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Mechanisms of Ageing and Development 128 (2007) 383–391
Low auxotrophy-complementing amino acid concentrations reduce
yeast chronological life span
Pedro Gomes, Belem Sampaio-Marques, Paula Ludovico 1,Fernando Rodrigues 1, Cecılia Leao *
Life and Health Sciences Research Institute (ICVS), School of Health Sciences,
University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Received 21 December 2006; received in revised form 27 April 2007; accepted 30 April 2007
Available online 3 May 2007
Abstract
In the yeast Saccharomyces cerevisiae, interventions resembling caloric restriction, either by reduction of glucose or non-essential amino acid
content in the medium, prolong life span and retard aging. Here we have examined the role of auxotrophy-complementing amino acid
supplementation of S. cerevisiae strains in determining yeast chronological life span and stress resistance. The results obtained from cells cultured
in standard amino acid concentrations revealed a reduced final biomass yield and premature aging phenotypes. These included shorter life span and
indicators of oxidative stress, together with a G2/M cell cycle arrest and the appearance of a sub-G0/G1 population pointing to the occurrence of a
specific cell death programme under starvation of essential amino acids. In order to overcome this starvation, five times higher amino acid
concentrations were supplied to the medium as has already been commonly used by few laboratories. Such cultures reached more than five-fold
higher final biomass yield in stationary phase and the early aging phenotypes were abrogated. Furthermore, in a long-lived yeast strain lacking
TOR1, there was no positive effect of amino acid supplementation on longevity. On the contrary, amino acid supply had a positive effect on
chronological life span of RAS2 deleted cells. This study may provide novel insights into the role of essential nutrients and their effect on aging
process and raises the warning that the positive effects of caloric restriction on life span maybe restricted to non-essential nutrients. Moreover, the
severe consequences on cell physiology, life span and stress resistance induced by essential amino acid imbalances presents a note of caution for
those still using standard amino acid concentrations for studies with auxotrophic yeast strains.
# 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Yeast; Chronological life span; Auxotrophy; Amino acids; Oxidative stress; Cell cycle
1. Introduction
Aging research in yeast and other model organisms has been
devoted primarily to the identification of genes that regulate life
span, in part due to the high degree of conservation in the
molecular mechanisms that govern the aging process. A
number of genes have been implied in determining yeast
replicative life span (Jazwinski, 2001), whereas only a few
genes have been associated to chronological aging (Fabrizio
et al., 2001; Longo, 2003; Powers et al., 2006). However, the
environmental factors that extend yeast longevity remain
* Corresponding author. Tel.: +351 253 604823; fax: +351 253 604862.
E-mail address: [email protected] (C. Leao).1 These authors contributed equally to this work.
0047-6374/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mad.2007.04.003
largely unknown. Nevertheless, the aging phenotypes are the
result of gene–environment interactions. Thus far, the only
environmental interventions known to increase life span in
yeast are caloric restriction (Jiang et al., 2000; Lin et al., 2000),
high osmolarity (Kaeberlein et al., 2002) and mild heat stress
(Shama et al., 1998).
Caloric restriction is well established as an experimental
intervention that retards aging and extends median and
maximal life span in a broad range of species (for reviews,
see Kirkwood and Shanley, 2005; Koubova and Guarente,
2003; Masoro, 2000) and in mammals it also slows the
progression of a variety of age-related disorders (Hursting et al.,
1994; Lane et al., 1999; Stern et al., 2001). Among the many
theories that have emerged to explain the mechanisms by which
caloric restriction extends life span, oxidative damage is the
leading hypothesis for aging-related deterioration in function
P. Gomes et al. / Mechanisms of Ageing and Development 128 (2007) 383–391384
(Balaban et al., 2005; Masoro, 2000; Sohal and Weindruch,
1996). Another potentially important mechanism consists of
the cellular response to nutrients mediated through broadly
conserved nutrient-sensing pathways that can extend life span
when signaling through these pathways is reduced or abrogated.
