Ammonium is toxic for aging yeast cells, inducing death and shortening of the chronological lifespan.

Abstract

Here we show that in aging Saccharomyces cerevisiae (budding yeast) cells, NH(4) (+) induces cell death associated with shortening of chronological life span. This effect is positively correlated with the concentration of NH(4) (+) added to the culture medium and is particularly evident when cells are starved for auxotrophy-complementing amino acids. NH(4) (+)-induced cell death is accompanied by an initial small increase of apoptotic cells followed by extensive necrosis. Autophagy is inhibited by NH(4) (+), but this does not cause a decrease in cell viability. We propose that the toxic effects of NH(4) (+) are mediated by activation of PKA and TOR and inhibition of Sch9p. Our data show that NH(4) (+) induces cell death in aging cultures through the regulation of evolutionary conserved pathways. They may also provide new insights into longevity regulation in multicellular organisms and increase our understanding of human disorders such as hyperammonemia as well as effects of amino acid deprivation employed as a therapeutic strategy.

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Ammonium Is Toxic for Aging Yeast Cells, Inducing
Death and Shortening of the Chronological Lifespan
Ju ´lia Santos1,2, Maria Joa ˜o Sousa3*., Cecı ´lia Lea ˜o1,2.
1Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, 2ICVS/3B’s - PT Government Associate Laboratory,
Braga/Guimara ˜es, Portugal, 3Molecular and Environmental Research Centre (CBMA)/Department of Biology, University of Minho, Braga, Portugal
Abstract
Here we show that in aging Saccharomyces cerevisiae (budding yeast) cells, NH4+induces cell death associated with
shortening of chronological life span. This effect is positively correlated with the concentration of NH4+added to the culture
medium and is particularly evident when cells are starved for auxotrophy-complementing amino acids. NH4+-induced cell
death is accompanied by an initial small increase of apoptotic cells followed by extensive necrosis. Autophagy is inhibited
by NH4+, but this does not cause a decrease in cell viability. We propose that the toxic effects of NH4+are mediated by
activation of PKA and TOR and inhibition of Sch9p. Our data show that NH4+induces cell death in aging cultures through
the regulation of evolutionary conserved pathways. They may also provide new insights into longevity regulation in
multicellular organisms and increase our understanding of human disorders such as hyperammonemia as well as effects of
amino acid deprivation employed as a therapeutic strategy.
Citation: Santos J, Sousa MJ, Lea ˜o C (2012) Ammonium Is Toxic for Aging Yeast Cells, Inducing Death and Shortening of the Chronological Lifespan. PLoS
ONE 7(5): e37090. doi:10.1371/journal.pone.0037090
Editor: Matt Kaeberlein, University of Washington, United States of America
Received December 13, 2011; Accepted April 17, 2012; Published May 15, 2012
Copyright: ? 2012 Santos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Fundac ¸a ˜o para a Cie ˆncia e Tecnologia (FCT), Portugal Grant PTDC/AGR-ALI/102608/2008. JS has a fellowship from FCT
(SFRH/BD/33314/2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: mjsousa@bio.uminho.pt
. These authors contributed equally to this work.
Introduction
Starvation of an exponentially growing yeast culture for a given
nutrient usually results in the growth arrest of cells in the culture in
an unbudded state as they exit the cell cycle. Under extreme
starvation conditions such as culturing in water, cells can attain a
quiescent state and are able to survive for long periods. The time
cells survive in this non-dividing state, known as chronological life
span (CLS), is dependent on pre-culture conditions, reaching a
maximum for cells grown on respiratory carbon sources and
allowed to reach stationary phase [1]. Several nutrient signaling
pathways have been implicated in the regulation of yeast CLS,
mainly TOR (target of rapamycin), PKA (protein kinase A) and
Sch9p [2]. In Saccharomyces cerevisiae, TOR signaling responds to
nitrogen and possibly to carbon sources. This pathway controls
cell growth by activating anabolic processes and inhibiting
catabolic processes and mRNA degradation [3–5]. Inactivation
of TORC1 (TOR complex 1) or other members of the TOR
pathway is accompanied by phenotypic changes characteristic of
starved cells, protects against stress, and leads to extension of the
longevity of non-dividing yeast [6,7].
The PKA pathway also plays a major role in the control of
metabolism, stress resistance, cell cycle, growth, and transcription.
It is highly regulated by the nutrient composition of the medium,
in particular by the presence of a rapidly fermentable sugar and
other essential nutrients such as amino acids and phosphate or
ammonium [8,9]. Addition of a rapidly fermentable sugar triggers
activation of adenylate cyclase (Cyr1p) and a rapid increase in
cAMP levels. This increase boosts the activity of the cAMP-
dependent PKA by displacing the regulatory subunit Bcy1p from
the catalytic subunits Tpk1p, Tpk2p and Tpk3p. PKA affects
several downstream targets, thereby allowing cells to make the
necessary adaptations for fermentative growth. These adaptations
include upregulation of glycolysis, stimulation of cell growth and
cell cycle progression, downregulation of stress resistance and
gluconeogenesis, and mobilization of the reserve carbohydrate
glycogen and the stress protector trehalose [10,11]. Down-
regulation of the PKA pathway by starvation of an essential
nutrient causes growth arrest and subsequent entrance into G0.
Cells in G0 acquire a variety of characteristics such as
accumulation of the carbohydrates trehalose and glycogen,
induction of stress-responsive element- and postdiauxic shift-
controlled genes, induction of autophagy and increased stress
resistance [8,12]. Mutations in components of PKA pathway
confer chronological life span extension [13].
The protein kinase Sch9 also plays an important role in
nutrient-mediated signaling. It acts in parallel with the PKA
pathway and is directly phophorylated by TORC1, mediating
many of the TORC1-regulated processes [10,14]. Recent studies
revealed that Sch9p also acts independently of TORC1, and can
even exert opposite effects to TORC1 in the adaptation to stressful
conditions [15].
It has previously been shown that, in the absence of other
nutrients, adding glucose to cells suspended in water can cause
cells to exit the quiescent state and commit to an apoptotic cell
death program that includes production of reactive oxygen species
(ROS), RNA and DNA degradation, membrane damage, nucleus
fragmentation and cell shrinkage [16]. Chronological aging of
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yeast cells in medium also results in a loss of viability with
increasing time accompanied by morphological and biochemical
characteristics of both apoptosis and necrosis [17,18]. In the
present work, we aimed to identify other nutrient signals that could
induce cell death of chronological aging yeasts and the signaling
pathways involved. Ammonium (NH4+) is a nitrogen source
commonly used for yeast growth and it is usually not toxic.
Production of ammonia in yeast colonies has even been described
as a mechanism of protection from cell death during colony
development [19]. To our knowledge, only one report in the
literature refers to NH4+toxicity in yeast, which was observed in
steady-state chemostat cultures limited for potassium [20].
We have found that decreasing the concentration of NH4+in
the culture medium increases yeast CLS. Furthermore, we have
extensively characterized for the first time a cell death process
induced by NH4+in yeast cells. NH4+induced loss of cell viability
in aging S. cerevisiae cultures either in nutrient-depleted culture
medium or upon transfer to water with NH4+. This effect was
particularly significant for cells starved for auxotrophic-comple-
menting amino acids, but not completely starved for nitrogen. We
also determined that activation of PKA stimulated NH4+- induced
cell death, consistent with the observation that deficiency in
upstream components of the cAMP PKA pathway partially
reverted the toxic effect of ammonium. Deletion of TOR1 also
significantly rescued NH4+- induced cell death and decreased
PKA activation. In contrast, SCH9 deletion abolished PKA
activation in response to NH4+but did not revert the decrease
in cell viability. This indicates that PKA inactivation cannot
protect cells from NH4+- induced cell death in the absence of
Sch9p, suggesting a potential role of Sch9p in cell survival.
