Aging Cell
(2007)
6
, pp649–662 Doi: 10.1111/j.1474-9726.2007.00326.x
© 2007 The Authors
649
Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
Blackwell Publishing Ltd
Calorie restriction extends the chronological lifespan of
Saccharomyces cerevisiae
independently of the Sirtuins
Daniel L. Smith, Jr, Julie M. McClure, Mirela Matecic and Jeffrey S. Smith
Department of Biochemistry and Molecular Genetics, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908, USA
Summary
Calorie restriction (CR) extends the mean and maximumlifespan of a wide variety of organisms ranging fromyeast to mammals, although the molecular mechanismsof action remain unclear. For the budding yeast
Saccharomyces cerevisiae
reducing glucose in the growthmedium extends both the replicative and chronologicallifespans (CLS). The conserved NAD
+
-dependent histonedeacetylase, Sir2p, promotes replicative longevity in
S. cerevisiae
by suppressing recombination within theribosomal DNA locus and has been proposed to mediatethe effects of CR on aging. In this study, we investigatedthe functional relationships of the yeast Sirtuins (Sir2p,Hst1p, Hst2p, Hst3p and Hst4p) with CLS and CR.
SIR2
,
HST2
, and
HST4
were not major regulators of CLS andwere not required for the lifespan extension caused byshifting the glucose concentration from 2 to 0.5% (CR).Deleting
HST1
or
HST3
moderately shortened CLS, but didnot prevent CR from extending lifespan. CR thereforeworks through a Sirtuin-independent mechanism in thechronological aging system. We also show that lowtemperature or high osmolarity additively extends CLSwhen combined with CR, suggesting that these stressesand CR act through separate pathways. The CR effect onCLS was not specific to glucose. Restricting other simplesugars such as galactose or fructose also extended lifespan.Importantly, growth on nonfermentable carbon sourcesthat force yeast to exclusively utilize respiration extendedlifespan at nonrestricted concentrations and providedno additional benefit when restricted, suggesting thatelevated respiration capacity is an important determinantof chronological longevity.Key words: calorie restriction; chronological aging;
HST
;lifespan,
SIR2
; yeast.
Introduction
As the human population continues to age, interventions that
improve the quality of life and delay the onset of age-related
diseases are highly desirable. Calorie restriction (CR) has been
known to delay the aging process in a variety of organisms for
over 70 years (McCay
et al
., 1935). This regimen of limiting
calories by manipulating the dietary intake of nutrients works
almost universally, having been tested with organisms ranging
from yeast to ongoing studies in monkeys (see Masoro, 2005
for review). However, the basic mechanisms by which CR
influences aging have been difficult to dissect.
Saccharomyces cerevisiae
is a budding yeast that is commonly
used to investigate the mechanism of CR-mediated lifespan
extension (Guarente & Picard, 2005). The replicative lifespan (RLS)
of this organism is defined as the number of buds (daughters)
that mother cells produce before senescing (Mortimer & Johnston,
1959). Many genes have been implicated in regulating RLS. One
of the most heavily studied examples, silent information
regulator
2
(
SIR2
) (Kaeberlein
et al
., 1999), encodes an NAD
+
-
dependent histone deacetylase that removes acetyl groups
from specific lysine residues on the tails of histones H3 and H4
(Imai
et al
., 2000; Landry
et al
., 2000b; Smith
et al
., 2000). This
deacetylation promotes transcriptional silencing at the
HML/HMR
silent mating-type loci, telomeres and the ribosomal DNA
(rDNA), where it also suppresses homologous recombination
between the tandemly repeated rDNA genes (see Rusche
et al
.
2003; Buck
et al
. 2004 for reviews). Inter-repeat rDNA recom-
bination results in the production of extrachromosomal rDNA
circles (ERC), which are self-replicating episomes that naturally
accumulate to high levels in replicatively old mother cells, inducing
their senescence through an uncharacterized mechanism
(Sinclair & Guarente, 1997). Deleting
SIR2
dramatically increases
the frequency of rDNA recombination, and the subsequent
overaccumulation of ERCs in mother cells results in premature
aging, while overexpressing
SIR2
lengthens RLS (Kaeberlein
et al
., 1999).
CR has been proposed to extend RLS by activating Sir2p
deacetylase activity, either through an increase in the intracellular
NAD
+
/NADH ratio (Lin
et al
., 2004), and/or a reduction in the
nicotinamide (NAM) concentration (Anderson
et al
., 2003). NAM
is a by-product of the deacetylation reaction and is also a potent
noncompetitive Sir2p inhibitor (Landry
et al
., 2000a; Bitterman
et al
., 2002). CR elevates the expression of a nicotinamidase,
Pnc1p, which limits the intracellular NAM concentration by
converting it to nicotinic acid, and thereby promoting Sir2p
deacetylase activity (Anderson
et al
., 2003; Gallo
et al
., 2004).