The target of rapamycin (TOR) is an evolutionarily conserved
serine/threonine kinase that integrates signals from nutrients, in
particular amino acids, to regulate cell growth (Martin and Hall,
2005; Schmelzle and Hall, 2000). The TOR signaling cascade
appears to have a central role in the process of life span
extension in response to nutrient availability (Kaeberlein et al.,
2005; Kapahi and Zid, 2004; Powers et al., 2006). Furthermore,
reduction of TOR signaling also enhances oxidative stress
resistance and induces the nuclear localization of stress
responsive transcription factors (Beck and Hall, 1999; Powers
et al., 2006).
The budding yeast Saccharomyces cerevisiae has been
extensively used as a model system for studying factors that
determine cellular longevity. The aging of mitotically active
cells in higher eukaryotes can be modeled by the replicative life
span of yeast mother cells (Sinclair, 2002; Tissenbaum and
Guarente, 2002), whereas aging of post-mitotic cells more
closely resembles the chronological survival of quiescent yeast
during the stationary phase of growth (Longo, 1999). Caloric
restriction in yeast has been imposed almost exclusively by
reducing the glucose content of the media (Anderson et al.,
2003; Lin et al., 2000). In one study, progressive reduction of
non-essential amino acids concentration has been shown to
promote yeast life span extension (Jiang et al., 2000).
Therefore, amino acids are at least one specific nutrient
through which caloric restriction modulates aging.
The nutritional status of the environment is an important
player in aging. Consequently, to study the aging process
without interferences of the metabolic/physiological status of
the cell, adequate culture media should be employed for cell
growth. In fact, when one or more of the nutrients for which
the strains are auxotrophic are supplemented insufficiently,
severe changes in physiology and growth behavior of a strain
can be expected. Indeed, as reported by Cakar et al. (2000),
leucine limitation in auxotrophic S. cerevisiae strains
translates into several phenotypic defects, namely reduced
growth, altered vacuolar morphology and cell cycle distribu-
tion. Following up on that study, we have hypothesized that
auxotrophic mutant strains subjected to insufficient supply of
the essential amino acids exhibit a reduction of chronological
life span, rather than an increase, observed for caloric
restriction, which might reflect inadequate nutrients to support
Table 1
Saccharomyces cerevisiae strains used in this study
Strain Relevant genotype and/or phenotype
CEN.PK113-7D MATa, prototrophic
CEN.PK2-1C MATa his3D1 leu2D0 lys2D0 ura3D0
BY4742 MATa his3D1 leu2D0 lys2D0 ura3D0
TOR1 BY4742; MATa his3D1 leu2D0 lys2D0
RAS2 BY4742; MATa his3D1 leu2D0 lys2D0
viability. Our results demonstrate that an intervention
unrelated to reduction in caloric content of the media, i.e.,
increased amino acid supplementation of yeast strains, is
accompanied by lower oxidative damage and chronological
life span extension by a mechanism that seems to involve the
TOR signaling pathway.
2. Materials and methods
2.1. Strains, growth conditions and general techniques
The yeast strains used in this study are listed in Table 1. Strains were
maintained in YEPD agar medium consisting of 0.5% yeast extract, 1%
peptone, 2% glucose and 2% agar. All experiments were performed in
synthetic complete (SC) medium containing 0.67% yeast nitrogen base
without amino acids (Difco Laboratories, Detroit, MI) and 2% glucose as
carbon source, supplemented with the appropriate amino acids and bases for
which the strains were auxotrophic. The regular amino acid concentration
(1�) corresponds to those routinely described in the literature (Pronk, 2002;
Sherman, 1991) as sufficient to satisfy auxotrophic requirements for these
amino acids (10 mg/l histidine, 10 mg/l lysine, 60 mg/l leucine and 20 mg/l
tryptophan). High amino acid supplementation corresponds to five-fold the
regular amino acid content (5�) that has also been used by several other
laboratories (Longo et al., 1996; Madeo et al., 2002; Fabrizio et al., 2004;
Powers et al., 2006). The nitrogenous base uracil was held constant at 100 mg/
l. In a standard experiment, overnight cultures were grown in either media and
inoculated into flasks with volume/medium ratio of 3:1 and grown at 30 8Cwith shaking at 150 rpm. Growth was monitored by measuring the turbidity of
the culture at 640 nm (OD640) on a spectrophotometer and viability was
determined by counting colony-forming units (CFUs) after 2 days of incuba-
tion at 30 8C on YEPD agar plates. For glucose measurements, cell culture
supernatants were collected and centrifuged in Spin-X filter columns
(0.22 mm) for 5 min at 13,000 rpm. Supernatant fluids were stored at
�20 8C before analysis by HPLC.
