Increased Life Span due to Calorie
Restriction in Respiratory-Deficient Yeast
Matt Kaeberlein1*, Di Hu2, Emily O. Kerr2, Mitsuhiro Tsuchiya2, Eric A. Westman2, Nick Dang2, Stanley Fields1,3,
Brian K. Kennedy2*
1 Departments of Genome Sciences and Medicine, University of Washington, Seattle, Washington, United States of America, 2 Department of Biochemistry, University of
Washington, Seattle, Washington, United States of America, 3 Howard Hughes Medical Institute, University of Washington, Seattle, Washington, United States of America
A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced
glucose in the growth medium leads to activation of the NADþ–dependent histone deacetylase Sir2. One mechanism
proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels
of NADþor NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in
which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for
nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the long-
lived BY4742 strain background. Here we have determined the requirement for respiration and the effect of
nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratory-
deficient cells independent of strain background, and moreover, suppresses the premature mortality associated with
loss of mitochondrial DNA in the short-lived PSY316 strain. Addition of nicotinamide to the medium dramatically
shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the
replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1)
demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide
inhibits life span extension by CR through a Sir2-independent mechanism, and (3) suggest that CR acts through a
conserved, Sir2-independent mechanism in both PSY316 and BY4742.
Citation: Kaeberlein M, Hu D, Kerr EO, Tsuchiya M, Westman EA, et al. (2005) Increased life span due to calorie restriction in respiratory-deficient yeast. PLoS Genet 1(5): e69.
Calorie restriction (CR) has been shown to slow aging in
evolutionarily divergent species, including yeast, worms, flies,
and rodents [1–5]. In addition to increasing longevity, CR is
reported to cause additional phenotypes, including increased
resistance to oxidative stress [6–8], enhanced DNA damage
repair [9,10], decreased levels of oxidatively damaged proteins
[11–13], improved glucose homeostasis and insulin sensitivity
[14–16], altered levels of apoptosis , and delayed onset of a
number of age-related diseases [18–21]. Although it has been
known for more than 70 y that calorie restriction can increase
life span in mammals , a mechanistic understanding of
this phenomenon has remained elusive. It seems clear that
nutrient and growth factor responsive pathways, such as those
mediated by insulin, IGF-1, TOR, and Akt, are likely to
represent important conduits through which these signals
affect the aging rate. CR mediates enhancement of stress
response pathways in mammals [23,24], and signaling through
the insulin-like pathway in worms coordinates expression of a
variety of antioxidant, chaperone, and anti-bacterial stress
response proteins [25–27]. Similarly, TOR-mediated regula-
tion of translational machinery appears to play a role in the
response to nutrient deprivation in yeast , worms [29,30],
flies , and mammals . Finally, models postulating a role
for Sir2-like protein deacetylases in CR-mediated life span
extension have gained popularity for yeast , flies , and
mammals, as well [4,35].
In the budding yeast Saccharomyces cerevisiae, CR can be
imposed by reducing the concentration of glucose in the
growth medium, resulting in a 20%–40% increase in
replicative life span in multiple strain backgrounds
[33,36,37]. In addition, genetic models of CR include deletion
of the gene coding for hexokinase, HXK2, and mutations that
decrease signaling through the cAMP-dependent protein
kinase, PKA, such as deletion of the genes coding for the
glucose sensing proteins GPA2 or GPR1, and temperature-
sensitive alleles of adenylate cyclase (cdc35–1) or the RAS-
associated GTPase (cdc25–10) .
CR has been proposed to increase yeast replicative life span
by a mechanism involving activation of Sir2 , an NADþ–
dependent histone deacetylase [38–40] that inhibits the
formation of extrachromosomal rDNA circles (ERCs) .
ERCs are self-replicating DNA molecules that accumulate in
the mother-cell nucleus with age and are thought to cause
senescence . Overexpression of Sir2 increases life span in
multiple strain backgrounds [36,41,43], and deletion of Sir2
shortens life span by about 50% [41,44]. CR fails to increase
the life span of short-lived sir2D cells , consistent with the
idea that CR could be acting by a mechanism involving Sir2.
Received July 7, 2005; Accepted October 24, 2005; Published November 25, 2005
Copyright: ? 2005 Kaeberlein 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.
Abbreviations: CR, calorie restriction; ERC, extrachromosomal rDNA circles; ORF,
open reading frame; SC, synthetic complete
Editor: Stuart Kim, Stanford University School of Medicine, United States of America
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
edu (MK), email@example.com (BKK)
A previous version of this article appeared as an Early Online Release on October
25, 2005 (DOI: 10.1371/journal.pgen.0010069.eor).
PLoS Genetics | www.plosgenetics.orgNovember 2005 | Volume 1 | Issue 5 | e690614
The question of how CR might activate Sir2 has been a
source of considerable controversy . Saccharomyces cerevisiae
is a facultative anaerobe that, under standard laboratory
growth conditions (2% glucose), generates ATP largely by
fermentation. Under conditions of reduced glucose, such as
CR, S. cerevisiae shifts from fermentation to respiration,
resulting in increased transcription of respiratory genes and
a higher rate of oxygen consumption . In models put forth
by Lin et al., this metabolic shift results in activation of Sir2,
either through increased cellular NADþ or decreased
cellular NADH . Alternatively, Anderson et al. have
reported that CR does not alter NADþlevels , but leads
to enhanced expression of PNC1 and a reduction in cellular
nicotinamide . Since nicotinamide is an inhibitor of the
Sir2 deacetylation reaction, its decreased concentration
could result in enhanced Sir2 activity [50,51]. Overexpression
of Pnc1 suppresses the effect of exogenously added nicoti-
namide on Sir2-dependent silencing at HM loci, telomeres,
and rDNA ; there are conflicting reports, however, on
whether Pnc1 overexpression alters Sir2 activity at endoge-
nous levels of nicotinamide [49,52].