NH4+-induced cell death has been implicated in a number of
different human disorders that are accompanied by hyperammo-
nemia [21]. However, the precise molecular mechanisms trigger-
ing NH4+-induced cell death in these disorders are not known. In
addition, deprivation of essential amino acids has been employed
as a strategy in cancer therapy but resistance has often been found
[22]. Our results enhance our understanding of longevity
regulation in multicellular organisms. They also suggest that S.
cerevisiae might serve as a useful model for the identification of
signaling pathways and new therapeutic targets for the referred
human disorders.
Results
NH4+causes loss of survival in chronologically aged yeast
cells
The chronological life span (CLS) of S. cerevisiae is strongly
affected by the concentration of the auxotrophy-complementing
amino acid in the medium. Cells of the auxotrophic S. cerevisiae
strain BY4742 cultured with an insufficient supply of essential
amino acids display reduced lifespan compared with cells grown
with increased amino acid supplementation in the medium [23].
In the present work we observed that BY4742 cells grown with
insufficient supply of amino acids grow less than those without this
restriction, and neither glucose (as previously reported [23]) nor
NH4+are completely depleted (Fig. S1A). We first asked whether
manipulating the ammonium concentration in the culture medium
might affect CLS as previously described for glucose [24].
Reducing the starting concentration of (NH4)2SO4in the medium
five- or fifty-fold (from 0.5% to 0.1 and 0.01%, respectively)
improved the survival of chronological aging cells cultured with
amino acid restriction (Figure 1). In contrast, when the initial
(NH4)2SO4concentration in the culture medium, either with or
without restriction of amino acids, was increased to 1%, there was
a decrease in cell survival, although loss of cell viability was much
faster for cells grown with amino acid restriction (Figure 1).
We sought additional insights into this phenomena by asking
whether increased NH4+could account for the loss of cell viability,
as reported in earlier studies [16] which showed that adding
glucose to yeast suspensions in water also causes cells to rapidly
die. Cells were grown in SC 2% glucose plus 0.5% (NH4)2SO4,
with or without amino acid restriction in the medium for 72 hours
and then transferred to water without NH4+(pH 7.0), water with
NH4+(pH 7.0), or to the depleted medium as a control. It has
been shown that medium acidification limits survival of yeast cells
during chronological aging in SC medium and that the longer
survival observed in water can be, at least in part, attributed to the
differences in pH [25–27]. To assess whether acidification could
play a role in the NH4+-induced loss of cell viability, we measured
cell survival in media without adjusting pH (pH 2.6–2.9 due to
culture acidification) or adjusted to pH 7.0 (see schematic of
methodology in Figure S2). When cells were transferred to water
or to depleted medium that was either adjusted or not adjusted to
pH 7.0, there was no significant pH variation during the entire
experiment. As shown in Figure 2, cells grown with or without
amino acid deprivation exhibited a longer CLS after they were
transferred to water compared to cells transferred to depleted
culture medium that maintained a pH of 2.6–2.9, although loss of
cell viability again occurred much faster for cells grown with
amino acid restriction. In all cases, addition of NH4+to water
reduced cell survival in proportion to its concentration, mimicking
its effect in the depleted media. Furthermore, the NH4+-induced
reduction in CLS observed in water positively correlated with the
concentration of NH4+in the growth medium, which indicates
that culture conditions pre-determined the cellular response to
NH4+. Furthermore, transferring cells cultured with insufficient
supply of amino acids with 1%, 0.5% or 0.1% (NH4)2SO4to the
respective exhausted medium adjusted to pH 7.0 did not lead to a
significant difference in CLS relative to the CLS of cells in the
exhausted acidic medium (Figure 2C, 2D and 2E). In contrast,
Figure 1. Ammonium stimulates CLS shortening. Survival of S.
cerevisiae stationary phase cells grown in media supplemented with low
(open symbols) and high (dark symbols) concentrations of auxotrophy-
complementing amino acid, and supplemented with 0.01% (%); 0.1%
(n); 0.5% (e,X) or 1% (#,N) ammonium sulphate. In all the cultures,
starting cell density was about 3.86107cells/ml. Values are means 6
SEM (n=3). P,0.001. Statistical analysis was performed by two-way
ANOVA.
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cells cultured under amino acid restriction conditions with the
lowest (NH4)2SO4 concentration (0.01%) after transfer to the
respective exhausted medium adjusted to pH 7.0 exhibited an
extended CLS (Figure 2F). Similar results were obtained with cells
cultured without amino acid restriction (Figure 2A and 2B), which
is consistent with results previously described for similar conditions
[26].
To further explore the toxic effects of NH4+during chronolog-
ical aging under amino acid restriction conditions, a conventional
nitrogen starvation protocol [28] was adapted to accommodate the
following conditions in SC glucose starvation medium: i) lack of
the auxotrophy-complementing amino acids and presence of
NH4+(aa-starved cells) or ii) lack of the auxotrophy-complement-
ing amino acids and of NH4+(N-starved cells). Cells were grown to
mid exponential phase in SC medium with 2% glucose and then
starved for 24 hours in both types of starvation media. Cells were
subsequently transferred to water (pH 7.0), with and without
NH4+or to the respective 24 hour starvation medium (final
pH 2.7–2.9) that was or was not adjusted to pH 7.0 (see scheme of
methodology in Figure S2). The initial pH did not significantly
change during the assay, except for cells transferred to starvation
media at pH 7.0, which reached a final pH around 5.0.
Both aa-starved and N-starved cells survived for a longer period
of time in water relative to those in starvation medium (pH 2.7–
2.9). Addition of NH4+to water induced a rapid loss of cell
viability and shortening of CLS for aa-starved cells (Figure 2G and
2H). Cells cultured in starvation medium that was adjusted to pH
of 7.0 also exhibited a rapid decrease in cell viability, indicating
that the NH4+effect is not due to the acidification of the medium.
In contrast to aa-starved cells, N-starved cells survived for a longer
period when transferred to the starvation medium adjusted to
pH 7.0. To eliminate the possibility that the reduced survival of
aa-starved cells induced by the addition of (NH4)2SO4to water
was due to sulphate and not to ammonium, the same experiment
was performed in water to which NH4OH was added instead of
(NH4)2SO4, and similar results were obtained (Figure S1B).
As discussed in the Introduction, NH4+toxicity was previously
described in steady-state chemostat cultures of yeast under limiting
potassium concentration [20]. To determine if the ammonium
toxicity we observed in our experiments depends on potassium
concentration, we repeated our experiments after adding potas-
sium to water at a concentration that according to this earlier
study abolished NH4+toxicity. In fact, addition of potassium did
not alter the NH4+-induced loss of cell viability (Figure S1C).
Taken together, our results (summarized in Table 1) suggest that
(i) NH4+in the culture medium has a substantial concentration-
dependent inhibitory effect on CLS indicated by a significant
increase in cell survival when the starting NH4+concentration in
the medium is reduced; (ii) the CLS of cells cultured to stationary
phase with amino acid restriction or starved for auxotrophy-
complementing amino acids and subsequently transferred to water
is significantly shortened by the addition of NH4+and (iii)
acidification of the medium does not promote the observed
decrease in cell survival, in contrast to what is observed at the
lowest NH4+concentration and in cells grown without amino acid
restriction.