2.2. Chronological life span
For chronological aging experiments, cultures were inoculated from fresh
overnight cultures and every day an aliquot of the culture was plated to complete
YEPD medium to score viability. The CFUs on day 1 are considered to denote
100% survival. The assay is performed until less than 0.01% of the culture is
viable (Fabrizio et al., 2001; Fabrizio and Longo, 2003; Longo, 1999).
2.3. Stress resistance assays
Acute stress assays were conducted in yeast cells grown until late expo-
nential phase in SC medium supplemented with regular or high amino acid
concentrations. For oxidative or acid stress resistance assays, cells were
harvested and suspended (107 cells/ml) in fresh medium (with regular amino
acid content) followed by treatment with 0, 0.5, 1, 1.5 and 2 mM of hydrogen
peroxide (H2O2) (Madeo et al., 1999) or 0, 140, 160, 180 and 200 mM of acetic
acid (Ludovico et al., 2002) and incubated for 200 min, as described. Viability
was assessed at the beginning and end of the stress assays. Heat-shock
resistance was performed in cells diluted to an OD640 equivalent to
Source
P. Koetter (Frankfurt)
P. Koetter (Frankfurt)
Euroscarf collection
ura3D0; YJR066w::kanMX4 Euroscarf collection
ura3D0; YNL098c::kanMX4 Euroscarf collection
P. Gomes et al. / Mechanisms of Ageing and Development 128 (2007) 383–391 385
107 cells/ml and incubated at 48 8C for 60 min. Viability was monitored before
and during the heat-shock, at selected time points. In all stress assays, viability
was determined by CFU counting after 2 days of incubation at 30 8C.
2.4. Determination of intracellular reactive oxygen species (ROS)
accumulation
The presence of free intracellular radicals or strongly oxidizing molecules
(ROS) were detected with dihydrorhodamine 123 (DHR) (Molecular Probes,
Eugene, OR) as previously described (Madeo et al., 1999). Briefly, aliquots
were taken at selected time points and DHR was added at 15 mg/ml of cell
culture from a 1 mg/ml stock solution in ethanol for 2 h at 30 8C. Cells were
then washed twice in PBS and viewed with an Olympus BX61 epifluorescence
microscope equipped with a rhodamine optical filter.
2.5. Cell cycle analysis
For cell cycle analysis samples were collected at defined culture times by
centrifugation and the analysis was performed as previously described
(Almeida et al., 2006). Briefly, at the desired time points, cells were harvested,
washed and fixed with ethanol (70%, v/v) for 30 min at 4 8C followed by
sonication, treatment with RNAse for 1 h at 50 8C in sodium citrate buffer
(50 mM sodium citrate, pH 7.5) and subsequent incubation with proteinase K
(0.02 mg/107 cells). DNA was then labeled with SYBR Green (Molecular
Probes) diluted in Tris-EDTA (pH 8.0) and incubated overnight at 4 8C. Before
cytometric analysis, samples were diluted 1:4 in sodium citrate buffer. The
percentage of cells in each phase of the cell cycle was determined offline with
ModFit LT software.
2.6. Flow cytometric measurements
All flow cytometric experiments were carried out using an EPICS XL-MCL
(Beckman–Coulter, Hialeah, FL) flow cytometer equipped with a 15 mWargon-
ion laser emitting at 488 nm. The green fluorescence was collected through a
488 nm blocking filter, a 550 nm long-pass dichroic with a 525 nm band-pass.
An acquisition protocol was defined to measure forward scatter (FS log), side
scatter (SS log), green fluorescence (FL1 log) and red fluorescence (FL3 log) on
a four-decade logarithmic scale. Data (minimum of 30,000 cells per sample
acquired at low flow rate) were analyzed with the Multigraph software included
in the system II software for the EPICS XL/XL-MCL version 1.0 and statistical
analysis was carried out with the windows Multiple Document Interface for
Flow Cytometry (WinMDI 2.8).
2.7. Reproducibility of the results
The results presented are mean values of at least three independent assays.
When possible, data are presented as means � standard deviation. Statistical
evaluations were carried out using independent samples t-tests. For all tests, the
level of statistical significance was set at P < 0.05.