More recently, we have questioned the importance of Sir2
in life span extension by CR [28,53]. In a long-lived strain
background, BY4742, CR increases life span to a greater
extent in cells lacking both Sir2 and the replication fork
barrier protein Fob1 than in wild-type cells . Based on this
observation, and the fact that deletion of SIR2 shortens life
span by approximately 50%, we proposed a model whereby
the inability of CR to increase life span in sir2D FOB1 cells is
explained as an indirect effect, resulting from the hyper-
accumulation of ERCs . Deletion of FOB1 in a sir2D
background suppresses the hyperaccumulation of ERCs, as
well as the longevity defect , thus allowing CR to
dramatically increase the life span of cells in the absence of
Much of the early work suggesting a link between CR and
Sir2 was carried out in the PSY316 strain background
[33,37,46,47,49,50,54], a strain in which, paradoxically, over-
expression of Sir2 fails to increase life span . Guarente
and Picard  have proposed that CR might act via different
mechanisms in the BY4742 and PSY316 strain backgrounds.
In order to further clarify the molecular mechanism(s)
underlying replicative life span extension by calorie restric-
tion in yeast, we have sought to directly test key components
of the models for Sir2-dependent CR in both of these strains.
Here, we examine in detail the role of respiratory metabolism
in life span extension by CR, finding that (1) respiration is not
required for life span extension by CR; and (2) CR suppresses
the enhanced early mortality, only apparent in PSY316, due
to loss of mitochondrial DNA. In contrast, exogenous
addition of nicotinamide partially, but not completely, blocks
Sir2-independent life span extension by CR.
Life Span Extension by CR Is Independent of Respiration in
A central facet of the Sir2-dependent models for life span
extension by CR is that a metabolic shift from fermentation
to respiration in response to CR results in activation of Sir2
[46,47]. Since Sir2 is not required for life span extension by
CR in BY4742 , we wished to determine whether
respiration was also dispensable. The effect of CR in the
absence of respiratory capacity was examined using rho0cells,
which completely lack mitochondrial DNA. Rho0yeast lack
three cytochrome c oxidase subunits (COX1, COX2, and
COX3), three ATP synthase subunits (ATP6, ATP8, and
ATP9), and apocytochrome b (CYTB), and are therefore
incapable of respiratory metabolism [56,57]. BY4742 rho0
cells were generated (see Materials and Methods), and the
absence of mitochondrial DNA was verified by staining cells
with DAPI (Figure 1A). Lack of respiratory capacity in rho0
cells was confirmed by inability to grow on the non-
fermentable carbon source, glycerol (Figure 1B). As previ-
ously observed , replicative life span under standard
conditions (2% glucose) was not altered by loss of mitochon-
drial DNA in this strain (Figure 1C). CR (0.05% glucose)
significantly enhanced the life span of both wild-type and
rho0cells to a comparable degree. Thus, respiration is not
required for life span extension by CR in the long-lived
BY4742 strain background.
Life Span Extension by CR Is Independent of Respiration in
Previous work indicating that life span extension by CR is
dependent on respiration was carried out in the PSY316
strain background, in which it was reported that deletion of
the gene coding for cytochrome c1, CYT1, prevents life span
extension by CR . In order to address whether life span
extension by CR in mutants incapable of respiratory
metabolism is specific to BY4742, we generated respiratory-
deficient cyt1D and rho0variants in the PSY316 background
(Figure 2A and 2B) and measured life span on medium
containing either 2% or 0.05% glucose. As in BY4742 rho0
cells, CR significantly increased both mean and maximum life
span in PSY316 rho0(Figure 2C) and cyt1D cells (Figure 2D).
Unlike the case in BY4742 in which deletion of mitochon-
drial DNA has no effect on life span, PSY316 rho0variants
exhibited a profound increase in early mortality under
standard growth conditions. This phenotype was not
observed in cyt1D cells, indicating that loss of mitochondrial
DNA, rather than general respiratory deficiency, is respon-
sible for the life span defect. Deletion of CYT1 in cells lacking
mitochondrial DNA, however, resulted in a short life span
comparable to that of rho0cells (Figure 2E), demonstrating
that rho0is epistatic to cyt1D for this phenotype. As observed
for rho0cells, CR more than doubled the short life span of
PLoS Genetics | www.plosgenetics.org November 2005 | Volume 1 | Issue 5 | e690615
Calorie Restriction in Yeast
Calorie restriction slows aging and increases life span in nearly every
organism studied. The mechanism by which this occurs is one of the
most important unanswered questions in biogerontology. One
popular theory, based on work from the budding yeast Saccha-
romyces cerevisiae, proposes that calorie restriction works by causing
a metabolic shift toward increased mitochondrial respiration,
resulting in activation of a family of proteins known as Sirtuins.