Consequently, in subsequent experiments we employed aa-
starved cells to address the mechanisms underlying cell death
induced by NH4+.
NH4+induces apoptosis and necrosis in association with
the reduction in CLS in amino acid starved yeast cells
To determine the mechanism by which cell death occurs in
association with the reduction in CLS induced by NH4+, several
standard markers of cell death were examined in aa-starved cells
transferred to water alone or to water containing NH4+after the
pH was adjusted to 7.0 in both cases. Increased ROS accumu-
lation is a common event in many cell death scenarios, both
apoptotic and necrotic [29–31]. We measured the accumulation of
reactive oxygen species (ROS) using the fluorescent probe
dihydrorhodamine 123 (DHR, which preferentially detects
H2O2). DHR signals increased with time either in the absence
or presence of NH4+, but this increase occurred more rapidly in
cells incubated with NH4+, peaking at day 2 (Figure 3A). In
contrast, levels of ROS detected using dihydroethidium (DHE,
which preferentially detects O22), were not significantly different
in the absence or presence of NH4+. The shorter CLS induced by
NH4+was also accompanied by an increase in the number of cells
exhibiting chromatin condensation and nuclear fragmentation
(Figure 3B) and by the emergence of a population of cells with a
sub G0/G1 content of DNA that increased over time (Figure 3C
and S3A). Incubation with NH4+also resulted in an increase in
TUNEL positive cells, although this occurred in a relatively small
percentage of the total population (Figure 3D). Furthermore,
staining with annexin V and PI was used to identify apoptotic and
necrotic cells [30]. In this double staining approach, annexin V
binds phosphatidylserine of the plasma membrane whereas PI,
being a membrane-impermeable stain, assesses loss in membrane
integrity. Annexin V+/PI2 staining shows cells with phosphati-
dylserine exposed on the outer surface of the plasma membrane in
the absence of a loss in membrane integrity and therefore cells are
considered apoptotic, while PI+ cells are necrotic. Cells transferred
to water containing NH4+exhibited a small increase in Annexin V
staining in the absence of PI staining during the first few days
(Figure 3E). However, after day 2 these cells exhibited extensive
permeabilization of the plasma membrane evidenced by PI
staining, which indicates they were mostly undergoing necrosis.
Necrosis was confirmed by the observation of the nucleus-cytosolic
translocation of Nhp6Ap (Figure 3G), the yeast homologue of
human chromatin bound non-histone protein HMGB1 (high
mobility group Box 1) whose nuclear release is considered a
marker of necrosis [17]. Also consistent with necrosis, we observed
a significant decrease in ATP content in these cells beginning on
the first day of assays (Fig. S3B) which may have limited energy
consuming apoptotic processes. Furthermore, NH4+-induced cell
death was not prevented by cycloheximide (Figure S4D),
indicating that death is not dependent on de novo protein synthesis.
Together these data point to an initial apoptotic cell death induced
by NH4+followed by a rapid secondary necrosis.
To clarify the mechanism(s) of cell death induced by NH4+, we
employed strains from which genes coding for the yeast
metacaspase (Yca1p), apoptosis inducing factor (Aif1p), mitochon-
drial cyclophylin (Cpr3p) and calpain (Rim13p) had been deleted.
Loss of cell viability induced by NH4+in aa-starved cells in water
was not altered by deletion of either YCA1 or AIF1 (Figure S5A
and S5B). Therefore, cell death does not depend on Yca1p or
Aif1p, which are key factors in several yeast apoptotic processes
[32,33]. Strains deleted in RIM13 and CPR3 coding for the yeast
orthologs of mammalian proteins previously associated with
necrotic phenotypes [29] displayed loss of cell viability induced
by NH4+in water similar (cpr3D) or higher (rim13D), when
compared to wild type strain (Figure S5C and S5D), indicating
that those genes are not associated with the NH4+sensitivity
phenotype. In agreement with the results obtained with the cpr3D
mutant, loss of cell viability induced by NH4+in aa-starved wild
type cells in water was also not altered by simultaneous incubation
with cyclosporine, an inhibitor of mitochondrial cyclophylin
(Figure S5E). We observed an increase in death induced by
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NH4+in the rim13D mutant, suggesting that instead of mediating
cell death, Rim13p, belonging to the calpain family of cysteine
protease that are activated by Ca2+[34], may protect against cell
death. Consistent with the involvement of calpain activity, an
increase in the intracellular calcium concentration was observed in
the presence of NH4+(Figure 3F).
In summary, although cell death induced by NH4+in aa-starved
cells was accompanied by chromatin condensation and DNA
fragmentation, both of which suggest an apoptotic process, the
subsequent loss of membrane integrity points to accelerated
necrosis at later times that may be partially rescued by calpain.
Autophagy is not a key player in NH4+-induced cell death
Autophagy is regulated by nitrogen availability via the major
nutrient signalling pathways, which also regulate CLS [6,35].
Therefore, we asked whether autophagy might be required for the
NH4+-induced decrease in CLS. ATG8 codes for a protein
essential for autophagosome assembly and its expression is up-
regulated by nitrogen starvation shortly after autophagy induction
[36]. Thus, we monitored Atg8p levels in cells starved for amino
acids (aa- starved cells), before and after transfer to water with or
without NH4+(Figure 4A). As expected, autophagy was induced in
control cells completely starved for nitrogen (N-starved cells).
Autophagy was not induced, however, in aa-starved cells before
they were transferred to water, although autophagy was detected
in both aa- and control N-starved cells after transfer to water in the
absence of NH4+. Importantly, the presence of NH4+in water
inhibited induction of autophagy in aa-starved cells but not in
control N-starved cells.
To evaluate the impact of inhibiting autophagy on cell viability,
we used a mutant in the TOR pathway (tor1D). Tor1p associates
with Tor2p and three other proteins to form the TORC1
complex, which negatively regulates autophagy [10]. The tor1D
mutant also did not exhibit autophagy either after amino acid
starvation or upon transfer to water with NH4+(Figure 4B).
However, there was a significant reduction in NH4+toxicity in this
mutant (Figure 4C), thus excluding inhibition of autophagy as a
causal factor in NH4+-induced cell death. To address this point
further, we employed wortmannin, an inhibitor of PI3-kinases that
blocks autophagy, as well as a mutant deficient for ATG8 (Figure
S4). atg8D aa-starved cells in water with NH4+displayed loss of cell
viability similar to that of wild type (WT) cells (Figure S4A).
Addition of wortmannin to aa-starved WT cells incubated in water
with NH4+also had no effect in cell survival (Figure S4B).
Furthermore, NH4+-induced cell death was not observed in atg8D
N-starved cells (Figure S4C). These results indicate that although
NH4+inhibits autophagy, autophagy inhibition is not the cause of
the NH4+-induced cell death observed in aa-starved cells.
PKA and TOR regulate the ammonium-induced reduction
in the CLS of amino acid-starved yeast cells
CLS is under the control of both TOR, Sch9p and PKA
signalling pathways [6]. The absence of autophagy inhibition as a
causal factor in NH4+-induced cell death led us to hypothesize
that NH4+toxicity might be mediated by PKA activation instead.