3. Results
3.1. Limitation of essential amino acids leads to early
yeast growth arrest with unbalanced cellular
performances: biomass yield, stress response and
chronological life span
To investigate the physiological influence of essential amino
acid concentrations, two auxotrophic yeast strains, BY4742 and
CEN.PK2-1C, were grown in synthetic complete (SC) medium
supplemented with standard (1�) or five times higher (5�)
amino acid concentrations, that are already commonly used by
few laboratories (Longo et al., 1996; Madeo et al., 2002;
Fabrizio et al., 2004; Powers et al., 2006). Cells grown under
those conditions were tested for stress response and long-term
survival in stationary phase. As shown in Fig. 1A and B, both
strains when cultured in media with 5� amino acid content
reached a higher than five-fold cell density at stationary phase
when compared to cultures in 1� amino acids. To support the
hypothesis that growth limitation was occurring as a
consequence of insufficient amino acid supplementation, we
monitored glucose consumption in culture supernatants by
HPLC. The yeast cells entered stationary phase after
approximately 24 h, when glucose was still in excess in strains
grown in medium with 1� amino acids (Fig. 1C and D). In
contrast, the glucose was exhausted in cultures supplemented
with 5� amino acids and in the prototrophic strain
concomitantly with stationary phase entry (Fig. 1C and D).
These results further strengthen the idea that cells grown in 1�amino acid concentrations undergo essential amino acid
starvation. The prototrophic CEN.PK 113-7D strain, which
does not require amino acid supplementation attained similar
final cell density in SD medium with or without amino acid
supply (Fig. 1B; data for amino acid supplementation is not
shown). Moreover, the final cell density of both auxotrophic
and prototrophic yeast strains was similar in rich YEPD
medium (data not shown).
To explore whether the restriction of essential amino acids
has other physiological repercussions, we evaluated the cell
response to different stress agents. Cell survival kinetics of
BY4742 and CEN.PK strains were evaluated under oxidative
(H2O2), acid (acetic acid) or heat (48 8C) stress conditions. To
differentiate the effects of culturing cells in different amino acid
concentrations from the inherent stress resistance associated
with stationary phase (Herman, 2002), stress resistance assays
were performed at late exponential growth phase. The results
show that cells of BY4742 and CEN.PK2-1C strains cultured in
high amino acid concentrations (5�) presented a significantly
higher resistance to oxidative stress than those cultured in
standard amino acid concentrations (Fig. 2A and D). In
contrast, both strains responded to acid stress in a similar
fashion, independently of nutritional status (Fig. 2B and E).
Regarding heat stress resistance, it seems that the yeast genetic
background has strong influence on cell responses (Fig. 2C and
F). In fact, while in the BY4742 strain amino acid starvation has
no effect on heat stress resistance, in the CEN.PK genetic
background results are concordant with an increased sensitivity
of amino acid starved cells to heat stress (Fig. 2C and F).
Altogether, these observations are consistent with a protective
role of amino acids in the response to oxidative and thermal
stress, but not to acid stress.
Taking into account the important role played by both
oxidative stress and nutritional status on long-term survival, we
further focused our study on the effects of essential amino acid
starvation on chronological life span. Our results show that the
chronological life span of both auxotrophic strains grown in 5�amino acids was markedly extended relative to the 1� amino
acid concentrations (Fig. 1E and F), the effect being strongly
marked on cells from BY4742 genetic background. Moreover,
when comparing the results in the CEN.PK genetic background
one can highlight that the auxotrophic cells grown in 5� amino
Fig. 1. Standard essential amino acid concentrations reduce final biomass yield and chronological life span. Growth curves (A and B), glucose consumption (C and D)
and chronological life span (E and F) of the auxotrophic strains BY4742 and CEN.PK2-1C cultured in synthetic medium with regular (1�) or high (5�) amino acid
content. The prototrophic strain CEN.PK113-7D was grown in the absence of amino acids. All strains were grown at 30 8C in shake flasks in a defined synthetic
medium with 2% glucose as the carbon source. Cultures were inoculated from exponentially growing cultures on the same medium. The life span experiments shown
are representative and were repeated three times with similar results.
