Aging Cell (2003) 2, pp73–81
Blackwell Publishing, Ltd
REVIEW
The chronological life span of Saccharomyces cerevisiae
The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo
Paola Fabrizio and Valter D. Longo
Andrus Gerontology Center, Division of Biogerontology and
Department of Biological Sciences, University of Southern
California, Los Angeles, USA
Summary
Simple model systems have played an important role in
the discovery of fundamental mechanisms of aging. Stud-
ies in yeast, worms and fruit flies have resulted in the
identification of proteins and signalling pathways that
regulate stress resistance and longevity. New findings
indicate that these pathways may have evolved to
prevent damage and postpone aging during periods of
starvation and may be conserved from yeast to mammals.
We will review the yeast S. cerevisiae model system with
emphasis on the chronological life span as a model system
to study aging and the regulation of stress resistance in
eukaryotes.
Key words: aging; bcl-2; insulin-like signalling; ras; sir;
SOD; stress resistance; yeast.
Replicative vs. chronological life span
The classic approach to the study of aging in yeast is based on
the measurement of replicative life span. Mother cells of the
yeast S. cerevisiae reproduce asymmetrically by originating buds
(daughter cells). Daughter cells are smaller than mothers and
can be easily recognized and removed by micromanipulation
after budding occurs. Replicative (budding) life span is defined
as the total number of daughter cells generated by a mother
cell (Mortimer, 1959). Replicative senescence can be caused by
the accumulation of extrachromosomal ribosomal DNA circles
(ERCs) in old mother cells (Sinclair et al., 1997). ERCs are self-
replicating units produced in the nucleolus by rDNA homolo-
gous recombination, which segregate asymmetrically to the
mother cell during cell division. The silent information regulator
proteins Sir2, Sir3, Sir4, which control chromatin silencing at
different DNA sites, can regulate ERCs production and aging
(Kaeberlein et al., 1999). In particular, the NAD-dependent
histone deacetylase Sir2 inhibits rDNA recombination and ERCs
accumulation (Kaeberlein et al., 1999). Increasing the dosage
Correspondence
Valter D. Longo, Division of Biogerontology Andrus Gerontology Center,
University of Southern California, 3715 McClintock Avenue, Los Angeles, CA
90089-0191, USA. E-mail: vlongo@usc.edu
Accepted for publication 30 September 2002
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
of Sir2 extends the replicative life span whereas the deletion of
the SIR2 gene decreases replicative longevity (Kaeberlein et al.,
1999). Notably, overexpression of the homologue of SIR2 extends
longevity in C. elegans, suggesting that this conserved gene
may affect both replicative senescence and chronological aging
in other eukaryotes (Tissenbaum & Guarente, 2001).
Other genes that regulate replicative aging have been iden-
tified. In particular, RAS1 and RAS2 show opposite roles in
replicative aging. The deletion of RAS1 extends, whereas the
deletion of RAS2 shortens replicative longevity (Sun et al., 1994).
As described later in this review, Ras may play opposite roles in
the regulation of replicative and chronological longevity since
the deletion of RAS2 extends the chronological life span. The
LAG1 and LAG2 genes are also implicated in the control of rep-
licative life span. The deletion of each of these genes separately
decreases life span by 40% and 50%, respectively. Conversely,
their overexpression increases the number of buds generated
by yeast mother cells (D’Mello N et al., 1994). The mechanisms
behind Lag1/ Lag2 life span extension are still unknown. How-
ever, Lag1 has been recently implicated in ceramide synthesis
and a possible role of this function in regulation of aging has
been proposed (Guillas et al., 2001). Replicative aging has been
linked to the retrograde response, which is activated by an intra-
cellular signalling pathway from the mitochondrion to the
nucleus and leads to the transcription of several genes encoding
for metabolic enzymes. The retrograde response is usually
induced in yeast strains that have lost part of the mitochondrial
DNA and consequently do not have fully functional mitochon-
dria (petite strains). These strains show a significant life span
extension, which depends upon the activation of the retrograde
response. In fact, the deletion of the RTG2 gene, which is a
downstream mediator of the retrograde response, abolishes the
life span extension effect (Kirchman et al., 1999).
Calorie restriction (CR), an intervention that lengthens life
span in several organisms including mammals, also extends
yeast replicative life span (Lin et al., 2000). This effect is medi-
ated by Sir2 and requires increased respiration rates. In fact,
calorie restriction failed to extend the life span of a petite strain
lacking cytochrome c1 (Lin et al., 2002). Intriguingly, the elim-
ination of the electron transport chain appears to have opposite
effects on replicative longevity since in some strains it also
promotes replicative life span extension (Kirchman et al., 1999).
We have developed a different system to measure yeast
longevity based on the chronological longevity of a population of
non-dividing yeast (chronological life span) (Longo et al., 1996;
Fabrizio et al., 2001; Longo & Fabrizio, 2002). The chronological
life span is monitored either during a hypometabolic phase
(stationary phase) or during a high-metabolic state (post-diauxuc
phase). Studies of the high-metabolism life span have been
73
74 The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo
crucial in establishing a link between stress resistance and
longevity and have provided evidence for the conservation of por-
tions of the pathways that regulate life span in phylogenetically
distant eukaryotes (Fabrizio et al., 2001).
Although the relationship between the mechanisms that
regulate the replicative and chronological life span are poorly
understood, several studies suggest that these two survival
paradigms are related: (1) passages through stationary phase
decrease the replicative life span of yeast (Ashrafi et al., 1999).