The Sir2p activation by CR is proposed to extend RLS through
the suppression of rDNA recombination and ERC accumulation
Correspondence
Jeffrey S. Smith, Department of Biochemistry and Molecular Genetics,
University of Virginia Health System, Jordan Hall, Box 800733, Charlottesville,
VA 22908, USA. Tel.: 434-243-5864; fax: 434-924-5069;
e-mail: [email protected]
Accepted for publication
24 May 2007
Calorie restriction extends yeast chronological lifespan, D. L. Smith
et al.
© 2007 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
650
(Lin
et al
., 2002). However, independent experiments revealed
that CR extended RLS independently of
SIR2
when rDNA
recombination (ERC formation) was simultaneously prevented
by deleting the
FOB1
(fork block 1) gene (Defossez
et al
., 1999;
Kaeberlein
et al
., 2004). As its name implies, Fob1p blocks DNA
replication forks moving through the rDNA in an opposing
direction to that of transcription by RNA polymerase I (Kobayashi
& Horiuchi, 1996). The stalled forks are potential sources of
DNA breaks that can lead to homologous recombination and ERC
generation. The Sir2p-related proteins, Hst1p and Hst2p, have
also been implicated in CR-mediated extension of RLS by sup-
pressing rDNA recombination (Lamming
et al
., 2005), although
the role of these ‘Sirtuins’ in mediating CR effects on yeast RLS
remains controversial (Kaeberlein
et al
., 2006; Sinclair
et al
., 2006).
Metazoans such as
Drosophila melanogaster
and
Caenorhabditis elegans
are also responsive to CR (Guarente &
Picard, 2005), although unlike yeast, most cells in fully grown
metazoan organisms are nondividing.
SIR2
orthologs have been
shown to influence lifespan and to at least partially function in
the CR response in both of these organisms (Rogina & Helfand,
2004; Guarente & Picard, 2005; Wang & Tissenbaum, 2006).
Yet, ERCs have not been reported to accumulate in aging
Drosophila
or
C. elegans
, and do not appear to accumulate in
dividing mammalian cells (Marciniak
et al
., 1998). This raises
the important questions of whether the mechanisms of CR-
mediated lifespan extension in dividing and nondividing cells
are different, and whether Sirtuins mediate the CR effect in cells
that do not accumulate ERCs.
Yeast longevity can also be measured as the number of days
that nondividing cells maintain viability in a saturated culture
or while resuspended in water (Fabrizio & Longo, 2003). This
is called the chronological lifespan (CLS). Viability is measured
by the ability of cells to produce a colony when transferred to
fresh media. Importantly, because of the dynamics of a liquid
culture, greater than 95% of cells in the population are
replicatively young, less than five generations old. These cells
are quiescent (G
0
) but are metabolically active throughout the
course of the experiment (Fabrizio & Longo, 2003), much like
postmitotic tissues in an adult multicellular organism. With the
large overlap of biochemical pathways between yeast and
other organisms, the yeast CLS system is posited as a model for
analyzing the effects of metabolism on longevity in postmitotic
cell types (MacLean
et al
., 2001; Chen
et al
., 2005). In this study,
we have adapted the yeast CLS system to investigate the
mechanisms of the Sirtuins and CR in regulating longevity in
nondividing cells. We also investigate how variations in types
of carbon source nutrients impact lifespan to begin dissecting
the mechanism of a Sirtuin-independent CR pathway.
Results
CR extends CLS independently of SIR2
The CLS system involves growing yeast strains into stationary
phase and then monitoring cell viability over time. An earlier
study reported that ERCs do not accumulate in cells grown into
stationary phase (Ashrafi
et al
., 1999). We were therefore
interested in using this system to test whether
SIR2
or the other
Sirtuins play any role in regulating lifespan in a situation where
cells are not dividing and not accumulating ERCs. We were also
interested in determining whether they function in mediating
CR-induced lifespan extension.
Traditional CLS assays are performed by growing liquid yeast
cultures into stationary phase in shaking flasks, and then
calculating the number of colony-forming units (CFU) per mL
as the culture ages. This method is accurate but labor intensive
and requires a great deal of space in shaking incubators, making
it cumbersome to analyze multiple strains or growth conditions
at one time. As an alternative assay, we grow small 10 mL yeast
cultures in test tubes that rotate on a roller drum, rather than
vigorously shaking in a flask. At the appropriate time-points,
aliquots of the cultures are serially diluted tenfold in sterile water
and then spotted onto rich YPD medium to give a semiquanti-
tative indication of the CFUs (Fig. 1A).