P. Gomes et al. / Mechanisms of Ageing and Development 128 (2007) 383–391386
acid content display identical life span to that of the
prototrophic strain. These results indicate that essential amino
acid starvation triggered by 1� amino acid concentrations
routinely used in supplemented media induces shortening life
span of auxotrophic yeast strains.
3.2. TOR1 disruption abrogates cell cycle alterations and
ROS accumulation observed during chronological life span
reduction induced by essential amino acid starvation
Taking into consideration the finding that essential amino
acid starvation induces growth arrest and premature cell death,
we monitored cell cycle distribution during growth and
chronological life span. This analysis was performed along
time until reaching 50% survival, which allows a confident
determination of cell cycle alterations discarding the biased
contributions of death cells, since, due to technical limitations,
it is not possible to discriminate viable and non-viable cells.
The results obtained show that while for cells cultured in 5�
amino acid content cells arrest growth in G0/G1 phases
(P < 0.05) for cells cultured in 1� amino acid content an arrest
in G2/M phases of cell cycle was observed (P < 0.05) (Fig. 3A
and C). In addition, we also observed the emergence of a
population of sub-G0/G1 cells, indicative of reduced DNA
content, usually assigned as apoptotic cells (Fig. 3A and C). In
contrast, yeast cells cultured in 5� amino acid concentrations
displayed a cell cycle profile compatible with a situation of
carbon and energy source limitation where the sub-G0/G1
population was absent (Fig. 3B and D). The cell cycle
distribution of the prototrophic CEN.PK 113-7D strain closely
resembled that of the auxotrophic CEN.PK2-1C grown on high
amino acid content (Fig. 3E).
Having in mind the higher resistance of cells cultured in 5�amino acids to H2O2 (Fig. 2A and D), and the important role of
reactive oxygen species (ROS) production in yeast chronolo-
gical life span (Fabrizio et al., 2004; Reverter-Branch et al.,
2004), we then asked whether the latter is accompanied by
higher intracellular ROS concentration. The results (Fig. 4)
Fig. 2. Protective role of essential amino acid supplementation in the cellular response to oxidative and thermal stress. Survival rate of BY4742, CEN.PK2-1C and
CEN.PK113-7D cultures treated by short exposure to (A–D) oxidative stress (0–2 mM H2O2, 200 min), (B–E) acid stress (0–200 mM acetic acid, 200 min) or (C–F)
heat shock (48 8C, 1 h). Cells were grown for 24 h in synthetic medium, with regular (1�) or high (5�) amino acid content (BY4742 and CEN.PK2-1C) or in the
absence of external amino acid supplementation (CEN.PK113-7D). Dilutions were then spotted onto YEPD agar plates to measure survival; 100% corresponds to the
number of CFUs at time 0. Data represent the mean � standard deviation of three independent experiments (* indicates statistically significant differences compared
with cultures supplemented with 1� amino acids).
Fig. 3. Amino acid starvation induces cell cycle arrest at G0/G1 phases and the appearance of a sub-G0/G1 population. Cell cycle profile of (A and B) BY4742, (C
and D) CEN.PK2-1C and (E) CEN.PK113-7D strains. All strains were grown at 30 8C in synthetic medium, with regular (1�) or high (5�) amino acid content
(BY4742 and CEN.PK2-1C) or in the absence of external amino acid supplementation (CEN.PK113-7D). Results are presented as the percentage of cells in each
phase of cell cycle.
P. Gomes et al. / Mechanisms of Ageing and Development 128 (2007) 383–391 387
Fig. 4. Essential amino acid supplementation results in lower ROS accumulation. Cells from chronologically aged cultures (1 day and 4 day of cultivation) on
synthetic medium were analyzed for accumulation of ROS visualized by DHR staining and viewed through rhodamine channel. The corresponding phase contrast
displays are shown. All strains were grown at 30 8C in shake flasks on a defined synthetic medium, with regular (1�) or high (5�) amino acid content (BY4742 and
CEN.PK2-1C) or in the absence of external amino acid supplementation (CEN.PK113-7D). Bars, 10 mm.
P. Gomes et al. / Mechanisms of Ageing and Development 128 (2007) 383–391388
indicate that ROS accumulation in auxotrophic strains is
markedly reduced in cells grown in 5� amino acid content. The
low levels of ROS in the prototrophic strain are also correlated
with its ability to endure oxidative damage (Fig. 2D). These
results point to an increased intracellular level of ROS induced
by essential amino acid starvation that culminates in the
shortening of yeast cell longevity.