The longer the yeast are incubated in stationary phase, the
shorter their replication potential once they are switched to
fresh medium. (2) Mutations that decrease the activity of the
PKA pathway increase both types of life span ( Longo, 1997; Lin
et al., 2000; Fabrizio et al., 2001). (3) The deletion of SOD1,
which encodes for cytoplasmic superoxide dismutase, or of
both SOD1 and SOD2 (mitochondrial superoxide dismutase)
dramatically reduces yeast chronological life span and decreases
replicative life span (Laun et al., 2001; Longo et al., 1996).
Extensive studies on the role of the PKA pathway in both
aging paradigms showed that decreasing its activity extends
both types of life span. However, whereas the effect of the
cAMP/ PKA pathway on chronological life span requires Msn2/
Msn4 and Sod2 and is associated with increased stress resist-
ance, its effect on replicative life span appears to be independent
from stress resistance and dependent on the expression of the
Sir2 deacetylase (Lin et al., 2000). Therefore, the chronological and
replicative life spans appear to be regulated by overlapping but
distinct mechanisms.
Chronological life span: survival in the post-
diauxic and stationary phases
Yeast and other micro-organisms have evolved to survive under
adverse conditions commonly encountered in the wild, such as
starvation. In fact, most micro-organisms are estimated to survive
in a low-metabolism stationary phase under nutrient-depleted
conditions (Werner-Washburne et al., 1996). In the wild, yeast
organisms are likely to exit stationary phase only during the rare
periods when all the nutrients required for growth become avail-
able. For this reason we perform most of our experiments in either
a medium containing a limited amount of nutrients [synthetic
dextrose complete (SDC)] or in water. Wild type yeast incubated
in SDC medium survive for 6 –7 days at high metabolic rates and
then begin to die rapidly. When yeast grown in SDC are switched
to water between days 1 and 5, metabolic rates decrease and
survival is extended (see below). However, since long-lived
mutants isolated by incubation in SDC also live longer when
incubated in water, we believe that analogous pathways and
mechanisms regulate survival in both paradigms. Yeast grown
and incubated in the nutrient-rich YPD medium also survive for
months in a low-metabolism stationary phase. However, it is not
clear whether YPD medium allows some growth to occur during
the supposedly ‘stationary’ phase (see survival in water/ YPD).
To understand how yeast age and identify conserved path-
ways that regulate longevity in many eukaryotes, it is important
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
to reproduce conditions similar to those under which these
pathways have evolved. Although the chronological life span
paradigm may appear to be a starvation phase that does not
resemble the life span of higher eukaryotes, non-dividing yeast
are not starving but are slowly utilizing the nutrients stored
intracellularly at the end of the growth phase (see below). An
environment that lacks nutrients may not be common for certain
mammals but it is very common for micro-organisms. In some
circumstances even mammals have learned how to respond to
long periods of starvation. For example, black bears and Turkish
hamsters alternate between a high- and a low-metabolism
hibernation phase, in which stored nutrients are utilized to
survive. The longer the period Turkish hamsters spend under
hibernation the longer the life span (Lyman et al., 1981). Similarly,
yeast forced to enter the low-metabolism stationary phase by
incubation in water survive longer than yeast grown and incu-
bated in SDC medium, which maintain high metabolic rates.
In addition to the high-metabolism post-diauxic life span
(SDC), and the low-metabolism stationary phase, under partic-
ularly severe starvation conditions, diploid S. cerevisiae can form
haploid spores that may survive for years in a dormant state.
Yeast spores may be the equivalent of the worm dauer larva,
which also live much longer than adult worms (Riddle, 1988;
Guarente, 2001). The food supply determines whether worms
grow and become metabolically active adults or exit develop-
ment at the L2 larva stage to enter the low-respiration dauer
larva stage. Although most yeast diploid organisms enter and
remain in stationary phase and only a minority of diploid organ-
isms form spores (Codon et al., 1995), all of our life span studies
are performed using haploid strains which behave similarly to
diploid cells under most conditions but do not sporulate.
Survival in SDC: post-diauxic phase
Most of our chronological longevity studies are performed by
monitoring survival in the high-metabolism post-diauxic phase
(SDC medium) (Fig. 1A). The SDC studies are started by diluting
6 −1
overnight cultures to an initial density of 1–2 10 cells mL
(OD600 of 0.1– 0.2) in 10 –50 mL of synthetic complete medium
containing 2% glucose (SDC) as well as a four-fold excess of
the supplements Trp, Leu, Ura and His. The SDC medium con-
tains glucose, yeast nitrogen base, agar, ammonium sulphate
(nitrogen source), sodium phosphate, vitamins, metals and salts.
Yeast cultures are incubated at 30 °C in flasks with a volume/
medium ratio of 5 : 1, shaking at 220 r.p.m. After approxi-
mately 10 h of exponential growth, the glucose concentration
in the medium reaches very low levels and yeast switch from a
fermentation- to a respiration-based metabolism. After this
switch, called ‘diauxic shift’, yeast catabolize the ethanol accu-
mulated during the fermentative phase and obtain most of the
energy from mitochondrial oxidative phosphorylation. When
yeast organisms are incubated in SDC, the diauxic shift is
followed by a post-diauxic phase, in which growth continues
slowly until approximately 48 h, and then stops. In the post-
diauxic phase metabolic rates remain high until day 5 – 6 (day 0

Fig. 1 (A) Chronological life span of wild type
DBY746 incubated in either minimal synthetic
medium (SDC) or water. (B) Metabolic rates
determined by oxygen consumption
measurements of strain DBY746.
= dilution day) ( Fig. 1B). The final density reached at day 3 varies
from strain to strain and is usually between 7 and 15 (OD 600).