Trehalase is a target of PKA regulation and its activity has been
extensively used to monitor PKA activation [11]. As shown in
Figure 5A, trehalase activity was much higher in aa-starved cells
upon transfer to water with NH4+than in the same cells without
Figure 2. Ammonium stimulates cell death of S. cerevisiae associated with a shortening of the CLS. A, B, C, D, E and F. Survival of S.
cerevisiae stationary phase cells grown in media supplemented with low (L-AA) and high (H-AA) concentrations of auxotrophy-complementing amino
acid, and supplemented with (F) 0.01%; (E) 0.1%; (A, C) 0.5% or (B, D) 1% ammonium sulphate (AS). After 72 hours of growth, cells were transferred to:
(e) water (pH 7.0); (#) water with 0.5% (NH4)2SO4(pH 7.0); (n) water with 1% (NH4)2SO4(pH 7.0); (N) exhausted medium; (X) exhausted medium
(pH 7.0). Values are means 6 SEM (n=3–5). G and H - Survival of (H) nitrogen starved cells (N-) or (G) amino acid-starved cells (aa-), after transfer to:
(e) water (pH 7.0); (#) water with 0.5% (NH4)2SO4(pH 7.0); (N) starvation medium; (X) starvation medium (pH 7.0). In all the cultures, starting cell
density was about 3.86107cells/ml. Values are means 6 SEM (n=8). P,0.001 (aa-starved H2O vs aa-starved 0.5% (NH4)2SO4). Statistical analysis was
performed by two-way ANOVA.
doi:10.1371/journal.pone.0037090.g002
Table 1. Values of Area under the survival curve (AUC) of strain BY4742 cultured in different medium composition.
Cell culture or pre-incubation conditions
High-AALow-AA aa-starved
N-starved
(NH4)2SO4
0.5%
(NH4)2SO4
1%
(NH4)2SO4
0.01%
(NH4)2SO4
0.1%
(NH4)2SO4
0.5%
(NH4)2SO4
1%
(NH4)2SO4
0.5%
Aging assay in:Medium
Sc
1205613 117562 805647207612 11565 7165 18667 27263
Medium
Sc pH7
1439633120265121462 1756574697462 24963 72561
H2O13756871347629139065 916625 745642446644 72361067962
(NH4)2SO4
0.5%
92367795165 706623 385620271613133612 188610 588616
(NH4)2SO4
1%
53562 5526102n.d.n.d. 1206109067n.d. n.d.
n.d. – not determined. Cells were grown in SC media supplemented with low (Low-AA) or high (High-AA) concentrations of auxotrophy-complementing amino acid and
with 0.01%; 0.1%; 0.5% or 1% (NH4)2SO4for 72 hours; or cells were grown in SC media until O.D. 1–1.5, harvested and resuspended in Nitrogen-starvation medium (N-)
or in amino acid-starvation medium (aa-) for 24 hours. The aging assays were performed by resuspending cells from the different culture conditions in their respective
exhausted medium, exhausted medium pH 7, or in 0.5 and 1% (NH4)2SO4, pH 7.
doi:10.1371/journal.pone.0037090.t001
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NH4+or in N-starved cells (negative control) under both
conditions. In support of the hypothesis that activation of PKA
increases sensitivity to NH4+, addition of cAMP increased cell
death in the presence of NH4+in N-starved cells and had no effect
on aa-starved cells, which display high PKA activity in the absence
of added cAMP (Figure 5B and 5C). In addition, deletion of RAS2,
a regulator of PKA activity through the stimulation of cAMP
production, caused a partial reversion of the NH4+sensitivity
phenotype of aa-starved cells (Figure 5E). The NH4+permease
Mep2 (and Mep1 to a lesser extent) function as sensors for NH4+-
induced activation of PKA, whereas Mep3p, the other member of
the family of NH4+transporters, does not [28,37]. As shown in
Figure 5E, the mep2D and mep1D strains exhibited a decrease in
NH4+-induced death in aa-starved cells, although this decrease
was significant only in mep2D. This is in agreement with the more
predominant role of Mep2p in PKA signalling. In order to identify
the specificity of the signalling process through PKA, we also
tested the effects of deleting the genes that code for the three
isoforms of the catalytic subunit of this kinase, TPK1, TPK2 and
TPK3. Only deletion of TPK1 caused a significant reversion of the
NH4+-induced decrease of the CLS, whereas no differences were
detected for strains deficient in TPK2 and TPK3 (Figure 5F).
Sch9p is a protein kinase with high sequence homology to
Tpk1, 2, 3 kinases and regulates cell metabolism in response to
several nutritional signals, such as nitrogen and carbon source
[38]. Sch9p shares many targets with PKA and TORC1, and
different interactions between these pathways, either cooperating
or antagonizing, have been described [15]. Data from Figure 5D
show that sch9D aa-starved cells underwent increased cell death
upon transfer to water plus NH4+and that the lack of Sch9p
reduced survival after cells were transferred to water. These results
Figure 3. Ammonium-induced cell death was accompanied by an initial small increase of apoptotic cells followed by extensive
necrosis. Cell death markers measurements in aa-starved cells of S. cerevisiae, upon transfer to: (X) water (pH 7.0) or (e) water with 0.5% (NH4)2SO4
(pH 7.0). (A) ROS accumulation, (B) chromatin condensation and fragmentation, (C) appearance of Sub-G0/G1 peak, (D) TUNEL staining, (E) Annexin/PI
positive staining and (F) calcium accumulation. (G) Fluorescence microscopy of aa-starved cells (day 0, 1 and 3) expressing Nhp6A–EGFP, upon
transfer to water (pH 7.0) or water with 0.5% (NH4)2SO4(pH 7.0). Scale bars, 10 mm. In all the cultures, starting cell density was about 3.86107cells/ml.
Values are means 6 SEM (n=3). H2O vs 0.5% (NH4)2SO4: (A) P,0.001; (C) P,0.001; (D) P,0.01; (E) P,0.001; (F) P,0.01. Statistical analysis was
performed by two-way ANOVA.
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suggest that pathways regulated by Sch9p are important for
survival under these conditions
To evaluate the dependence of PKA activation on Sch9p,
Tor1p, and Mep2p, trehalase activity was measured in aa-starved
cells of the corresponding deletion mutants. Trehalase activity was
similar in all strains before or after transfer to water. However, in
the presence of NH4+, trehalase activity decreased in tor1D and in
sch9D cells and was almost completely undetectable in the latter
strain (Figure 5A). These results establish that NH4+signalling to
PKA requires Tor1p and Sch9p. However, the opposite cell death
phenotypes of sch9D compared to tor1D and tpk1D cells observed in
aa-starved cells in the presence of NH4+suggest that the role of
Sch9p in the process is essentially independent of the TOR-PKA
pathway.
Hog1p is a kinase that regulates and is regulated by Sch9p and
mediates stress response independently of PKA and TOR
pathways [39]. To assess whether Hog1p might play a role in
resistance to the toxic effects of NH4+mediated by Sch9p, we
examined the effects of NH4+in a hog1D strain. Like sch9D cells,
hog1D strains were more sensitive to the toxic effects of NH4+,
which suggests that Sch9p may be signaling Hog1p to mediate
increased resistance.
Metabolism of NH4+is not required for NH4+-induced cell
death
The role of Mep2p in signaling PKA activation in response to
NH4+in nitrogen starvation medium is not dependent on the
metabolism of NH4+[28]. Therefore, we next asked whether
NH4+toxicity that leads to a reduction in CLS under our
experimental conditions might be signaled directly by NH4+or
perhaps requires that it be metabolized. In yeasts, the first step of
NH4+assimilation is mediated by NADPH-dependent glutamate
dehydrogenase, which converts a-ketoglutarate to glutamate,
which can be further metabolized to glutamine by glutamine
synthetase. Glutamine synthetase activity was higher in N-starved
cells than in aa-starved cells, indicating that the activity of this
enzyme was not related to the higher toxicity of NH4+(Table S1).