This study demonstrates that life span extension by calorie
restriction does not require respiration and occurs even in cells
completely lacking mitochondrial DNA. Interestingly, calorie restric-
tion protects yeast cells against a severe longevity defect associated
with absence of mitochondrial DNA, suggesting the possibility that
the consequences of age-associated mitochondrial dysfunction
might be alleviated or prevented by calorie restriction.
cyt1D rho0cells, which contain both nuclear and mitochon-
drial mutations that prevent respiration.
Our observation that CR increased life span in PSY316 cells
lacking CYT1 is in contrast to a previous report . A
potential explanation for this difference is that 0.5% glucose
was used for CR in the prior study, rather than the 0.05%
glucose concentration used in this study. We therefore
measured the life span of respiratory deficient rho0and
cyt1D cells grown on 0.5% glucose. As we observed for cells
grown on 0.05% glucose, growth on 0.5% glucose increased
the life span of rho0and cyt1D cells, although the magnitude
of life span extension is reduced relative to 0.05% glucose
(Figure 3A). Thus, the use of a non-optimal level of CR may
have precluded detection of life span extension by CR in
cyt1D mutants in the prior study.
We also examined the effect of CR on Sir2 activity in
respiratory-deficient PSY316 cells. The PSY316AUT variant
has both URA3 and ADE2 marker genes integrated near
telomeres, thus allowing for efficient determination of Sir2-
dependent telomeric silencing in response to genetic or
environmental perturbations . As previously reported,
increased Sir2 activity can be achieved by overexpression of
SIR2 in the PSY316 strain background , significantly
enhancing telomere silencing and survival on FOA, while
inhibition of Sir2 by addition of 5 mM nicotinamide to the
medium decreased telomere silencing (Figure 3B). CR,
however, had no detectable effect on Sir2-dependent silenc-
ing at telomeres. Similarly, respiratory deficiency caused by
deletion of CYT1 also fails to impact Sir2-dependent silencing
at either 2% or 0.05% glucose (Figure 3C). A decrease in
survival on FOA was observed in rho0cells relative to wild-
type or cyt1D cells at 2% glucose (Figure S1); however, CR
failed to result in a detectable increase in Sir2 activity in
respiratory deficient or respiratory competent cells. There-
fore, we find no evidence that a metabolic shift from
fermentation toward respiration is involved in life span
extension by CR or that Sir2 is activated in response to CR.
Nicotinamide Blocks Life Span Extension by CR
CR has also been proposed to activate Sir2 by reducing
cellular pools of nicotinamide, a Sir2 inhibitor . Addition
of 5 mM nicotinamide to the medium prevents life span
extension by CR in wild-type mother cells ; however,
interpretation of this experiment is complicated by the fact
that, like deletion of SIR2, high levels of nicotinamide result
in a dramatically shortened life span. We took advantage of
the fact that deletion of FOB1 suppresses the short life span
and ERC hyperaccumulation phenotypes associated with
deletion or inhibition of Sir2  to ask whether nicotina-
mide could inhibit the longevity effect of calorie restriction,
even in the absence of Sir2. As expected, growth on 5 mM
nicotinamide reduced the life span of wild-type cells to a level
not significantly different from that of sir2D cells (Figure 4A).
The very short life span of sir2D cells or nicotinamide-treated
wild-type cells is most likely due to the hyperaccumulation of
ERCs in cells lacking Sir2 activity [36,41]. Also as expected,
nicotinamide had no effect on the life span of sir2D fob1D
double mutants (Figure 4B), because Sir2 is already absent
from these cells. CR dramatically increased the life span of
sir2D fob1D double mutants, but, unexpectedly, addition of
nicotinamide decreased the magnitude of life span extension
Figure 1. Respiration Is Not Required for Life Span Extension by CR in
(A) BY4742 rho0strains lack mitochondrial DNA. DAPI staining of BY4742
(i) wild-type or (ii) rho0cells grown under standard conditions (2%
glucose) and calorie-restricted (iii) wild-type or (iv) rho0cells (CR¼0.05%
(B) BY4742 rho0strains are unable to grow on glycerol as the sole carbon
source. (i) BY4742 wild-type or (ii) rho0cells on YEP supplemented with
2% glucose or 3% glycerol.
(C) CR increase life span in BY4742 rho0cells. Replicative life span analysis
for BY4742 wild-type and rho0cells on 2% glucose and 0.05% glucose
(CR). Mean life span is shown in parentheses.
PLoS Genetics | www.plosgenetics.org November 2005 | Volume 1 | Issue 5 | e690616
Calorie Restriction in Yeast
conferred by CR (Figure 4B). Thus, Sir2-independent life
span extension by CR is partially prevented by nicotinamide.
Role of Respiration and Nicotinamide in Life Span
Extension by CR
Sir2 is dispensable for life span extension by CR in yeast
. It remains possible, however, that under some con-
ditions, CR might be mediated by Sir2. Central to this
possibility is the premise that CR results in activation of Sir2.
One mechanism by which CR has been hypothesized to
activate Sir2 involves altered levels of the nicotinamide
adenine dinucleotide cofactors NADþand NADH, resulting
from a metabolic shift toward increased respiration in
response to CR [46,47]. The other mechanism by which CR
has been suggested to activate Sir2 is through a reduction in
nicotinamide levels .