The mean survival of wild type strains depends on their genetic
background and ranges from 6–7 days (DBY746/SP1) to 15–
20 days (S288C/BY4700). A diauxic-shift-like switch from
fermentation to respiration may also occur after reducing the
glucose concentration in the medium from 2 to 0.5%. This form
of calorie restriction increases respiration rates and causes an
extension of the yeast replicative life span (Lin et al., 2002).
In a standard post-diauxic experiment we monitor survival by
measuring the ability of an individual yeast cell/organism to
form a colony (colony forming units or CFUs) within 3 days of
plating onto YPD plates. CFUs are normally monitored until at
least 99.9% of the population dies. The number of CFUs at day
3 is considered to be the initial survival (100% survival) and is
used to determine the age-dependent mortality. Day 3 was
selected considering that in our wild type strains DBY746 and
SP1 the population density does not normally increase after day
3, suggesting that the great majority of the cells have stopped
dividing. Other methods have been used in our laboratory to
confirm that the loss of CFUs correlates with death (see Viability
loss section).
The ‘post-diauxic phase’ differs for organisms incubated in
SDC and those incubated in rich YPD medium (Werner-Wash-
burne et al., 1996). In fact, incubation in YPD promotes a 6 –
7 day post-diauxic phase characterized by slow growth and low
respiration, followed by entry into a non-dividing hypometa-
bolic stationary phase in which organisms are highly resistant
to multiple stresses and survive for up to 3 months. By contrast,
incubation in SDC triggers the entry into an alternative post-
diauxic phase in which only minimal growth occurs after 48 h
and metabolic rates remain high until the population begins
to die.
Survival in water/YPD: stationary phase
Yeast incubated in YPD or water survive much longer than yeast
grown and maintained in SDC medium (Fig. 1A). In fact, the
mean life span of strains DBY746 and SP1 in water is approx-
imately three times longer than in SDC (15–20 days). Our exper-
iments are usually repeated in water to simulate an alternative
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo 75
environment that may be commonly encountered by yeast in
the wild. Instead, we do not monitor survival in YPD since this
rich medium may promote growth after the culture reaches the
maximum density. After more than 99% of wild type DBY746
and SP1 yeast incubated in SDC die, in about 30% of the studies
a better-adapted subpopulation is able to grow back by utilizing
the nutrients released by dead cells (unpublished results). A
similar phenomenon called ‘gasping’ is observed for populations
of bacteria (Zambrano & Kolter, 1996). By contrast, incubation
of yeast in YPD appears to promote major increases in viability
before the majority of the population has died, suggesting
that some growth may occur when viability is high. Growth
would create a mixed population containing both young and
old organisms, which would invalidate the survival studies.
For life span studies in water, yeast are grown and incubated
for 3 days in SDC, and are then washed with sterile distilled
water and re-suspended in sterile water. Viability is monitored
by measuring CFUs every 2 days. The cells are washed three times
with water every 2 days to remove all the nutrients released by
dead yeast. Incubation in water and the removal of nutrients
released by dead organisms minimizes the chance of growth
during long-term survival in stationary phase. Notably, the life
span in the post-diauxic and stationary phases (water) appear
to be regulated by similar mechanisms since the ras2∆ and sch9∆
mutants survive longer than wild type yeast in both SDC and water.
Incubation in water decreases metabolic rates and may increase
life span simply by promoting a slower rate of senescence.
Oxidative damage and longevity in yeast
Micro-organisms have played a key role in the characterization
of anti-oxidant enzymes and identification of the sources and
targets of reactive oxygen species. Like mammalian cells, S. cer-
evisiae expresses a cytosolic CuZn superoxide dismutase (Sod1)
and catalase (Ctt1), as well as a mitochondrial MnSOD (Sod2).
By monitoring the chronological life span of yeast mutants lack-
ing specific genes it was possible to understand how different
anti-oxidant enzymes affect cell damage and aging. Cytoplas-
mic and mitochondrial superoxide dismutases, but not catalase
and metallothionein, are required for long-term survival (Longo
et al., 1996) ( Fig. 2). Sod2 is required under both low and normal
76 The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo
Fig. 2 Chronological survival of wild type DBY746 (j) and mutants lacking
catalase T (h), CuZnSOD (m), MnSOD (d) or both CuZnSOD and MnSOD
(r) in normal aeration.
oxygen conditions, whereas cytoplasmic Sod1 is mainly required
under normal aeration (Longo et al., 1996). The expression of
human SOD1 in yeast sod1 null mutants completely reverses
the survival defects, suggesting that the function of this enzyme
is conserved from yeast to mammals. In fact, these results are
consistent with studies performed using mice lacking sod1.
Although sod1 knockout mice showed few abnormalities,
cultured fibroblasts obtained from these mice were 80 times more
sensitive to the superoxide generator paraquat than wild type
cells and grew poorly in air (Huang et al., 1997), indicating that,
as reported for yeast, Sod1 is only required under a high
concentration of oxygen or superoxide.
Unlike most experimental organisms, yeast have the ability
to grow either by respiration utilizing non-fermentable carbon
sources (ethanol, lactate), or by fermentation utilizing glucose
(low respiration). We have exploited this feature to determine
whether mitochondrial damage precedes death. We have called
the percentage of live yeast able to utilize non-fermentable
carbon sources and respiration to grow the Index of Respiratory
Competence (IRC). Using the IRC, it was possible to define the
sequence of events that lead to the death of mutants lacking
mitochondrial SOD (sod2) and to explore the role of mitochon-
drial damage in the death of wild type yeast in stationary phase.
