration of DNA down to small fragments and nucleotides would be consistent with the observed leaking of the TUNEL signal from the nucleus to the cell lumen and the nonsignificant decrease in overall TUNEL staining of day 6 cells, respectively. Starvation of auxotrophic yeast cells on selective medium leads to a burst in ROS load The phenomenon of apoptosis is closely related to the concentration of reactive oxygen species in mammals [38] and yeast [6]. These poisonous substances, formed in any organism exposed to molecular oxygen, can cause cell damage, but can also act as a mediator of apoptosis [15,39]. Because we observed that lysine starvation massively

triggers apoptotic cell death, it was self-evident to examine the ROS load of both respiration-competent and rho0 cells during lysine starvation. We cultivated lysine-auxotrophic yeast cells on lysinefree synthetic complete drop out medium for 6 days. Cell viability was monitored by use of the red dye phloxine B that accumulates in dead cells. The load of ROS was examined with the help of dihydrorhodamine-123, a nonfluorescent substance, which is oxidized by ROS to the yellow fluorescent rhodamine-123 [40]. Exponentially growing cells of all three strains showed very few signs of oxidative stress (Fig. 3, day 0). Only a very small subfraction of dead (phloxine Bstained) cells exhibited a higher amount of reactive oxygen species. This phenotype changed drastically when the cells were transferred to lysine-free selective medium. After 1 day of lysine starvation, as much as 100% of the cells sustained considerable amounts of oxygen radicals, visualized by strong rhodamine-123 fluorescence (Fig. 3, day 1). Simultaneously, the number of phloxine-B-stained cells increased to 20% for both haploid strains and to 37% for the diploid strain (Table 1). Continuing starvation led to a constantly high burden of ROS in cells of all three strains while the number of dead cells increased from about 50% at day 3 to about 60% at day 6 (Fig. 3, days 3 and 6; Table 1). Little difference between the strains was discovered. Respirationdeficient cells were as prone to oxidative cell stress as

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Fig. 3. Accumulation of ROS during lysine starvation. The ROS load of DHR-123-stained cells (yellow rhodamine-123 fluorescence) and corresponding ratio of phloxine-B-stained dead cells (red) was determined for haploid (panel A) and diploid (panel B) respiration-competent cells and for rho0 cells (panel C). All pictures were taken with constant shutter time and excitation light intensity. Insets in the day 0 fluorescence images were shot with fourfold shutter time. The experiments were performed three times with similar outcome. This figure shows one representative set of results. Scale bar = 30 Am.

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respiration-competent cells. Despite the complete loss of mitochondrial DNA and thus loss of many mitochondrial functions, rho0 cells were capable of producing a wild-typelike amount of ROS (Fig. 3, panel C).

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We also analyzed the ROS content of EH150 cells starved for histidine and also observed a sharp increase from a very low basal level of ROS to a constantly high level of ROS from day 1 to 3 of this alternative amino

Fig. 4. Close-up views of changes in localization and intensity of the ROS burden during starvation for lysine. The ROS load of the cells was determined by DHR-123 staining (yellow rhodamine-123 fluorescence). Corresponding transmitted light images (dead cells stained red by phloxine B) are displayed in the lower row of each panel. Proliferating cells (day 0) exhibit a very low intensity of rhodamine fluorescence (shutter opened 4 times longer than for day 3 and 6 photos). Respiration-competent haploid (panel A) and diploid cells (panel B) show a strict localization of these radicals inside the vacuoles, while the cytoplasm remains unstained. The cytoplasm of day 0 rho0 cells (panel C) contains ROS. DHR-123 images of day 1 were obtained with twice the exposure time of day 3 and day 6 images. Scale bar = 10 Am.

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acid starvation (Supplementary Fig. 2 and Supplementary Table 1). This behavior of lysine- or histidine-starved cells is partly consistent with results of carbon source starvation of yeast: Slower but persistent induction of ROS formation was found as well as an induction of apoptosis [10]. We presume that the conditions on an expired rich medium are a bit milder than during starvation for an essential amino acid. The stronger impact of amino acid starvation could be due to more serious consequences of the abnormal imbalance in amino acid pools. After transfer to lysine-free medium, the auxotrophic cells finish ongoing cell cycles or perform a maximum of one additional cell division at the expense of internal lysine reserves before they typically enter a G0/G1 cell cycle arrest [25,34]. Probably, the anabolism (and the cell cycle) runs into an unforeseen severe shortage of lysine, which results in a hampered protein synthesis and cellular stress. Concerning the relationship between ROS accumulation and apoptosis, we have to consider that throughout exposure to amino acid starvation, the frequency of TUNEL-positive cells was clearly lower (Table 1) than the frequency of ROSburdened cells (virtually 100%, Fig. 3). Up to about 40% of the starving cells remained viable until the end of the experiment in spite of a high ROS load. Therefore, it is unlikely that the accumulation of ROS at large was a consequence of apoptotic processes. Rather, apoptosis could have been triggered by the considerable ROS burden. However, apoptotic cells might experience a further amplification of the ROS load, as we consistently observed the strongest rhodamine 123 fluorescence in slightly phloxine-B-stained cells, which probably had started phloxine B accumulation very recently and thus were in the process of losing their metabolic viability (Fig. 4).

(Fig. 4, right column). This last change seemed to be symptomatic for cell death as most cells exhibiting these structures were stained red with phloxine B. Unexpectedly, proliferating rho0 cells resembled starving respiration-competent cells (Fig. 4C). Unlike the vacuolespecific ROS staining of respiration-competent cells, the vacuoles of the rho0 cells were dark, while the cytoplasm exhibited differing intensities of DHR staining with some bright grains. Upon starvation for lysine, the cytoplasmic ROS concentration increased similar to the phenotype of the respiration-competent cells. Thus, a strong source of ROS has to exist in rho0 cells. Presently, we do not know if the rudimentary mitochondria present in these respirationdeficient cells could be this source. A ROS contamination could be due to deregulated electrons from the truncated respiratory chain, enhanced by decreased expression of antioxidative enzymes in respiration-deficient cells [41]. In addition to the effect of the increased overall ROS load, the change in localization in mitochondrially intact cells from day 0 to day 1 or 3 probably intensifies the biological impact, as it starts from a very low level in sensitive cell compartments. The amount of oxidative damage to cell structure is expected to be very low in healthy, proliferating respiration-competent cells, but dramatically increased in starving, stressed cells. This damage could lead to programmed and accidental cell death.

Acknowledgment This work was supported by a grant from the Herzfeldersche Familienstiftung to E.H.

Appendix A. Supplementary data Morphological aspects of the ROS load Localization of the potentially dangerous ROS molecules could play a key role for damage, mutagenesis, and cell death. During the analyses of the overall oxygen radical load of the cells, we observed a remarkable shift in the preferred localization of intracellular ROS of haploid and diploid respiration-competent cells. After exponential growth, a very low basic concentration of ROS with a distinct localization was detected (Fig. 4, left column): Respiration-competent cells showed a weak rhodamine-123 staining inside the (partly fragmented) vacuoles (Figs. 4A, B). The cytoplasm was completely free of detectable radicals. This phenotype shifted during the first 3 days of starvation to a strong fluorescent staining of the cytoplasm while the vacuole staining receded into the background. Due to the burst of ROS-dependent fluorescence of the cytoplasm, which outshined the darker areas, it was not possible to determine if the vacuolar ROS level was decreased or whether it remained constant. Later on, the staining changed into a granular, filamentous pattern without visible vacuoles

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2004. 07.025.

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