An important test of the respiration-dependent model for
CR is whether CR can increase life span in cells that are
incapable of respiration. Contradictory to the prediction
from this model, we find that respiration is dispensable for
enhanced longevity in response to CR. Growth on reduced
glucose resulted in increased life span in two distinct models
of respiratory deficiency, cyt1D and rho0(see Figures 1C and
2C–2E). These data, combined with the observation that
inhibition of Sir2 cannot account for the effect of nicotina-
mide on life span extension by CR (Figure 4B), call into
question the proposed molecular explanations for activation
of Sir2 in response to CR. Further, we find no evidence that
Figure 2. Respiration Is Not Required for Life Span Extension by CR in PSY316
(A) PSY316AUT rho0strains lack mitochondrial DNA. DAPI staining of PSY316 (i) wild-type or (ii) rho0cells grown under standard conditions (2% glucose)
and calorie-restricted (iii) wild-type or (iv) rho0cells (CR ¼ 0.05% glucose).
(B) PSY316AUT rho0strains are unable to grow on glycerol as the sole carbon source. (i) PSY316AUT wild-type, (ii) cyt1D rho0, (iii) cyt1D, or (iv) rho0cells
on YEP supplemented with 2% glucose or 3% glycerol.
(C) CR increases life span in PSY316AUT rho0cells. Replicative life span analysis for PSY316AUT wild-type and rho0cells on 2% glucose and 0.05%
glucose (CR). Mean life span is shown in parentheses.
(D) CR increases the life span of cyt1D cells. Replicative life span analysis for PSY316AUT wild-type and cyt1D cells on 2% glucose and 0.05% glucose
(CR). Mean life span is shown in parentheses.
(E) CR increases the life span of cyt1D rho0cells. Replicative life span analysis for PSY316AUT wild-type and cyt1D rho0cells on 2% glucose and cyt1D
rho0cells on 0.05% glucose (CR). Mean life span is shown in parentheses.
PLoS Genetics | www.plosgenetics.org November 2005 | Volume 1 | Issue 5 | e690617
Calorie Restriction in Yeast
Sir2 activity is altered either by CR or by respiratory
deficiency, as measured by Sir2-dependent transcriptional
silencing near telomeres (see Figure 3B and 3C). This result
does not rule out the possibility that CR specifically enhances
Sir2 activity at the rDNA; however, like the case at telomeres,
Sir2-dependent silencing of a modified URA3 marker gene
inserted into the rDNA is not enhanced by CR (J. Smith,
personal communication). Thus, we propose that life span
extension by CR occurs through a conserved Sir2-independ-
ent, respiration-independent mechanism (Figure 5).
It should be noted that our results do not contradict
previous findings that increased respiration correlates with
replicative life span in PSY316. Overexpression of the HAP4
transcription factor, which results in transcriptional up-
regulation of respiratory genes and increased oxygen
consumption, or overexpression of a mitochondrial NADH
oxidoreductase, are reported to increase life span in PSY316
[46,47]. It remains possible that these interventions do indeed
activate Sir2 by altering levels of NADþor NADH, as
proposed. Alternatively, these interventions may behave as
genetic mimics of CR, increasing life span through a Sir2-
Our data suggest that high levels of nicotinamide can alter
the response of yeast cells to CR. How might nicotinamide
interfere with life span extension by CR? We can imagine at
least three possible models. First, nicotinamide could partially
block CR by inhibiting the activity of the other yeast Sirtuins
(Hst1–4). This model seems unlikely because CR increases the
life span of yeast cells lacking both Sir2 and either Hst1, Hst2,
Hst3, or Hst4, and CR increases the life span of a sir2D fob1D
hst1D hst2D quadruple mutant by greater than 50% (unpub-
lished data). Second, nicotinamide could specifically interfere
with the longevity benefits of CR, but through a mechanism
unrelated to Sirtuin action. Nicotinamide, conventionally
classified as a vitamin, participates in many biological
processes distinct from Sirtuins , and could conceivably
alter the activity of any NADþ–binding protein in the cell.
Third, a reduction in nicotinamide levels conferred by CR
might be important to offset detrimental effects, resulting
from growth on reduced glucose medium, that are themselves
unrelated to replicative aging, but may shorten life span to an
extent that it masks life span extension by CR. Further study
will be required to distinguish between these models.
Mitochondrial Defects, CR, and Longevity
Defects in mitochondrial function cause several human
diseases, and mutation of mitochondrial DNA has been
suggested to result in age-associated phenotypes in mammals
[60–62]. Yeast provides a unique model in which to study the
phenotypic consequences of mutation to the mitochondrial
genome. With respect to replicative life span, complete
deletion of the mitochondrial genome (rho0) results in
different phenotypic outcomes depending on the genetic
background of the strain [58,63]. Indeed, we report here that
rho0cells of BY4742 have a life span comparable to that of
wild-type cells, whereas, rho0cells of PSY316 are extremely
short-lived (compare Figure 1C with Figure 2C). Presumably,
this difference is the result of polymorphisms present in the
nuclear genomes of these strains. Interestingly, in the PSY316
strain background, a nuclear mutation (cyt1D) that prevents
respiration results in a life span comparable to that of wild-
type cells (see Figure 2D). Thus, the short life span of PSY316
Figure 3. Effect of Glucose Concentration on Life Span and Sir2 Activity
in Respiratory-Deficient Mutants
(A) Mean replicative life span is significantly increased in PSY316 AUT
cyt1D and rho0cells as the glucose concentration is decreased to either
0.5% or 0.05%, relative to life span on 2% glucose. *p , 0.05, **p , 0.01.