In both sod2 mutants and wild type yeast, death was preceded
by a decrease in IRC suggesting that loss of mitochondrial
function preceded death (data not shown). The characterization
of yeast sod2 null mutants also resulted in the identification of
mitochondrial aconitase and succinate dehydrogenase, both
4Fe−4S cluster binding proteins, as the primary targets of
mitochondrial superoxide (Longo et al., 1999). These results are
consistent with studies in sod2 knockout mice in which the
activity of mitochondrial aconitase was reduced by more than
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
67% in the heart and brain, and succinate dehydrogenase was
reduced by more than 65% in the heart and skeletal muscle
(Melov et al., 1998). As expected, in sod2 mice the cells most
severely affected by the increased concentration of mitochondrial
superoxide were post-mitotic. The similarities between non-dividing
yeast and mice lacking either SOD1 or SOD2 suggest that the
chronological life span of yeast is a valuable model system for
mechanisms of oxidative damage in mammalian cells, particularly
post-mitotic cells.
The overexpression of CuZnSOD has been convincingly
shown to increase the life span of fruit flies. (Orr & Sohal, 1994;
Parkes et al., 1998; Sun & Tower, 1999.) To test whether the
overexpression of anti-oxidant enzymes could also extend the
life span of yeast, strains expressing several fold higher concen-
trations of these antioxidant enzymes were generated. The
overexpression of SOD1 and SOD2 together increased chrono-
logical survival by 30% whereas the overexpression of each SOD
alone had a significant but more modest effect (Fabrizio et al.,
2003). These results are in agreement with the age-dependent
reversible inactivation of the mitochondrial enzyme aconitase,
which is caused by superoxide (Fabrizio et al., 2001).
Yeast has served as a powerful model system in understanding
the function and mechanism of action of mammalian proteins.
The anti-apoptotic human Bcl-2 protein was overexpressed in
yeast sod mutants and wild type yeast to investigate its mech-
anism of action and to understand whether yeast may have
components of a programmed cell death pathway. Human
Bcl-2 partially reversed several defects of yeast lacking superoxide
dismutases, consistent with the demonstrated anti-oxidant func-
tion of Bcl-2 in mammalian cells. Bcl-2 overexpression increased
long-term viability and growth in 100% oxygen of sod1 and
sod1sod2 mutants (Longo et al., 1997). Bcl-2 also increased
survival in wild type cells, raising the possibility that portions of
an apoptotic pathway are present in yeast. This hypothesis was
recently supported by yeast studies in which the expression of
the human pro-apoptotic protein Bax causes cell death, which can
be reversed by coexpression of Bcl-2 (Matsuyama et al., 1998;
Shaham et al., 1998). Furthermore, a caspase-related protease
and several features of apoptosis were recently identified in
yeast (Madeo et al., 1999; Madeo et al., 2002).
Viability loss and starvation in the post-diauxic
and stationary phases
Yeast viability in most of our experiments is measured by count-
ing the number of colonies generated after plating an aliquot
of the culture onto YPD plates. In theory, each viable cell should
divide and form a colony (CFU). We tested whether the loss of
CFU correlates with the loss of viability in the post-reproductive
phase. We measured the concentration of proteins released into
the medium by dead and damaged wild type DBY746-plasmid
controls and by SOD1SOD2 double overexpressors. The increase
in protein concentration in the medium of both strains began
48 h after the loss of CFUs and continued for 7 days. In agreement
with the CFU measurements, the increase in protein concentration

in the medium of SOD1SOD2 overexpressors was delayed by
2 days compared with controls (Fabrizio et al., 2003). The release
of protein was lower in SOD1SOD2 overexpressors than in wild
type controls throughout the study. To characterize further the
chronological life span, we also performed confocal microscopy
and two-photon confocal microscopy in aging organisms.
Three- to 7-day-old DBY746 yeast were stained with FUN-1,
which stains intravacuolar structures only in metabolically active
organisms with an intact plasma membrane. Yeast were also
stained with Calcofluor, a marker of fungal cell wall. FUN-1 staining of
DBY746 and sch9∆ mutants suggests that the loss of membrane
integrity and metabolic activity correlates with the decrease in
CFUs (Fabrizio et al., 2003). At day 7 the majority of sch9∆
mutants were marked with fluorescent intravacuolar structures
whereas the majority of wild type organisms produced a diffuse
green cytoplasmic fluorescence (unpublished results). Taken
together, these results suggest that during the chronological life
span, the ability to reproduce and form a colony correlates with
death. However, further studies are necessary, considering that
it may be difficult to distinguish between a damaged organism
surviving at very low metabolic rates and a dead organism.
Since most of the extracellular nutrients are internalized
during the post-diauxic and stationary phases, it is often believed
that stationary phase yeast starve to death. However, the pub-
lished data and our own results suggest that yeast organisms
are not dying by starvation. During the long-term survival in
stationary phase, yeast produce energy mainly by catabolizing
the glycogen accumulated in the late exponential growth. Using
strain S288C, Lillie & Pringle (1980) showed that the reserve
carbohydrates glycogen and trehalose are available after an
incubation of 90 days in rich YPD medium (stationary phase).
Our studies of the high-metabolism post-diauxic phase also
suggest that death is not caused by a depletion of reserve
nutrients. Glycogen levels adjusted for population density are
not significantly different between day 3 and day 7 (SDC medium),
when only 30% of the organisms are viable (unpublished results).
Thus, it would be surprising if yeast were maintaining high
concentrations of the major reserve carbohydrate but were
dying of starvation. The mean life span of strains DBY746 and
SP1 is more than 15 days in water but less than 7 days in SDC
medium. An extended survival in the complete absence of
nutrients (water) is also not consistent with death by starvation.
Furthermore, the switch from expired medium to water during
the high death phase halts cell death, suggesting that dying
organisms have not depleted reserve nutrients (unpublished results).