We also tested the effect of NH4+in both aa-starved and N-starved
cells in the presence of the glutamine synthetase inhibitor
methionine sulfoximine. No significant differences in loss of cell
viability were observed (Figure S6A and S6B), further supporting
the hypothesis that the toxic effect of NH4+does not require that it
be metabolized. Glutamate dehydrogenase activity at T0 was
higher in N-starved cells than in aa-starved cells, but incubation in
water with or without NH4+led to a decrease in its activity
(Figure 6A). In contrast, glutamate dehydrogenase activity
increased approximately 3-fold in aa-starved cells incubated in
the presence of NH4+. We asked whether a-ketoglutarate
depletion or glutamate accumulation, which might result from
the higher glutamate dehydrogenase activity, could be the cause of
NH4+toxicity. Adding a-ketoglutarate to the medium did not alter
the toxic effects of NH4+(Figure S6C), whereas adding glutamate
resulted in more rapid loss in cell viability, even in the absence of
NH4+(Figure 6B). Furthermore, the non-metabolizable NH4+
analogue methylamine also induced cell death in aa- but not N-
starved cells (Figure S6D). In agreement with these results, the
NH4+toxicity observed in SC media cultures (Fig. 1) was also not
associated with a significant NH4+metabolization, as depicted
from the levels of (NH4)2SO4along time (Fig. S1A).
Taken together, these results suggest that although glutamate
could play a role in NH4+-induced cell death to some extent,
NH4+-induced shortening of CLS does not appear to require that
it be metabolized.
Discussion
Our studies demonstrate that at high concentrations, NH4+,
which is a commonly employed source of nitrogen in laboratory
yeast cultures, induces cell death in association with a reduction in
CLS. The toxic effects of NH4+correlate with NH4+concentration
Figure 4. Ammonium inhibits autophagy induction in aa-
starved cells of S. cerevisiae. Western-blot analysis of Atg8p levels
present in: (A) wild-type (WT) aa-starved or N-starved cells, upon
transfer to water or water with 0.5% (NH4)2SO4; and in (B) WT and tor1D
aa-starved cells, upon transfer to water or water with 0.5% (NH4)2SO4.
(C) Survival of aa-starved cells of wild-type and tor1D mutant, upon
transfer to water or water with 0.5% (NH4)2SO4. In all the cultures,
starting cell density was about 3.86107cells/ml and the initial pH was
adjusted to 7.0. Values are means 6 SEM (n=3–4). (C) P,0.001 (H2O vs
0.5% (NH4)2SO4). Statistical analysis was performed by two-way ANOVA.
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in the culture medium and were enhanced in cells starved for
auxotrophy-complementing amino acids. Addition of NH4+to
cultures after they were transferred to water reduced cell survival,
indicating that NH4+alone could also induce loss of cell viability as
observed in culture media. Overall the results suggest NH4+is a
factor accounting for the loss of cell viability in aging cells.
Although some of the toxic effects of NH4+were accompanied by
markers for apoptosis, NH4+-induced cell death was predomi-
Figure 5. Ammonium reduces CLS of S. cerevisiae through the regulation of both PKA and Sch9 activities. (A) Trehalase activity of wild-
type (WT) N-starved cells and WT and mutant (mep2D, sch9D and tor1D) aa-starved cells, before transferred to water (T0h) and after 2 hours in water
(T2h H2O) or water with 0.5% (NH4)2SO4(T2h NH4+). Survival of (B) wild-type aa-starved cells or (C) N-starved cells, after transfer to: (X) water (pH 7.0);
(e) water with 0.5% (NH4)2SO4(pH 7.0); (m) water (pH 7.0) supplemented with cAMP (4 mM); (n) water with 0.5% (NH4)2SO4(pH 7.0) supplemented
with cAMP (4 mM). Survival in water (pH 7.0) or water with 0.5% (NH4)2SO4(pH 7.0) of aa-starved cells of: (D) WT, sch9D and hog1D; (E) WT, mep1D,
mep2D and ras2D; (F) WT, tpkD mutants (tpk1D, tpk2D or tpk3D). In all the cultures, starting cell density was about 3.86107cells/ml and the initial pH
was adjusted to 7.0. Values are means 6 SEM (n=3–4). (A) *P,0.05 (WT H2O vs WT 0.5% (NH4)2SO4), (WT 0.5% (NH4)2SO4vs tor1D 0.5% (NH4)2SO4);
**P,0.01 (WT 0.5% (NH4)2SO4vs sch9D 0.5% (NH4)2SO4); (D) P,0.05 (WT 0.5% (NH4)2SO4vs hog1D 0.5% (NH4)2SO4), P,0.001 (WT H2O vs sch9D H2O);
(E) P,0.05 (WT 0.5% (NH4)2SO4vs ras2D 0.5% (NH4)2SO4); P,0.01 (WT 0.5% (NH4)2SO4vs mep2D 0.5% (NH4)2SO4); P,0.05 (WT 0.5% (NH4)2SO4vs
mep1D 0.5% (NH4)2SO4); (F) P,0.01 (WT 0.5% (NH4)2SO4vs tpk1D 0.5% (NH4)2SO4). Statistical analysis was performed by two-way ANOVA.
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nantly necrotic at later time points. Our data suggest that NH4+
causes an initial apoptotic cell death followed by a fast secondary
necrosis. Necrosis due to ATP depletion has been reported in
other cell death scenarios, namely in tumor cells under metabolic
stress [40]. This appears to be the case in NH4+-induced necrosis,
since ATP depletion was observed in cells incubated in water with
NH4+(Figure S3B), which might block ATP-dependent apoptosis
and thus trigger necrosis. The results obtained with the deletion
mutant rim13D point to a protective function of the protease
calpain in this cell death process.
As discussed in the Introduction, cells in G0 acquire a variety of
characteristics including induction of autophagy [8,12]. Previous
studies showed that cells starved for auxotrophic amino acid
markers in otherwise complete medium fail to properly arrest in
G0 [41]. In accordance, aa-starved cells in our study also do not
seem arrested in G0 (indicated by a failure to induce autophagy). It
should be noted that this failure to induce autophagy by aa-starved
cells was sustained when cells were transferred to water containing
NH4+in the absence of other nutrients. This is in contrast with
that observed in G0 arrested N-starved cells transferred to water
where NH4+could not activate PKA or inhibit autophagy.
Although autophagy was inhibited by NH4+in aa-starved cells,
inhibition of autophagy by deletion of ATG8 did not induce the
NH4+sensitivity phenotype in N-starved cells, suggesting that
autophagy inhibition is not responsible for the loss of cell viability
and shorter CLS induced by NH4. We also assessed whether
activation of PKA could be inducing replication stress, a
mechanism responsible for cell aging under different conditions
[42]. This could be the case, at least to some extent, since there
was a slight increase in the number of budded cells (evaluated by
bright field microscopy) for aa-starved (16%) conditions relative to
the control N-starved cells (8%).
In contrast to what has been described for aging cells that reach
stationary phase due to carbon limitation [43], we observed that
autophagy mutants did not exhibit increased cell death after they
were transferred to water, indicating that autophagy is not a key
player in cell survival in water when the cells were previously
starved for amino acids or nitrogen. It was recently shown that
ATG genes are important for removing ROS and for maintaining
mtDNA and mitochondrial function [44]. This may explain the
lack of dependence of cell survival on autophagy in our
experimental conditions, as the production of ROS was relatively
low. Hence, the cell physiological state resulting from different
culture conditions influences not only life span extension [2], but
also the cellular processes essential for its regulation.