To directly compare the accuracy and resolution of the colony
counting method vs. the spot test method (Fabrizio
et al
., 2001;
Reverter-Branchat
et al
., 2004), we used the BY4741 strain
background, which has been shown to have a relatively long
CLS compared to other strain backgrounds (data not shown;
Piper
et al
., 2006). BY4741 and a
sir2
∆
mutant version (DSY233)
were inoculated (in triplicate) into 10 mL of synthetic complete
(SC) medium (Burke
et al
., 2000) containing either 2% (non-
restricted, NR) or 0.5% glucose (CR condition) and grown into
stationary phase at 30
°
C. At the indicated days following
inoculation, aliquots were removed, tenfold serially diluted,
and spotted onto rich YPD agar plates for the spot test assay
(Fig. 1A). In parallel, the culture aliquots were appropriately
diluted and spread onto YPD plates to allow for colony counting
(Fig. 1B). There was little variation in viability between the
triplicate cultures using either measurement. As a result, several
interesting conclusions can be drawn from this experiment.
First, both measurements clearly showed that the CR growth
condition extended the lifespans of BY4741 and the
sir2
∆
mutant. By day 20, both strains were at least 100-fold more
viable in the 0.5% glucose cultures.
SIR2
is therefore not
required for the CR effect on CLS. Second, deleting
SIR2
did
not shorten the CLS in 2% glucose as it previously did for
replicative lifespan (Kaeberlein
et al
., 1999). Instead, there was
a slight increase in viability for the
sir2
∆
mutant that became
observable at day 14 for the colony counting assay and then
remained between five- and tenfold higher throughout the later
time-points (Fig. 1B). For the spot test assay, the slightly longer
lifespan for the
sir2
∆
mutant was apparent at day 20, when
there were approximately five- to tenfold more viable cells in
the
sir2
∆
cultures compared to BY4741 (Fig. 1A). After day 23,
there were not enough viable cells in the cultures to yield
colonies at the dilutions used for the spot tests. The improved
viability for the
sir2
∆
mutant was also observed in the CR
cultures. By day 30, the
sir2
∆
mutant was approximately five-
to tenfold more viable as measured by both assays. As both
Calorie restriction extends yeast chronological lifespan, D. L. Smith
et al.
© 2007 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
651
measurements of viability gave similar results, especially for the
detection of CR effects, we have used the spot test assay for
subsequent experiments in this study. As with the traditional
CFU method of measuring CLS, small, but significant, differences
in lifespan can be detected using the spot test assay when
comparing strains or growth conditions within an individual
experiment. When comparing data from different experiments,
however, only large differences in lifespan such as the CR effect
are considered significant.
To confirm the lack of an ERC effect on a typical CLS experi-
ment, we generated a congenic set of strains (all
MAT
α
) in
which
SIR2
,
FOB1
or both were deleted. The absence of
FOB1
suppresses ERC formation, extends RLS (Defossez
et al
., 1999)
and suppresses the short RLS caused by a
sir2
∆
mutation
(Kaeberlein
et al
., 1999). As shown in Fig. 2A, deleting
SIR2
again slightly extended the maximum lifespan compared to WT,
although with this set of strains the
sir2
∆
mutant had lower
viability at days 7 and 9 compared to WT. The
fob1
∆
mutation
had no effect on lifespan compared to the WT control (Fig. 2A),
and the
sir2
∆
fob1
∆
double mutant had a lifespan that was
equivalent to the
sir2
∆
mutant. Unlike the case with RLS, the
yeast CLS is therefore not strongly influenced by mutations that
Fig. 1 Calorie restriction extends yeast CLS. (A) Spot test assay for cell viability over time. WT (BY4741) and sir2∆ (DSY233) strains were inoculated into SC medium containing initial glucose concentrations of 2% (NR) and 0.5% (CR) and allowed to grow into late stationary phase. Following inoculation, aliquots of the cultures were serially diluted tenfold at the indicated time points, spotted onto YPD plates and incubated for 3 days to allow for colony growth. The cultures were inoculated in triplicate, and lifespan results for each culture are shown up to day 30. (B) Quantitative CLS assay. Aliquots from the same cultures were diluted in water and plated onto YPD plates such that single colonies could be counted and the colony forming units (CFU) per ml calculated. Standard deviations between the cultures are shown.
Calorie restriction extends yeast chronological lifespan, D. L. Smith
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© 2007 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
652
affect ERC production such as
sir2
∆
and
fob1
∆
. However, as
most cells in a stationary phase population are relatively young,
we cannot rule out the possibility that a minor population of
very old cells could be chronologically affected by ERC accumu-
lation. Small variations in the mean lifespan between various
sir2
∆
isolates have previously been observed (Fabrizio
et al
.,
2005), implying that the lower viability for the
sir2
∆
mutant in
Fig. 2A (days 7 and 9) may not be significant.