Finally, overexpression of both SOD1 and SOD2 extends survival
by 30% but does not affect metabolic rates. Thus, it is difficult
to reconcile this role of increased protection against oxidative
stress in extending survival with death by starvation, as increased
investment in maintenance would require additional energy.
Although these results suggest that starvation does not play a
major role in the chronological life span of yeast, further studies
are required to rule out this possibility. We are also currently
testing the effect of the lack of amino acid biosynthetic enzymes
and of the depletion of amino acids on the survival of various
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo 77
strains. Our preliminary results suggest that chronological life
span (SDC) is not increased by adding to the expired medium
the amino acids that strains SP1 and DBY746 are unable to
biosynthesize (data not shown).
The identification of pathways that regulate
stress resistance and longevity
The deletion of RAS2 increases stress resistance and doubles
the chronological life span of yeast (Longo, 1997; Fabrizio et al.,
2003). The identification of Ras mutations that increase Sod
activity and longevity resulted in the description of the first
eukaryotic longevity pathway (Longo, 1997). These results and
the concurrent identification of genes that regulate longevity in
worms and flies led to the hypothesis that in organisms ranging
from yeast to humans, longevity is regulated by a similar signal
transduction pathway that modulates Sod activity through the
activation of stress resistance transcription factors (Longo, 1997,
1999). The role of ras2 mutations in extending longevity by
activating stress resistance transcription factors Msn2/Msn4 and
SODs (Longo, 1997) was recently conclusively demonstrated by
showing that Msn2/Msn4 and Sod2 are required for longevity
extension in ras2 mutants (Fabrizio et al., 2003). As described
in the next section, the yeast Ras2 and Sch9 pathways share
remarkable similarities with the worm insulin/IGF-I-like pathway
and with the mammalian insulin/IGF-I pathway. Notably, Ras
functions in the mammalian insulin/IGF-I signalling pathway but
has not been implicated in the worm longevity pathway.
Our strategy to isolate long-lived mutants combined the
selection of stress-resistant mutants with transposon mutagenesis
(Ross-Macdonald et al., 1999). Because of the link between stress
resistance and survival in higher eukaryotes, after generating a
population of transposon-mutagenized yeast, we performed a
preselection for mutants that were (a) thermotolerant (52 °C,
1 h) or (b) resistant to oxidative stress (1 mM paraquat, 9 –10 days).
Four thousand thermotolerant and 40 oxidative stress resistant
mutants were isolated and their mean post-diauxic survival was
measured and compared with that of the wild type strain.
Mutants with life span significantly longer than wild type were
identified and their mutated alleles were cloned as described
(Ross-Macdonald et al., 1999). From the mutants isolated, the
ones that carried transposon insertions in the CYR1 gene and
in the promoter region of the SCH9 gene were the two longest
lived (Fabrizio et al., 2001). These were also the only two
mutants isolated independently in the thermotolerance and
oxidative stress selection. These two genes encode for adenylate
cyclase and for the Sch9 serine/threonine protein-kinase,
respectively.
Cyr1 and Sch9 function in parallel signalling pathways (Ras /
adenylate cyclase and Sch9 pathways) are both involved in medi-
ating the glucose/nutrients-dependent response including the
stimulation of growth and glycolysis and the down-regulation
of stress resistance, glycogen accumulation and gluconeogen-
esis. Since the transposon insertion in both CYR1 and SCH9
genes appears to reduce the activity of the corresponding
78 The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo
proteins, we conclude that the down-regulation of the glucose/
nutrients signalling pathways extends yeast chronological life
span (Fabrizio et al., 2001).
The deletion of RAS2, which encodes for an upstream regulator
of Cyr1, doubles the yeast life span (Longo, 1997; Fabrizio et al.,
2003). To verify that the effect of Sch9 and Ras2 on the life
span regulation is not dependent on the yeast background we
deleted SCH9 and RAS2 in another strain (SP1) and measured the
chronological life span of the corresponding strains. The life span
extension of the deletion mutants in the SP1 background was
very similar to their counterparts in the DBY746 background
(Fig. 3).
Stress resistance, superoxide dismutases and
longevity
Several studies support a major role for multiple stress resistance
in the regulation of longevity in yeast: (a) to identify long-lived
mutants we preselected for heat /oxidative stress-resistant yeast
and isolated mutants that are highly resistant to both heat and
oxidative damage and live up to three times longer than wild
type; (b) the deletion of RAS2 increases longevity as well as
thermotolerance and resistance to oxidative stress; (c) the life
span extension of the ras2 and cyr1 mutants was shown to be
mediated by stress-induced transcription factors Msn2/Msn4
(Fabrizio et al., 2001); (d) the deletion of the SOD2 gene, encoding
for the mitochondrial superoxide dismutase dramatically reduced
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
Fig. 3 Chronological survival of long lived ras2∆
and sch9∆ mutants originated in the (A) SP1 and
the (B) DBY746 background.
the life span extension of the ras2, cyr1 and sch9 mutants
(Fabrizio et al., 2003); and (e) the up-regulation of the heat shock
response produced by decreasing the activity of the Hsp90
chaperone extends the life span (Harris et al., 2001).
Superoxide resistance seems to be particularly important in
extending the chronological life span. In fact, mutants lacking
either SOD1 (cytoplasmic superoxide dismutase) or SOD2 or both
are short-lived compared to wild type (Fig. 2). Furthermore, our
studies on the activity of the mitochondrial enzyme aconitase
in post-diauxic yeast strongly suggest a role for superoxide
toxicity in aging (Longo et al., 1999). Aconitase is a citric acid cycle
enzyme, which contains a 4Fe−4S cluster that is particularly
sensitive to superoxide. An age-dependent reversible inactivation
of this enzyme caused by superoxide has been shown during
survival (Longo et al., 1999). This inactivation contributes to the
mitochondrial damage that precedes cellular death. Interestingly,
both cyr1 and sch9 mutants showed a delay in the aconitase
inactivation, confirming the role played by superoxide in aging
(Fabrizio et al., 2001) (Fig. 4).