Considering that TOR pathway is one of the major signaling
cascades involved in amino acid sensing in yeast (Wilson and
Roach, 2002) and that deletion of TOR1 (tor1D) induces
chronological life span extension (Powers et al., 2006), we
questioned the contribution of this pathway to the phenotype
observed. Therefore, the life span of yeast cells lacking TOR1
gene cultured with either 1� or 5� amino acid concentrations,
was evaluated in the BY4742 genetic background. In agreement
with previous reports, tor1D cells displayed a strong life span
extension, and were insensitive to further amino acid
supplementation (Fig. 5A and B).
Ras-cAMP pathway is an essential nutrient sensing system in
yeast, which is activated preferentially by glucose (Mbonyi et al.,
1988). The relative order of the TOR and Ras pathways has not
been definitely established. Previous studies have suggested that
the Ras proteins are functioning either independently, or
downstream of the TOR signaling pathway as another effector
branch to control an array of cellular processes (Budovskaya
et al., 2004; Schmelzle et al., 2004). To investigate how
abrogation of Ras pathway affects amino acid sensing, we
performed life span assays in yeast cells lacking RAS2 (ras2D)
gene in the BY4742 genetic background. In accordance with
previous report (Longo, 2003), ras2D induces chronological life
span extension similar to that observed for the wild type cells
supplemented with 5� amino acids (Fig. 5B). Interestingly, we
found that addition of amino acids under RAS2 deletion
conditions caused an additional increase in life span that was
comparable to that obtained for tor1D cells (Fig. 5B).
4. Discussion
Yeasts are known as prototrophic microorganisms; however,
a large number of auxotrophic yeast strains have been
developed and extensively used for molecular and cellular
studies. In these auxotrophic strains, amino acids are essential
nutrients for cell growth and most of the studies use standard
concentrations of these nutrients that are largely diffused in the
literature. However, higher amino acid concentrations (5�)
have already been used by few laboratories (Longo et al., 1996;
Madeo et al., 2002; Fabrizio et al., 2004; Powers et al., 2006).
Our results show that limitation of essential amino acids leads
to early yeast growth arrest with unbalanced cellular
performances that negatively affect biomass yield, stress
response and chronological life span. As expected, cells grown
in 1� essential amino acid concentrations display reduced
Fig. 5. Essential amino acid supplementation does not affect chronological life
span of yeast TOR1 mutant but has a positive effect on RAS2 deletion mutant.
Chronological survival in synthetic medium with regular (1�) or high (5�)
essential amino acid supplementation of cells lacking either (A) TOR1 or (B)
RAS2 compared to wild type cells with the same genetic background (BY4742).
P. Gomes et al. / Mechanisms of Ageing and Development 128 (2007) 383–391 389
growth and final biomass yield due to amino acid, rather than
glucose, limitation. Essential amino acid starvation has
physiological repercussions in the cell response to different
stress conditions, the results being consistent with a protective
role of amino acids in the response to oxidative and thermal
stress. If oxidative stress and the ability to respond
appropriately to it is important in aging (Balaban et al.,
2005; Sohal and Weindruch, 1996), then it follows that factors
that increase resistance to stress should have anti-aging benefits
and lead to enhanced life span. In line with this, increased
amino acid supplementation resulted in higher resistance to
H2O2 and lower ROS production/accumulation over time,
while causing life span extension.
Analysis of cell cycle profile revealed that cells cultured in
1� amino acid content, display an arrest/delay in G2/M phases
of cell cycle together with the appearance of a sub-G0/G1
population in contrast with the expected arrest in G0/G1 phases
regularly observed under carbon starvation. Moreover, either
prototrophic or auxotrophic (grown in 5� amino acids) aged
cells although dying by an apoptotic programme, do not present
the sub-G0/G1 population and G2/M arrest. These results point
to the triggering of a specific cell death programme under
starvation of auxotrophic-complementing amino acids that
promotes earlier appearance of the apoptotic phenotypes
commonly observed during chronological life span (Herker
et al., 2004; Fabrizio et al., 2004; Allen et al., 2006). These
earlier cell death phenotypes seems to be caused by the specific
absence of essential amino acids, within glucose containing
media, and not due to a general caloric restriction since
complete starvation (water) causes a chronological life span
extension (Longo et al., 1997; Fabrizio et al., 2005).