In yeasts, the TOR, Sch9p and PKA pathways are key players
in the regulation of CLS [6]. In our study, activation of PKA
correlates with sensitivity to NH4+, which is partially suppressed by
deletion of RAS2, indicating the RAS/Cyr1/PKA pathway is
involved in this process. Partial, but not complete, suppression of
these effects when RAS2 is deleted suggests either that the second
RAS isoform (RAS1) also participates in NH4+-induced PKA
activation or the existence of two pathways responsible for NH4+
toxicity, one that depends on RAS/Cyr1/PKA and one that is
independent of this pathway. In nitrogen starvation medium,
addition of NH4+directly signals PKA activation through Mep2p
and does not depend on its metabolization [28]. Our results show
that Mep2p is involved in NH4+-induced death but does not
appear to have a major role in PKA activation. Still, although
glutamate could somewhat mediate the effect of NH4+, CLS
shortening also seemed to be directly signalled by NH4+, as it was
not dependent on its metabolization to either glutamate or
glutamine.
The deletion of TPK1, but not of TPK2 or TPK3, encoding the
other two PKA isoforms, significantly reverted the NH4+-induced
death and shorter CLS. These results suggest that different
programmed cell death processes can be regulated by distinct PKA
isoforms, since Tpk3p has been reported to regulate apoptosis
induced by actin stabilization [45]. Our data are also in agreement
with previous results showing that CLS extension of glucose-
growth limited stationary phase cells depends on PKA inactivation
[46]. Our results indicate that PKA inactivation cannot extend cell
survival time in the absence of Sch9p, since we observed that
SCH9 deletion abolishes PKA activation in response to NH4+, but
does not rescue the shortening in CLS induced by NH4+.
Furthermore, the phenotype of aa-starved sch9D cells in the
presence of NH4+was the opposite of that of tor1D and tpk1D,
suggesting that the role of Sch9p in the process is essentially
independent of the TOR-PKA pathway mediated by a TORC1-
Sch9 effector branch. Instead, the two pathways likely regulate
Figure 6. Ammonium-induced loss of cell viability of S. cerevisiae does not depend on its metabolization. (A) Glutamate dehydrogenase
(GDH1) activity of aa-starved (aa-H2O and aa-NH4+) and N-starved cells (N-H2O and N-NH4+), before transferred to water (T0h) and after 2, 24 and
48 hours in water or water with 0.5% (NH4)2SO4. (B) Survival of wild-type aa-starved cells, in water or water with 0.5% (NH4)2SO4, supplemented or not
with glutamate (5 mg/ml). In all the cultures, starting cell density was about 3.86107cells/ml and the initial pH was adjusted to 7.0. Values are means
6 SEM (n=3–4). (A) ***P,0.001; **P,0.01 (B) P,0.05 (H2O vs H2O+Glut.). Statistical analysis was performed by two-way ANOVA.
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their downstream targets that are involved in NH4+-induced cell
death in an opposing manner. Consistent with this possibility, it
was reported that Sch9p positively regulates many stress-response
genes and genes involved in mitochondrial function, whereas the
same classes of genes are inhibited by the TOR1C pathway [15].
Data suggest that Sch9p may mediate survival in response to
NH4+through activation of Hog1p, the yeast closest homolog to p-
38 and c-JNK of mammalian cells [47]. Previous reports have
shown that sch9D yeast cells exhibit a longer CLS compared to
wild type cells, when aging in SC medium or after transfer from
this medium to water [46,48]. Differences in strain background
and/or in culture conditions may account for the discrepancy in
results [46,48–50]. Supporting this explanation it was also
previously reported that SCH9 deletion shortened the CLS
survival of S288c-based strains (as is the case of BY4742 strain
used in the present work) pregrown on glycerol [1].
In conclusion, here we have shown that NH4+induces cell death
in aging yeast in association with a reduction in CLS, both of
which are positively correlated with NH4+concentration in the
culture medium. Furthermore, these effects are enhanced in cells
starved for auxotrophy-complementing amino acids. As for the
mechanism involved (Figure 7), the results indicate that in aa-
starved cells NH4+activates PKA through both RAS and TOR/
Sch9p signalling cascades and leads to cell death increase with
predominant necrotic features. The mediation of NH4+effects
seems to involve the NH4+permeases Mep2 and (to a lesser extent)
Mep1 as sensors. Sch9p is also mediating survival in response to
NH4+possibly through activation of Hog1p. NH4+action on both
pathways culminates in the shortening of CLS.
As discussed in the introduction, NH4+is toxic for mammals,
and NH4+-induced cell death is involved in different human
disorders that are accompanied by hyperammonemia, such as
hepatic encephalopathy [21]. Here we extensively characterized
for the first time a cell death process induced by NH4+in yeast
cells. This process shares common features with NH4+-induced cell
death in brain cells. A better understanding of NH4+-induced cell
death in the yeast cell model can help clarify controversial issues
on NH4+toxicity associated to hyperamonemia that are not easy
to examine in more complex models. Our results show that the
effect of NH4+is not due to different levels of NH4+metaboliza-
tion, an open question for brain cells, but relies on the over-
activation of PKA and the TOR pathway and inhibition of Sch9p
(yeast closest homolog of mammalian Akt and S6K). On the other
hand, the mitogen activated protein kinase (MAPK) Hog1p was
associated with higher cell viability in the presence of NH4+
similarly to what was found for its human homolog p38 that
mediates endogenous cell protection in response to ammonium in
astrocytes [51]. Also, we observed that NH4+toxicity is higher in
non-arrested cells, which is consistent with the observation that
hyperammonemia presents with much more severe consequences
in the developing brain of newborns or infants than in adulthood.
Furthermore, our data link NH4+toxicity to amino acid limitation,
a situation that can also be present in hyperammonemic patients,
who are often on dietary protein restriction [52]. Further
experiments will be necessary to establish whether over-activation
of TOR and PKA pathways and inhibition of Sch9p is a widely
conserved mechanism in NH4+toxicity and induction of cell
death. We believe that our model can be useful in the elucidation
of conserved mechanisms and pathways of NH4+-induced cell
death and in identification of therapeutic targets for diseases
associated with hyperammonemia. Deprivation of essential amino
acids has been employed as a strategy in cancer therapy, but
resistance has often been found. Our results establishing that NH4+
can stimulate cell death in amino acid-deprived cells and suggests
that S. cerevisiae might serve as useful model for the identification of
signaling pathways for this disease. Furthermore, our finding that
NH4+decreases cell survival during aging through the regulation
of the evolutionary conserved pathways PKA and TOR also
enriches our understanding of longevity regulation in multicellular
organisms.