(B) Sir2 activity is not increased by CR but is responsive to increased
expression of Sir2 or to addition of exogenous nicotinamide. Transcrip-
tional silencing of the telomeric URA3 marker in PSY316AUT (WT) was
monitored by the survival of cells plated onto medium containing 5-FOA.
(C) Sir2 activity is not altered in respiratory deficient cyt1D cells and is
unaffected by CR. Transcriptional silencing of the telomeric URA3 marker
in PSY316AUT (WT) was monitored by the survival of cells plated onto
medium containing 5-FOA.
PLoS Genetics | www.plosgenetics.org November 2005 | Volume 1 | Issue 5 | e690618
Calorie Restriction in Yeast
rho0cells is apparently caused by loss of mitochondrial DNA
rather than a general consequence of respiratory deficiency.
Although the PSY316 rho0variant is extremely short-lived,
CR by growth on low glucose is capable of increasing the life
span of these cells by more than 100% (see Figure 3A). In fact,
CR increases life span of the rho0strain to a level that is
comparable to calorie-restricted wild-type cells. To the best
of our knowledge, this is the first indication, in any organism,
that CR has a beneficial effect on defects caused by deletion
of mitochondrial DNA. It will be of interest to understand the
molecular basis for this effect and to determine whether this
is a general feature of CR in multicellular eukaryotes.
Three competing models of life span extension by CR in
yeast have been put forward: (1) Sir2 activation through a
metabolic shift to respiration [46,47], (2) Sir2 activation by
decreased nicotinamide levels , and (3) Sir2-independent
life span extension [28,36]. Although CR can increase life
span by a Sir2-independent mechanism , it remains to
be determined whether either of the Sir2-dependent models
account for a portion of the longevity benefits of CR under
any conditions. We show here that in two different strain
backgrounds, one of which is the PSY316 strain background
used to generate the data supporting the Sir2-dependent
models, life span extension by CR does not require
respiration. We also show that the partial inhibition of CR
by addition of exogenous nicotinamide does not act
through Sir2. Thus, activation of Sir2 through a metabolic
shift to respiration or through depletion of intracellular
nicotinamide cannot explain CR-mediated increases in
Materials and Methods
Strains and media. Unless otherwise stated, all yeast strains were
derived from the parent strain for the haploid yeast open reading
frame (ORF) deletion collection , BY4742 (obtained from
Research Genetics, Invitrogen, Carlsbad, California, United States)
or from PSY316AUT . Strains used in this study are listed in Table
1. Gene disruptions were carried out by transforming yeast with PCR-
amplified deletion constructs containing 45 nucleotides of homology
to regions flanking the ORF to be deleted and either HIS3, LEU2, or
Figure 4. Effect of Nicotinamide on Life Span Extension by CR
(A) Nicotinamide shortens the life span of wild-type cells. Replicative life
span analysis for BY4742 wild-type and sir2D cells on 2% glucose
containing or lacking 5 mM nicotinamide (nic). Mean life span is shown in
(B) Nicotinamide partially prevents Sir2-independent life span extension
by CR. Replicative life span analysis for BY4742 wild type on 2% glucose,
along with sir2D fob1D double mutant cells on 2% glucose or 0.05%
glucose (CR) containing or lacking 5 mM nicotinamide (nic). Mean life
span is shown in parentheses.
Figure 5. Genetic Pathways Determining Replicative Life Span in Yeast
Sir2 and CR act in parallel pathways to slow aging. Both pathways are
affected by nicotinamide levels.
Table 1. Yeast Strains Used in This Study
MATa his3D1 leu2D0 lys2D0 ura3D0
BY4742 sir2D::HIS3 fobD::LEU2
MATa ura3–52 leu2-3,112 his3- 200 ade2–101 lys2–801
PSY316 TELVR::ADE2 TELVIIL::URA3
PSY316AUT cyt1D::HIS3 rho0
PLoS Genetics | www.plosgenetics.org November 2005 | Volume 1 | Issue 5 | e690619
Calorie Restriction in Yeast
URA3 amplified from pRS403, pRS405, or pRS406 , respectively.
In each case, the entire ORF of the deleted gene was removed. All
gene disruptions were verified by PCR. Medium used for life span
studies was YEP (2% bacto peptone, 1% yeast extract) supplemented
with filter-sterilized glucose at the designated concentration. For
nicotinamide supplementation experiments, nicotinamide was added
to YEP from a 500 mM nicotinamide (1003) filter sterilized stock
solution to a final concentration of 5 mM just prior to pouring plates.
Nicotinamide was obtained from Sigma (St. Louis, Missouri, United
Generation of rho0strains and verification by DAPI staining. The
rho0strains used for life span analysis were generated by treatment
with ethidium bromide. In each case, life span was determined for
more than one rho0isolate in order to verify the observed phenotype.