The age-dependent inactivation of mitochondrial aconitase
and the role of superoxide dismutases in extending the chrono-
logical life span in yeast indicate that superoxide and other
toxic oxygen species play a major role in aging. In 1956 Harman
proposed that oxygen species with one unpaired electron (free
radicals) may cause aging. The free radical theory of aging became
one of the most widely accepted theories after the overexpres-
sion of anti-oxidant enzymes was shown to extend longevity
 The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo 79

in signal transduction genes can extend longevity up to

three-fold (Clancy et al., 2001; Fabrizio et al., 2001; Tatar et al.,
2001). Therefore, increasing anti-oxidant protection appears to
be important but not sufficient for the major longevity extension
caused by mutations in glucose or insulin/IGF-I signalling
pathways.

From yeast to mammals

Fig. 4 Long lived mutants cyr1 and sch9 show a delay in the inactivation of
the superoxide-sensitive enzyme aconitase. Mitochondrial aconitase percent
reactivation after treatment of the yeast cell extracts from cultures at day 5
through 7 with agents (iron and Na2S) able to reactivate superoxide
inactivated [4Fe−4S] clusters.
and after most long-lived model organisms were shown to be
resistant to oxidative stress (Longo, 1999; Finkel & Holbrook,
2000). Although superoxide toxicity contributes to aging and
death in model organisms, mutations in the yeast Sch9/Cyr1
and in the worm and fly insulin/IGF-I-like pathways appear to
extend longevity by regulating the expression of many genes.
In fact, the overexpression of Sod1, Sod2 and catalase in yeast
and flies can extend longevity by up to 30%, whereas mutation
Thanks to studies by several laboratories, a significant proportion
of the genes that function in longevity pathways in yeast and
worms have been characterized. In yeast, the down-regulation
of glucose signalling by ras2, cyr1 and sch9 mutations increase
longevity and stress resistance (Fig. 5). In the cyr1 mutants
chronological life span extension is mediated by stress resistance
transcription factors Msn2 and Msn4, which induce the expres-
sion of genes encoding for several heat shock proteins, catalase
(C T T1), the DNA damage inducible gene DDR2, and SOD2 (Fig. 5)
(Thevelein & de Winde, 1999; Fabrizio et al., 2001). Analogously,
in worms, the inactivation of the insulin / IGF-I-like/daf-2 pathway
extends survival (Johnson, 1990; Kenyon et al., 1993) and increases
thermotolerance and anti-oxidant defences (Larsen, 1993;
Lithgow et al., 1995; Kimura et al., 1997), by activating stress
resistance transcription factor DAF-16 (Fig. 5) (Lin et al., 1997).
In yeast, chronological life span extension is associated with
decreased superoxide generation and aconitase inactivation in
the mitochondria (Fabrizio et al., 2001). Furthermore, life span
extension in ras2, cyr1 and sch9 mutants requires SOD2 and
the overexpression of superoxide dismutases extends longevity

Fig. 5 Regulation of stress resistance and longevity

extension in yeast and worms. In yeast, glucose and
other nutrients activate the Ras2/Cyr1/cAMP/PKA
and the Sch9 pathways. PKA down-regulates
transcription factors Msn2/Msn4, which induces
the expression of heat shock proteins, catalase,
MnSOD and other maintenance proteins.
Activation of Sch9 results in a major decrease in
stress resistance via unidentified mediators.
Mutations in RAS2, SCH9 and CYR1 increase
multiple stress resistance systems, decrease
mitochondrial superoxide levels, delay aconitase
inactivation and extend longevity. Analogously, in
worms, insulin/IGF-I-like signalling activates the
daf-2/age-1/AKT-1/AKT-2 pathway, which down-
regulate transcription factor DAF-16. The latter
activates several stress resistance proteins including
MnSOD and catalase. Mutations in daf-2 and age-
1 increase thermotolerance and oxidative stress
resistance and extend survival.

© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003

80 The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo
(Fabrizio et al., 2003). In worms, among the genes regulated by
the daf-2 pathway are mitochondrial MnSOD and several heat
shock proteins (Boy-Marcotte et al., 1998; Cherkasova et al., 2000).
The yeast Ras/Cyr1/PKA pathway down-regulates glycogen
storage and genes involved in the switch to the hypometabolic
stationary phase and to the dormant spore state (Werner-
Washburne et al., 1996; Boy-Marcotte et al., 1998). The worm daf-
2 pathway also down-regulates the storage of reserve nutrients
(fat and glycogen) and the switch to the hypometabolic dauer
larvae state (Fig. 5) (Kenyon et al., 1993; Morris et al., 1996;
Kimura et al., 1997). Thus, in addition to the high sequence
similarities between the yeast SCH9 and the worm AKT-1/AKT-2
genes, these distantly related organisms appear to regulate stress
resistance and longevity by modulating the activity of similar
proteins and pathways ( Fig. 5). Remarkably, life span regulation
of more complex organisms such as Drosophila and mice appears
to depend on the activity of insulin/IGF-I-stimulated pathways
similar to those identified in yeast and worms. These pathways
include serine/threonine kinases (Akt /PKB) and regulate the activity
of several stress-resistant proteins including SODs, catalase and
Hsps (Longo & Fabrizio, 2002).