Materials and Methods
Strains and growth conditions
Saccharomyces cerevisiae strain BY4742 (MATa his3D1 leu2D0
lys2D0 ura3D0) (EUROSCARF, Frankfurt, Germany) and the
respective knockouts in YAC1, AIF1, RIM13, RAS2, CPR3, ATG8,
SCH9, MEP1, MEP2, TPK1, TPK2, TPK3 and TOR1 genes, were
used. For experiments with stationary phase cells with or without
restriction of auxotrophy-complementing amino acids, cells were
cultured at 26uC, 150 rpm, for 72 hours, in defined minimal
medium (SC medium) containing 0.17% yeast nitrogen base
without amino acids and without ammonium sulphate (Difco, BD),
2% D-glucose; supplemented with 0.01%, 0.1%, 0.5% or 1%
ammonium sulphate, and with low (10 mg/l histidine, 10 mg/l
lysine, 60 mg/l leucine and 100 mg/l uracil) or high (50 mg/l
histidine, 50 mg/l lysine, 300 mg/l leucine and 100 mg/l uracil)
concentrations of essential amino acids. After 72 hours, cells were
collected by centrifugation and: A) resuspended in growth medium
(exhausted medium without adjusting pH - pH 2.9) with a cell
density of about 3.8610
medium (exhausted medium) with a cell density of about 3.8610
7cells/ml, with pH adjusted to 7.0.; C) resuspended in water
(pH 7.0), after being washed three times, at cell density of about
3.86107cells/ml; D) resuspended in water with ammonium
sulphate (0.5% or 1%, pH 7.0.), after being washed three times, at
cell density of about 3.86107cells/ml. Viability of stationary 3 day
7cells/ml, B) resuspended in growth
Figure 7. Proposed mechanism for the regulation of cell death
associated to CLS shortening induced by ammonium in amino
acid-starved yeast cells. NH4+activates PKA through both RAS and
TOR/Sch9p and leads to cell death increase with predominant necrotic
features associated to ATP depletion. Sch9p is mediating survival in
response to NH4+possibly through activation of Hog1p.
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old cultures was considered to be 100% of survival and this was
considered day 0 of the experiment. pH 7.0 was maintained
throughout the experiment in cultures with adjusted pH. For
experiments with aa- and N-starved cells, cells were first cultured
at 26uC and 150 rpm, in the defined minimal medium described
above, supplemented with 0.5% ammonium sulphate, appropriate
amino acids and base (50 mg/l histidine, 50 mg/l lysine, 300 mg/
l leucine and 100 mg/l uracil) and 2% D-glucose, to exponential
phase (OD600=1.0–1.5). These cells were harvested and resus-
pended in nitrogen-starvation medium (N-) containing 4% glucose
and 0.17% yeast nitrogen base without amino acids and
ammonium sulphate, or in amino acid-starvation medium (aa-)
containing the same components as N-starvation medium plus
0.5% ammonium sulphate. After 24 hours, cells were collected by
centrifugation and: A) resuspended in starvation medium (N- or
aa-) with a cell density of about 3.86107cells/ml, without adjusting
pH (pH 2.7); B) resuspended in starvation medium (N- or aa-) with
a cell density of about 3.86107cells/ml, with pH adjusted to 7.0;
C) resuspended in water (pH 7.0), after being washed three times,
at cell density of about 3.86107cells/ml; D) resuspended in water
with ammonium sulphate (0.5%, pH 7.0.), after being washed
three times, at cell density of about 3.86107cells/ml. Viability of
24 hours starved cultures was considered to be 100% of survival
and this was considered day 0 of the experiment. pH 7.0 was
maintained throughout the experiment in cultures with adjusted
pH. Cell viability was assessed by Colony Forming Units (CFU) at
day 0 (100% viability) and in subsequent days, as indicated, of
culture aliquots incubated for 2 days at 30uC on YEPD agar
plates. BY4742 strain was transformed with plasmids pUG35 and
pUG35- NHP6A-EGFP [17], kindly provided by Dr. Frank Madeo
(University of Gratz, Austria), and was cultured as described above
for aa-starved cells, in medium lacking uracil. The methodology of
aging experiments with stationary phase cells and with amino acid
(aa)- and nitrogen(N)-starved cells is schematically represented in
Figure S1.
Ammonium and ATP Determination
Ammonium in the culture media was quantified by Dr. Jose ´
Coutinho (University of Tra ´s-os-Montes e Alto Douro, Portugal)
as previously described [53].
ATP measurements were performed according to [54]. Briefly,
cells were collected by centrifugation and the pellet was frozen
with liquid nitrogen and stored at 280uC. For the ATP assay, the
pellet was mixed with 200 ml of 5% TCA and vortexed for one
minute, twice, with one minute interval on ice. This mix was
centrifuged for one minute, at 4uC, and 10 ml of the supernatant
were added to 990 ml of reaction buffer (25 mM HEPES, 2 mM
EDTA, pH 7.75). 100 ml of this mixture was added to 100 ml of
Enliten Luciferin/Luciferase Reagent (Promega) and lumines-
cence was measured on a ThermoScientific Fluoroskan Ascent FL.
Measurements of cell death markers
For the detection of chromatin changes, cells were stained with
4,6-diamido-2-phenyl-indole (DAPI, Sigma) according to [55].
DNA strand breaks were assessed by TUNEL with the ‘In Situ
Cell Death Detection Kit, Fluorescein’ (Roche Applied Science) as
described previously [55]. In both assays, and also for the nuclear
release of the necrotic marker Nhp6Ap–EGFP, cells were
visualized by epifluorescence in a Leica Microsystems DM-
5000B microscope, at least 300 cells of three independent
experiments being evaluated, with appropriate filter settings using
a 1006/1.3 oil-immersion objective. Images were acquired with a
Leica DCF350FX digital camera and processed with LAS AF
Leica Microsystems software. To measure DNA content, cells
were stained with SYBR Green I as described [56] and staining
was assessed by flow cytometry. Plasma membrane integrity was
assessed by incubating cells with 5 mg ml21propidium iodide (PI)
(Molecular Probes, Eugene, OR) for 10 minutes at room
temperature followed by flow cytometry measurements of PI-
stained cells. Intracellular reactive oxygen species were detected by
dihydrorhodamine(DHR)-123
(DHE) (Molecular Probes). For DHR, cells were incubated with
15 mg/mL of DHR-123 for 90 min at 30uC in the dark, washed
in PBS and evaluated by flow cytometry. For DHE, cells were
incubated with 5 mM and after incubation for 10 min at 30uC cells
were washed once with PBS and evaluated by flow cytometry.
Phosphatidylserine exposure was detected by FITC Annexin-V
(BD Pharmingen) as described previously [55]. Briefly, cell walls
were digested with 3% (v/v) glusulase (NEE-154 Glusulase;
Perkinelmer) and 7 U/ml lyticase (Sigma) for 40 minutes, at
28uC. For intracellular calcium measurements, cells previously
washed with PBS were stained with 10 mM FLuo3 AM (Molecular
Probes, Eugene, OR) for 2 hours at 30uC in the dark,
subsequently washed in PBS and assessed by flow cytometry.
Flow cytometry analysis of the above experiments was performed
in an EpicsH XLTM(Beckman Coulter) flow cytometer, equipped
with an argon ion laser emitting a 488 nm beam at 15 mW. The
green fluorescence was collected through a 488-nm blocking filter,
a 550-nmlong-pass dichroic and a525-nm bandpass. Red fluores-
cence was collected through a 488-nm blocking filter, a 590-
nmlong-pass dichroic and a620-nm bandpass. Thirty thousand
cells per sample were analyzed. Positive controls for apoptosis
involved treatment of cells with 160 mM acetic acid for
200 minutes, at pH 3 and 3 mM H2O2at pH 3. For the necrotic
marker Nhp6Ap–EGFP, no nuclear release was observed in the
presence of 3 mM H2O2.
staining or dihydroethidium
Treatments
Methionine sulfoximine (MSX,Sigma), an irreversible inhibitor
of glutamine synthetase, was dissolved in sterile water at a
concentration of 100 mM and stored at 4uC. MSX was added to
water (pH 7.0), and water with ammonium sulphate (0.5%,
pH 7.0), at the concentration of 1 mM. Wortmannin (Sigma), a
PI3K inhibitor, was added to water (pH 7.0), and water with
ammonium sulphate (0.5%, pH 7.0), at the concentration of 6 mM
or 23 mM. Glutamate was added to water (pH 7.0), and water with
ammonium sulphate (0.5%, pH 7.0), at the concentration of
5 mg/ml. Adenosine 39,59-cyclic monophosphate (cAMP, Sigma)
was added to aa-starvation or N-starvation medium or to water
(pH 7.0), and water with ammonium sulphate (0.5%, pH 7.0), at
the concentration of 4 mM. a-Ketoglutaric acid potassium salt
(Sigma) was added to water (pH 7.0), and water with ammonium
sulphate (0.5%, pH 7.0), at the concentration of 5 mg/ml.