In the case of PSY316AUT rho0, four different rho0isolates were
examined, and the severe shortening in life span was observed in all
four cases. Life span was also determined for spontaneously arising
PSY316AUT rho0cells, which showed a life span defect similar to that
of rho0cells generated by ethidium bromide. Absence of mitochon-
drial DNA was verified by fluorescence microscopy of log phase cells
stained with DAPI.
Replicative life span analysis. Replicative life span analysis was
carried out as described . For all life span experiments, strains
were coded such that the researcher performing the life span
experiment had no knowledge of the strain genotypes. Unless
otherwise stated, standard life span medium was YEP þ 2% glucose
(YPD) and CR medium was YEP þ 0.05% glucose. Life span
experiments in the presence of nicotinamide were carried out at a
final concentration of 5 mM nicotinamide in the plates. Cells were
grown on experimental medium for at least 8 h prior to micro-
dissection. Wilcoxon p-values were calculated using the MATLAB
‘‘ranksum’’ function, and strains are stated to have a significant
difference in life span for p , 0.05.
FOA telomere silencing assays. For the silencing experiment
shown in Figure 3B and Figure S1, three independent cultures were
inoculated from single colonies into liquid YPD for each genotype
and grown overnight. The next morning, each overnight culture was
diluted 1:100 into YPD or CR medium and grown for 4 h in a shaking
incubator. Cultures were then diluted to a cell density of approx-
imately 2 3 103cells/ml in water, and plated in 100-ll aliquots onto
synthetic complete (SC) or FOA medium, containing either 2% or
0.05% glucose, such that cells cultured in 2% glucose were plated
onto 2% glucose plates and cells cultured in CR medium were plated
onto 0.05% glucose plates (CR plates). Percent survival was calculated
as the number of colonies arising on FOA medium divided by the
number of colonies arising on SC medium. Nicotinamide silencing
experiments were carried out as above, except that after the
overnight culture, cells were preincubated for 4 h in YPD þ 5 mM
nicotinamide and plated onto SC þ 5 mM nicotinamide or FOA þ 5
For the silencing experiment shown in Figure 3C, cultures of wild-
type or cyt1D cells were inoculated from single colonies into liquid
YPD or CR medium. The next morning, each overnight culture was
diluted 1:1000 into fresh control or CR medium, such that cells grown
overnight in control medium were diluted in control medium and
cells grown overnight in CR medium were diluted into CR medium,
and grown for 8 h in a shaking incubator. Cell cycle division time for
BY4742 control cells was approximately 95 min and for BY4742 CR
cells was approximately 105 min. After outgrowth, cultures were then
diluted to a cell density of approximately 2 3 103cells/ml in water,
and plated in 100-ll aliquots onto SC or FOA medium, containing
either 2% or 0.05% glucose, such that cells cultured in 2% glucose
were plated onto 2% glucose plates and cells cultured in CR medium
were plated onto CR plates. Percent survival was calculated as the
number of colonies arising on FOA medium divided by the number
of colonies arising on SC medium.
Figure S1. CR Has No Effect on Sir2 Activity in Respiratory-
Competent or Respiratory-Deficient Cells
Transcriptional silencing of the telomeric URA3 marker in PSY316-
AUT was monitored by the survival of cells plated onto medium
Found at DOI: 10.1371/journal.pgen.0010069.sg001 (42 KB PDF).
We would like to thank J. Smith, D. Gottschling, and T. Powers for
helpful discussion. This work has been funded by a grant from the
Ellison Medical Foundation. Support for this work has also been
provided by the American Federation for Aging Research and the
University of Washington Nathan Shock Center of Excellence for the
Basic Biology of Aging. MK is supported by National Institutes of
Health training grant P30 AG013280. SF is an investigator of the
Howard Hughes Medical Institute. BKK is a Searle Scholar.
Competing interests. The authors have declared that no competing
Author contributions. MK, SF, and BKK conceived and designed
the experiments. MK, DH, EOK, MT, ND, and BKK performed the
experiments. MK and BKK analyzed the data. MK, DH, EAW, and
BKK contributed reagents/materials/analysis tools. MK and BKK
wrote the paper.
1. Walker G, Houthoofd K, Vanfleteren JR, Gems D (2005) Dietary restriction
in C. elegans: From rate-of-living effects to nutrient sensing pathways. Mech
Ageing Dev 126: 929–937.
2. Merry BJ (2005) Dietary restriction in rodents—Delayed or retarded ageing.
Mech Ageing Dev 126: 951–959.
3.Partridge L, Piper MDW, Mair W (2005) Dietary restriction in Drosophila.
Mech Ageing Dev 126: 938–950.
4. Sinclair DA (2005) Toward a unified theory of caloric restriction and
longevity regulation. Mech Ageing Dev 126: 987–1002.
5. Guarente L (2005) Calorie restriction and SIR2 genes: Towards a
mechanism. Mech Ageing Dev 126: 923–928.
6. Merry BJ (2004) Oxidative stress and mitochondrial function with aging—
The effects of calorie restriction. Aging Cell 3: 7–12.
7. Armeni T, Pieri C, Marra M, Saccucci F, Principato G (1998) Studies on the
life prolonging effect of food restriction: Glutathione levels and glyoxylase
enzymes in rat liver. Mech Ageing Dev 101: 101–110.