The analogous role of glucose or hormone/growth factor
signalling in stress resistance and aging in the major genetics model
systems suggests that longevity can be extended by similar
mechanisms in many organisms. Life span extension appears to
be caused by a shift from a reproductive phase to a non-reproductive
maintenance phase, accompanied by changes in the expression of
many genes normally induced by starvation. The yeast chronological
and replicative life span paradigms have provided evidence for
the conservation of longevity regulation in eukaryotic organisms.
These systems also play an important role in the dissection of the
fundamental molecular mechanisms leading to both chronol-
ogical and replicative aging in eukaryotes.
References
Ashrafi K, Sinclair D, Gordon JI, Guarente L (1999) Passage through sta-
tionary phase advances replicative aging in Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA 96, 9100 –9105.
Boy-Marcotte E, Perrot M, Bussereau F, Boucherie H, Jacquet M (1998)
Msn2p and Msn4p control a large number of genes induced at the
diauxic transition which are repressed by cyclic AMP in Saccharomyces
cerevisiae. J. Bacteriol. 180, 1044 –1052.
Cherkasova V, Ayyadevara S, Egilmez N, Reis RS (2000) Diverse
Caenorhabditis elegans genes that are upregulated in dauer larvae
also show elevated transcript levels in long-lived, aged, or starved adults.
J. Mol. Biol. 300, 433 – 448.
Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H, Hafen E, Leevers SJ,
Partridge L (2001) Extension of life-span by loss of CHICO, a Drosophila
insulin receptor substrate protein. Science 292, 104 –106.
Codon AC, Gasent-Ramirez JM, Benitez T (1995) Factors which affect
the frequency of sporulation and tetrad formation in Saccharomyces
cerevisiae baker’s yeasts [published erratum appears in Appl. Environ.
Microbiol 1995 April; 61 (4): 1677]. Appl. Environ. Microbiol. 61,
630–638.
D’Mello NP, Childress AM, Franklin DS, Kale SP, Pinswasdi C, Jazwinski SM
(1994) Cloning and characterization of LAG1, a longevity-assurance
gene in yeast. J. Biol. Chem. 269, 15451–15459.
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
Fabrizio P, Lee-Loung Liou, Moy V, Selverstone J, Butler Gralla VE, Longo
VD (2003) SOD2 functions downstream of sch9 to extend longevity
in yeast. Genetics in press.
Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD (2001) Regu-
lation of longevity and stress resistance by Sch9 in yeast. Science 292,
288 –290.
Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology
of ageing. Nature 408, 239–247.
Guarente L (2001) SIR2 and aging – the exception that proves the rule.
Trends Genet. 17, 391–392.
Guillas I, Kirchman PA, Chuard R, Pfefferli M, Jiang JC, Jazwinski SM,
Conzelmann A (2001) C26-CoA-dependent ceramide synthesis of
Saccharomyces cerevisiae is operated by Lag1p and Lac1p. EMBO J.
20, 2655 –2665.
Harris N, MacLean M, Hatzianthis K, Panaretou B, Piper PW (2001)
Increasing Saccharomyces cerevisiae stress resistance, through the
overactivation of the heat shock response resulting from defects in
the Hsp90 chaperone, does not extend replicative life span but can
be associated with slower chronological ageing of nondividing cells.
Mol. Genet. Genomics 265, 258 –263.
Huang T T, Yasunami M, Carlson EJ, Gillespie AM, Reaume AG, Hoffman EK,
Chan PH, Scott RW, Epstein CJ (1997) Superoxide-mediated cytotoxicity
in superoxide dismutase-deficient fetal fibroblasts. Arch. Biochem.
Biophys. 344, 424 – 432.
Johnson TE (1990) Increased life-span of age-1 mutants in Caenorhabditis
elegans and lower Gompertz rate of aging. Science 249, 908 –912.
Kaeberlein M, McVey M, Guarente L (1999) The SIR2/3/4 complex and
SIR2 alone promote longevity in Saccharomyces cerevisiae by two
different mechanisms. Genes Dev. 13, 2570 –2580.
Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R (1993) A C. elegans
mutant that lives twice as long as wild type. Nature 366, 461– 464.
Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G (1997) daf-2, an insulin
receptor-like gene that regulates longevity and diapause in Caenorhab-
ditis elegans. Science 277, 942–946.
Kirchman PA, Kim S, Lai CY, Jazwinski SM (1999) Interorganelle signaling
is a determinant of longevity in Saccharomyces cerevisiae. Genetics
152, 179 –190.
Larsen PL (1993) Aging and resistance to oxidative damage in
Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 90, 8905– 8909.
Laun P, Pichova A, Madeo F, Fuchs J, Ellinger A, Kohlwein S, Dawes I,
Frohlich KU, Breitenbach M (2001) Aged mother cells of Saccharo-
myces cerevisiae show markers of oxidative stress and apoptosis. Mol.
Microbiol. 39, 1166 –1173.
Lillie SH, Pringle JR (1980) Reserve carbohydrate metabolism in Saccha-
romyces cerevisiae: response to nutrient limitation. J. Bacteriol. 143,
1384–1394.
Lin SJ, Defossez PA, Guarente L (2000) Requirement of NAD and SIR2
for life-span extension by calorie restriction in Saccharomyces cerevisiae.
Science 289, 2126 –2128.
Lin K, Dorman JB, Rodan A, Kenyon C (1997) daf-16: an HNF-3/forkhead
family member that can function to double the life-span of
Caenorhabditis elegans. Science 278, 1319 –1322.
Lin SJ, Kaeberlein M, Andalis AA, Sturtz LA, Defossez PA, Culotta VC,
Fink GR, Guarente L (2002) Calorie restriction extends Saccharomyces
cerevisiae lifespan by increasing respiration. Nature 418, 344–348.