Cyclosporin A (Sigma) was added to water (pH 7.0), and water
with ammonium sulphate (0.5%, pH 7.0.), at the concentration of
120 mg/ml.
Western Blot analysis
Western blot analysis was performed according to [57]. For
Atg8p and Pgk1p detection, rabbit polyclonal anti-Aut7 (1:200;
Santa Cruz Biotech) and mouse monoclonal anti-PGK1 (1:5000;
Molecular Probes) were used, respectively, followed by Peroxidase-
AffiniPure Goat Anti-Rabbit IgG (1:10000; Jackson ImmunoR-
esearch).
Enzyme assays
Glutamine synthetase (Gs) assay was performed according to
[58]. Glutamate dehydrogenase activity was determined according
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to [59]. Briefly, cell extracts were prepared by adding to the cell
pellet a roughly equal volume of 0.5 mm diameter glass beads in
the presence of 0.1 M potassium phosphate buffer (pH 6.0),
followed by vigorous mixing during 1 minute intervals inter-
spersed with periods of cooling in ice. The NADP-dependent
GDH activity was determined by following the disappearance of
NADPH at 340 nm. Trehalase activity was determined according
to [60]. Briefly, crude enzyme extracts were obtained by
ressuspending the cell pellet in ice-cold 50 mM MES/KOH
buffer (pH 7.0) containing 50 mM CaCl2, and adding a roughly
equal volume of 0.5 mm diameter glass, followed by vigorous
mixing during 1 minute intervals interspersed with periods of
cooling in ice. The extracts were then dialyzed overnight at 4uC in
a dialysis cellulose membrane (Cellu Sep H1, Orange). The
dialyzed extract was then used to assess trehalase activity by
measuring the liberated glucose with glucose oxidase assay (GOD,
Roche).
Supporting Information
Figure S1
of S. cerevisiae with insufficient supply of amino acids,
and cell death induced by NH4OH or by (NH4)2SO4in the
presence of increased potassium concentration. (A)
Quantification of (NH4)2SO4in SC medium supplemented with
low concentrations of auxotrophy-complementing amino acids
and 0.5% (NH4)2SO4, during culture of wild-type cells; day 23
represents the day of culture inoculation and day zero represents
the beginning of aging experiments. (B) Survival of wild-type (WT)
aa-starved cells, in water or water with 0.5% NH4OH. (C)
Survival of wild-type (WT) aa-starved cells, in water, water with
0.5% (NH4)2SO4and water with 0.5% (NH4)2SO4supplemented
with 13 mM K2SO4. Values are means 6 SEM (n=3).
(TIF)
Ammonium levels in medium during culture
Figure S2
ments with the stationary phase cells and (B) experiments with aa-
and N-starved cells.
(TIF)
Scheme of the methodology used. (A) experi-
Figure S3
content. (A) Cell cycle histograms of aa-starved and N-starved S.
cerevisiae wild-type cells at day 0 and day 5 upon transfer to water
or water with 0.5% (NH4)2SO4, after a 24 hour period in
starvation (aa- and N-) media. (B) ATP content of aa-starved cells
(day 0, 1, 2 and 3) upon transfer to water or water with 0.5%
(NH4)2SO4. Values are means 6 SEM (n=3). (B) ***P,0.001 (T0
vs T1,2 and 3).
(TIF)
Effect of ammonium on the cell cycle and ATP
Figure S4
wortmannin and cycloheximide in NH4+- induced cell
death in S. cerevisiae. Survival of wild-type (WT) and atg8D
mutant (A) aa-starved or (C) N-starved cells, in water or water with
0.5% (NH4)2SO4. Survival of WT aa-starved cells, in water or
Effect of ATG8 deletion and of the inhibitors
water with 0.5% (NH4+)2SO4, supplemented with (B) wortmannin
(WN) or (D) cycloheximide (0.01%). Values are means 6 SEM
(n=3). (A); (B) and (D) P,0.001 (H2O vs 0.5% (NH4)2SO4).
Statistical analysis was performed by two-way ANOVA.
(TIF)
Loss of cell viability induced by NH4+in aa-
starved cells in water, of S. cerevisiae wild-type (WT)
and mutants deleted in the genes coding for the yeast
metacaspase (Yca1), the apoptosis inducing factor
(Aif1), mitochondrial cyclophylin (Cpr3) and calpain
(Rim13). Survival of (A) WT and yca1D, (B) WT and aif12D, (C)
WT and rim13D and (D) WT and cpr3D aa-starved cells, in water
or water with 0.5% (NH4)2SO4. (E) Survival of WT aa-starved
cells, in water or water with 0.5% (NH4)2SO4, supplemented or
not with cyclosporine A (CsA) (120 mg/ml). In all the cultures,
starting cell density was about 3.86107cells/ml and the initial pH
was adjusted to 7.0. Values are means 6 SEM (n=3–4). (A), (B),
(D) and (E) P,0.001 (H2O vs 0.5% (NH4+)2SO4); (C) P,0.001
(WT 0.5% (NH4)2SO4vs rim13D 0.5% (NH4+)2SO4). Statistical
analysis was performed by two-way ANOVA.
(TIF)
Metabolism of NH4+is not required for NH4+-
induced cell death in S. cerevisiae. Survival of wild-type
(WT) aa-starved cells (A) or N-starved cells (B), in water or water
with 0.5% (NH4)2SO4, supplemented with methionine sulfoximine
(MSX) (1 mM). (C) Survival of WT aa-starved cells, in water or
water with 0.5% (NH4)2SO4, supplemented with a-ketoglutarate
(a-KG) (5 mg/ml). (D) Survival of WT aa-starved or N-starved
cells, in water or water with 0.5% (NH4)2SO4, supplemented with
methylamine (MA) (30 mM). In all the cultures, starting cell
density was about 3.86107cells/ml and the initial pH was adjusted
to 7.0. Values are means 6 SEM (n=3–4). (A), (C) and (D)
P,0.001 (H2O vs 0.5% (NH4)2SO4). Statistical analysis was
performed by two-way ANOVA.
(TIF)
Figure S5
Figure S6
Table S1
N-starved cells of S. cerevisiae before (T0) and after
transfer to water or water with 0.5% (NH4)2SO4.
(DOCX)
Glutamine synthetase (GS) activity of aa- and
Acknowledgments
We thank Dr. Frank Madeo (Institute of Molecular Biosciences, University
of Graz, Austria) for the gift of the plasmid expressing the Nhp6A-EGFP
fusion and Dr J. Coutinho (University of Tra ´s-os-Montes e Alto Douro,
Portugal) for the determination of ammonium concentration.
Author Contributions
Conceived and designed the experiments: CL MJS. Performed the
experiments: JS MJS CL. Analyzed the data: JS MJS CL. Wrote the
paper: JS MJS CL.
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