8.De Cabo R, Cabello R, Rios M, Lopez-Lluch G, Ingram DK, et al. (2004)
Calorie restriction attenuates age-related alterations in the plasma
membrane antioxidant system in rat liver. Exp Gerontol 39: 297–304.
9. Guo Z, Heydari A, Richardson A (1998) Nucleotide excision repair of
actively transcribed versus nontranscribed DNA in rat hepatocytes: Effect
of age and dietary restriction. Exp Cell Res 245: 228–238.
10. Rao KS (2003) Dietary calorie restriction, DNA repair and brain aging. Mol
Cell Biochem 253: 313–318.
11. Yu BP (1996) Aging and oxidative stress: Modulation by dietary restriction.
Free Radic Biol Med 21: 651–668.
12. Youngman LD, Park JY, Ames BN (1992) Protein oxidation associated with
aging is reduced by dietary restriction of protein or calories. Proc Natl
Acad Sci U S A 89: 9112–9116.
13. Sohol RS, Weindruch R (1996) Oxidative stress, caloric restriction, aging.
Science 273: 59–63.
14. Masoro EJ, Katz MS, McMahan CA (1989) Evidence for the glycation
hypothesis of aging from the food-restricted rodent model. J Gerontol 44:
15. Masoro EJ, McCarter RJM, Katz MS, McMahan CA (1992) Dietary restriction
alters the characteristics of glucose fuel use. J Gerontol 47: B202–B208.
16. Kemnitz JW, Roecker EB, Weindruch R, Elson DF, Baum ST, et al. (1994)
Dietary restriction increases insulin sensitivity and lowers blood glucose in
rhesus monkeys. Am J Physiol 266: E540–E547.
17. Zhang Y, Herman B (2002) Ageing and apoptosis. Mech Ageing Dev 123:
18. Weindruch R, Walford RL (1988) The retardation of aging and disease by
dietary restriction. Springfield (Illinois): Charles C. Thomas. 436 p.
19. Jolly CA (2005) Diet manipulation and prevention of aging, cancer, and
autoimmune disease. Curr Opin Clin Nutr Metab Care 8: 382–387.
20. Mattson MP (2000) Emerging neuroprotective strategies for Alzheimer’s
disease: Dietary restriction, telomerase activation, and stem cell therapy.
Exp Gerontol 35: 489–502.
21. Saffrey MJ (2004) Ageing of the enteric nervous system. Mech Ageing Dev
22. McCay CM, Crowell MF, Maynard LA (1935) The effect of retarded growth
upon the length of life and upon ultimate size. J Nutr 10: 63–79.
23. Masoro EJ (1998) Influence of caloric intake on aging and on the response
to stressors. J Toxicol Environ Health B Crit Rev 1: 243–257.
24. Mattson MP, Duan W, Guo Z (2003) Meal size and frequency affect neuronal
plasticity and vulnerability to disease: Cellular and molecular mechanisms.
J Neurochem 84: 417–431.
25. Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, et al. (2003)
Genes that act downstream of DAF-16 to influence the lifespan of
Caenorhabditis elegans. Nature 424: 277–283.
PLoS Genetics | www.plosgenetics.org November 2005 | Volume 1 | Issue 5 | e690620
Calorie Restriction in Yeast
26. Kenyon C (2005) The plasticity of aging: Insights from long-lived mutants. Download full-text
Cell 120: 449–460.
27. Braeckman BP, Houthoofd K, Vanfleteren JR (2001) Insulin-like signaling,
metabolism, stress resistance and aging in Caenorhabditis elegans. Mech
Ageing Dev 122: 673–693.
28. Kaeberlein M, Powers RW, Steffen KK, Westman EA, Hu D, et al. (2005)
Regulation of yeast replicative life span by Tor and Sch9 in response to
nutrients. Science. In press.
29. Meissner B, Boll M, Daniel H, Baumeister R (2004) Deletion of the intestinal
peptide transporter affects insulin and TOR signaling in Caenorhabditis
elegans. J Biol Chem 279: 36739–36745.
30. Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, et al. (2003)
Genetics: Influence of TOR kinase on lifespan in C. elegans. Nature 426: 620.
31. Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, et al. (2004) Regulation
of lifespan in Drosophila by modulation of genes in the TOR signaling
pathway. Curr Biol 14: 885–890.
32. Sharp ZD, Bartke A (2005) Evidence for down-regulation of phosphoinosi-
tide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR)-
dependent translation regulatory signaling pathways in Ames dwarf mice.
J Gerontol A Biol Sci Med Sci 60: 293–300.
33. Lin SJ, Defossez PA, Guarente L (2000) Requirement of NAD and SIR2 for
life-span extension by calorie restriction in Saccharomyces cerevisiae. Science
34. Rogina B, Helfand SL (2004) Sir2 mediates longevity in the fly through a
pathway related to calorie restriction. Proc Natl Acad Sci U S A 101: 15998–
35. Guarente L, Picard F (2005) Calorie restriction—The SIR2 connection. Cell
36. Kaeberlein M, Kirkland KT, Fields S, Kennedy BK (2004) Sir2-independent
life span extension by calorie restriction in yeast. PLoS Biol 2: E296.
37. Kaeberlein M, Andalis AA, Fink GR, Guarente L (2002) High osmolarity
extends life span in Saccharomyces cerevisiae by a mechanism related to
calorie restriction. Mol Cell Biol 22: 8056–8066.