As it was referred in Section 1, the environmental factors that
extend yeast longevity remain largely unknown. The finding that
essential amino acid starvation reduces yeast chronological life
span further expands our knowledge of the environmental
determinants of aging. Nutrients are known to play a key role in
life span regulation, but the link between essential amino acid
sufficiency and chronological aging has not been addressed in
yeast. Previous studies have shown that nutrient starvation has a
positive effect in life span extension in yeast (Jiang et al., 2000;
Kaeberlein et al., 2005; Lin et al., 2000). In one study, Jiang et al.
(2000) showed that amino acid starvation induces life span
extension in yeast. In this report, we show that low concentra-
tions of auxotrophy-complementing amino acids cause a
decrease in yeast longevity. This discrepancy could be, at least
partly, explained by the fact that the authors have starved cells for
non-essential amino acids, while keeping all other nutrients
constant, and monitored replicative life span. To our knowledge,
there is only one previous work that reported replicative life span
extension resulting from higher nutrient availability (an increase
in glucose concentration from 2 to 20%), this effect being
mediated by the high osmolarity glycerol (HOG) response
pathway (Kaeberlein et al., 2002). One of the major functions of
the TOR signaling network seems to be the sensing of the levels
and/or quality of amino acids or other available nitrogen sources,
to control cell growth and life span (Martin and Hall, 2005). With
this notion in mind, we examined the effect of TOR1 deletion on
life span under 1� or 5� amino acid levels. The absence of a
synergistic effect on life span when 5� amino acid concentra-
tions is combined with TOR1 deletion is consistent with the
crucial known regulatory role of TOR pathway on amino
acid sensing.
As mentioned earlier, the relative order of TOR and Ras-
cAMP pathways signal has not been definitely established. Our
results show that blocking Ras signaling still allow TOR itself
or another TOR effector arm to respond to extracellular
nutrients. This conclusion is mainly supported by the
observation that amino acid supply had a positive effect on
chronological life span of RAS2 deleted cells. Altogether, these
results support the contribution of the TOR pathway in
mediating life span reduction upon supplementation with 1�essential amino acid concentrations. However, one should keep
in mind that disruption of TOR1, a signaling hub, might have
pleiotropic effects, and does not necessarily implicate TOR
itself in the observed effects. Therefore, the possibility that
other signaling cascades also participate in the response to
amino acids cannot be ruled out.
It is noteworthy that the results presented in this study have
immediate implications for studies using auxotrophic yeast
strains. As we demonstrated, an imbalance in the medium
essential amino acid concentrations might have dramatic
P. Gomes et al. / Mechanisms of Ageing and Development 128 (2007) 383–391390
consequences on several major cellular functions, including
cell growth, life span and stress resistance, and probably others
not addressed here. Caution should be exercised in the
interpretation and comparison of quantitative data from
auxotrophic yeast strains grown under different nutritional
conditions. To have a comprehensive evaluation of the role of
essential amino acids on cellular performance, it will be of
interest to further determine whether supplementation of single
amino acids, or small combinations of amino acids affect life
span and stress resistance.
In conclusion, we show here that in two different yeast strain
backgrounds, starvation for auxotrophic-complementing amino
acids leads to reduction of chronological life span. This study
may provide novel insights into the role of essential nutrients
and their effect on aging process and raises the warning that the
positive effects of caloric restriction on life span maybe
restricted to non-essential nutrients. Moreover, the severe
consequences on cell physiology induced by essential amino
acid starvation presents an alert for studies using auxotrophic
yeast strains.
Acknowledgements
We thank members of the Ludovico and Rodrigues labs for
assistance with figures, helpful discussions and comments.
Pedro Gomes and Belem Sampaio-Marques were financially
supported by fellowships from Fundacao para a Ciencia e
Tecnologia, Portugal (SFRH/BPD/13969/2003 and SFRH/BI/
15406/2005). This work was supported by a grant awarded by
Fundacao para a Ciencia e Tecnologia (POCI/BIA-BCM/
57364/2004).
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