Lithgow GJ, White TM, Melov S, Johnson TE (1995) Thermotolerance
and extended life-span conferred by single-gene mutations and
induced by thermal stress. Proc. Natl. Acad. Sci. USA 92, 7540 –7544.
Longo VD (1997) The Pro-Senescence Role of Ras2 in the Chronological
Life Span of Yeast. Thesis, University of California Los Angeles.
Longo VD (1999) Mutations in signal transduction proteins increase
stress resistance and longevity in yeast, nematodes, fruit flies, and
mammalian neuronal cells. Neurobiol. Aging 20, 479– 486.

Longo VD, Ellerby LM, Bredesen DE, Valentine JS, Gralla EB (1997)
Human Bcl-2 reverses survival defects in yeast lacking superoxide
dismutase and delays death of wild-type yeast. J. Cell Biol. 137, 1581–
1588.
Longo VD, Fabrizio P (2002) Regulation of longevity and stress resistance:
a molecular strategy conserved from yeast to humans? Cell. Mol. Life
Sci. 59, 903–908.
Longo VD, Gralla EB, Valentine JS (1996) Superoxide dismutase activity
is essential for stationary phase survival in Saccharomyces cerevisiae.
Mitochondrial production of toxic oxygen species in vivo. J. Biol.
Chem. 271, 12275 –12280.
Longo VD, Liou LL, Valentine JS, Gralla EB (1999) Mitochondrial super-
oxide decreases yeast survival in stationary phase. Arch. Biochem.
Biophys. 365, 131–142.
Lyman CP, O’Brien RC, Greene GC, Papagrangos ED (1981) Hyberna-
tional longevity in the Turkish hamster Mesocricetus brandti. Science
212, 668–670.
Madeo F, Frohlich E, Ligr M, Grey M, Sigrist SJ, Wolf DH, Frohlich KU
(1999) Oxygen stress: a regulator of apoptosis in yeast. J. Cell Biol.
145, 757–767.
Madeo F, Herker E, Maldener C, Wissing S, Lachelt S, Herlan M, Fehr M,
Lauber K, Sigrist SJ, Wesselborg S, Frohlich KU (2002) A caspase-related
protease regulates apoptosis in yeast. Mol Cell. 9, 911–917.
Matsuyama S, Xu Q, Velours J, Reed JC (1998) The Mitochondrial F0F1-
ATPase proton pump is required for function of the proapoptotic
protein Bax in yeast and mammalian cells. Mol. Cell. 1, 327–336.
Melov S, Schneider JA, Day BJ, Hinerfeld D, Coskun P, Mirra SS, Crapo
JD, Wallace DC (1998) A novel neurological phenotype in mice lacking
mitochondrial manganese superoxide dismutase [see comments].
Nature Genet. 18, 159 –163.
Morris JZ, Tissenbaum HA, Ruvkun G (1996) A phospatidylinositol-3-OH
kinase family member regulating longevity and diapause in Caenorh-
bditis elegans. Nature 382, 536 –539.
Mortimer RK (1959) Life span of individual yeast cells. Nature 183,
1751–1752.
© Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2003
The chronological life span of S. cerevisiae, P. Fabrizio and V. D. Longo 81
Orr WC, Sohal RS (1994) Extension of life-span by overexpression of
superoxide dismutase and catalase in Drosophila melanogaster.
Science 263, 1128 –1130.
Parkes TL, Elia AJ, Dickinson D, Hilliker AJ, Phillips JP, Boulianne GL (1998)
Extension of Drosophila lifespan by overexpression of human SOD1
in motorneurons. Nature Genet. 19, 171–174.
Riddle DL (1988) The Nematode C. Elegans. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press.
Ross-Macdonald P, Coelho PS, Roemer T, Agarwal S, Kumar A, Jansen R,
Cheung KH, Sheehan A, Symoniatis D, Umansky L, Heidtman M,
Nelson FK, Iwasaki H, Hager K, Gerstein M, Miller P, Roeder GS, Snyder M
(1999) Large-scale analysis of the yeast genome by transposon tagging
and gene disruption. Nature 402, 413– 418.
Shaham S, Shuman MA, Herskowitz I (1998) Death-defying yeast
identify novel apoptosis genes. Cell 92, 425– 427.
Sinclair DA, Mills K, Guarente L (1997) Accelerated aging and nucleolar
fragmentation in yeast sgs1 mutants. Science 277, 1313–1316.
Sun J, Kale SP, Childress AM, Pinswasdi C, Jazwinski SM (1994) Divergent
roles of RAS1 and RAS2 in yeast longevity. J. Biol. Chem. 269, 18638 –
18645.
Sun J, Tower J (1999) FLP recombinase-mediated induction of Cu/ Zn-
superoxide dismutase transgene expression can extend the life span
of adult Drosophila melanogaster flies. Mol. Cell. Biol. 19, 216 –228.
Tatar M, Kopelman A, Epstein D, Tu MP, Yin CM, Garofalo RS (2001) A
mutant Drosophila insulin receptor homolog that extends life-span
and impairs neuroendocrine function. Science 292, 107–110.
Thevelein JM, de Winde JH (1999) Novel sensing mechanisms and targets
for the cAMP-protein kinase A pathway in the yeast Saccharomyces
cerevisiae. Mol. Microbiol. 33, 904–918.
Tissenbaum HA, Guarente L (2001) Increased dosage of a sir-2 gene
extends lifespan in Caenorhabditis elegans. Nature 410, 227–230.
Werner-Washburne M, Braun EL, Crawford ME, Peck VM (1996) Stationary
phase in Saccharomyces cerevisiae. Mol. Microbiol. 19, 1159 –1166.
Zambrano MM, Kolter R (1996) GASPing for life in stationary phase.
Cell 86, 181–184.