38. Smith JS, Brachmann CB, Celic I, Kenna MA, Muhammad S, et al. (2000) A
phylogenetically conserved NADþ-dependent protein deacetylase activity
in the Sir2 protein family. Proc Natl Acad Sci U S A 97: 6658–6663.
39. Landry J, Sutton A, Tafrov ST, Heller RC, Stebbins J, et al. (2000) The
silencing protein SIR2 and its homologs are NAD-dependent protein
deacetylases. Proc Natl Acad Sci U S A 97: 5807–5811.
40. Imai S, Armstrong CM, Kaeberlein M, Guarente L (2000) Transcriptional
silencing and longevity protein Sir2 is an NAD-dependent histone
deacetylase. Nature 403: 795–800.
41. 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.
42. Sinclair DA, Guarente L (1997) Extrachromosomal rDNA circles—A cause
of aging in yeast. Cell 91: 1033–1042.
43. Michel AH, Kornmann B, Dubrana K, Shore D (2005) Spontaneous rDNA
copy number variation modulates Sir2 levels and epigenetic gene silencing.
Genes Dev 19: 1199–1210.
44. Kennedy BK, Austriaco NR, Zhang J, Guarente L (1995) Mutation in the
silencing gene SIR4 can delay aging in S. cerevisiae. Cell 80: 485–496.
45. Couzin J (2004) Scientific community. Aging research’s family feud. Science
46. Lin SJ, Kaeberlein M, Andalis AA, Sturtz LA, Defossez PD, et al. (2002)
Calorie restriction extends Saccharomyces cerevisiae life span by increasing
respiration. Nature 418: 344–348.
47. Lin SJ, Ford E, Haigis M, Liszt G, Guarente L (2004) Calorie restriction
extends yeast life span by lowering the level of NADH. Genes Dev 18: 12–16.
48. Anderson RM, Latorre-Esteves M, Neves AR, Lavu S, Medvedik O, et al.
(2003) Yeast life-span extension by calorie restriction is independent of
NAD fluctuation. Science 302: 2124–2126.
49. Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Sinclair DA (2003)
Nicotinamide and PNC1 govern lifespan extension by calorie restriction in
Saccharomyces cerevisiae. Nature 423: 181–185.
50. Bitterman KJ, Anderson RM, Cohen HY, Latorre-Esteves M, Sinclair DA
(2002) Inhibition of silencing and accelerated aging by nicotinamide, a
putative negative regulator of yeast sir2 and human SIRT1. J Biol Chem
51. Landry J, Slama JT, Sternglanz R (2000) Role of NAD(þ) in the deacetylase
activity of the SIR2-like proteins. Biochem Biophys Res Comm 278: 685–
52. Gallo CM, Smith DL Jr., Smith JS (2004) Nicotinamide clearance by Pnc1
directly regulates Sir2-mediated silencing and longevity. Mol Cell Biol 24:
53. Kaeberlein M, Kennedy BK (2005) Large-scale identification in yeast of
conserved ageing genes. Mech Ageing Dev 126: 17–21.
54. Kaeberlein M, Andalis AA, Liszt G, Fink GR, Guarente L (2004)
Saccharomyces cerevisiae SSD1-V confers longevity by a Sir2p-independent
mechanism. Genetics 166: 1661–1672.
55. Kaeberlein M, McDonagh T, Heltweg B, Hixon J, Westman EA, et al. (2005)
Substrate-specific activation of sirtuins by resveratrol. J Biol Chem 280:
56. Tzagoloff A, Myers AM (1986) Genetics of mitochondrial biogenesis. Annu
Rev Biochem 55: 249–285.
57. de Zamaroczy M, Bernardi G (1986) The primary structure of the
mitochondrial genome of Saccharomyces cerevisiae. Gene 47: 155–177.
58. Kaeberlein M, Kirkland KT, Fields S, Kennedy BK (2005) Genes determin-
ing yeast replicative life span in a long-lived genetic background. Mech
Ageing Dev 126: 491–504.
59. Williams AC, Ramsden DB (2005) Nicotinamide homeostasis: A xenobiotic
pathway that is key to development and degenerative diseases. Med
Hypotheses 65: 353–362.
60. Wallace DC (2001) A mitochondrial paradigm for degenerative diseases and
ageing. Novartis Found Symp 235: 247–266.
61. Wallace DC, Shoffner JM, Trounce I, Brown MD, Ballinger SW, et al. (1995)
Mitochondrial DNA mutations in human degenerative diseases and aging.
Biochim Biophys Acta 1271: 141–151.
62. Taylor RW, Turnbull DM (2005) Mitochondrial DNA mutations in human
disease. Nat Rev Genet 6: 389–402.
63. Kirchman PA, Kim S, Lai CY, Jazwinski SM (1999) Interorganelle signaling
is a determinant of longevity in Saccharomyces cerevisiae. Genetics 152: 179–
64. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, et al.
(1999) Functional characterization of the S. cerevisiae genome by gene
deletion and parallel analysis. Science 285: 901–906.
65. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Saccharomyces
cerevisiae. Genetics 122: 19–27.
PLoS Genetics | www.plosgenetics.org November 2005 | Volume 1 | Issue 5 | e690621
Calorie Restriction in Yeast