Increased
SIR2
dosage was previously shown to extend
replicative lifespan not only in yeast (Kaeberlein
et al
., 1999), but
also in worms and flies (Tissenbaum & Guarente, 2001; Rogina
& Helfand, 2004). To test for a possible
SIR2
dosage effect, we
integrated an extra copy of
SIR2
under control of its native
promoter at the
leu2
∆
1
locus of SY108 (related to BY4741) and
measured CLS in comparison with a control strain in which an
empty vector was integrated.
LEU2
was repaired due to the
recombination event, making the strains leucine prototrophs
(Leu
+
). As shown in Fig. 2B, the extra
SIR2 gene copy (2× SIR2)
had no effect on lifespan compared to the control (vector), even
though the steady state Sir2p protein level had increased
(Fig. 2C). The lack of an effect is significant because integrating
the same SIR2 construct into a BY4741-related rDNA silencing
reporter strain dramatically increased silencing of the mURA3marker located within the rDNA (Fig. 2D). Therefore, while Sir2p
is a limiting factor for rDNA silencing in the BY strain back-
ground, it is not limiting for CLS. It is also notable that making
the strains Leu+ altered the overall aging profile compared to the
leu2∆1 strains in panel (A), suggesting that specific amino acid
auxotrophies may influence CLS.
The HST1 and HST3 genes function in chronological longevity, but are not required for the CR effect on lifespan
NAM is a noncompetitive inhibitor of Sir2p and the other yeast
Sirtuins (Hst1p, Hst2p, Hst3p and Hst4p), and has been shown
to shorten RLS when added to the growth media at a concen-
tration of 5 mM (Landry et al., 2000a; Bitterman et al., 2002).
Based on the slightly longer lifespan of the sir2∆ mutant in
Fig. 1, we anticipated that supplementing the SC medium with
NAM would result in a similar small increase in CLS. However,
lifespan was modestly shorter than normal when 1 mM or 5 mM
NAM was added to the SC medium (Fig. 3A). We suspected that
NAM was inhibiting one or more of the other Hst proteins,
which was then causing the lifespan defect. To address this
possibility, we determined the CLS of strains that were deleted
for HST1, HST2, HST3 or HST4. As shown in Fig. 3B, the hst1∆and hst3∆ mutants had shorter lifespans than WT when grown
on 2% glucose, while the hst2∆ and hst4∆ mutants were
closer to normal. The short lifespan of the hst3∆ mutant could
potentially be caused by a genomic instability problem. Hst3p
and Hst4p were recently shown to specifically deacetylate lysine
56 of histone H3, which contributes to the maintenance of
genome integrity, with hst3∆ phenotypes being more severe
Fig. 2 SIR2 is not a major regulator of CLS. (A) Spot test CLS assay for WT (DSY278), sir2∆ (DSY281), fob1∆ (DSY291) and sir2∆ fob1∆ (DSY283) strains. (B) Spot test CLS assay showing the effect of integrating an extra copy of SIR2 into the BY4741-related strain background SY108. Either an empty LEU2 vector (pASC405) or the LEU2 SIR2 vector (pSB760) was integrated to introduce the SIR2 copy. (C) Western blot analysis of steady state Sir2 protein levels when an extra copy of SIR2 is integrated into the genome. α-Tubulin is used as the loading control. (D) rDNA silencing assay in which the extra copy of SIR2 is shown to dramatically strengthen repression of a URA3 reporter gene (mURA3) integrated within the rDNA tandem array.
Calorie restriction extends yeast chronological lifespan, D. L. Smith et al.
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653
than hst4∆ phenotypes (Celic et al., 2006). In congruence with
these observations, deletion of HST3 shortened RLS, while an
HST4 deletion showed no effect (Tsuchiya et al., 2006).
Hst1p acts as a transcriptional corepressor for vegetatively
repressed, meiosis-specific genes and for genes encoding
enzymes in the de novo NAD+ biosynthesis pathway, such as
BNA1 (Xie et al., 1999; Bedalov et al., 2003). Deleting HST1deregulates the de novo NAD+ biosynthesis pathway, causing
increased expression of several genes in the pathway, including
BNA1 (Bedalov et al., 2003). This deregulation could be
detrimental to chronological longevity. Consistent with this
idea, deleting the BNA1 gene of the de novo NAD+ synthesis
pathway extended the CLS (Fig. 4). More subtle effects were
observed for deletions of the NAD+ salvage pathway compo-
nents, NPT1 and PNC1 (Fig. 4). Regardless of the effects of
deleting the HST or NAD+ synthesis/salvage genes on CLS when
grown in 2% glucose, CR still clearly extended the lifespan of
each hst∆ mutant (Fig. 3B) or NAD+ salvage/synthesis mutant
(Fig. 4). CLS extension mediated by CR is therefore independent
of each Sirtuin, although we currently cannot rule out the
possibility of redundance.
CR does not extend CLS exclusively as a stress response
Glucose concentrations between 0.05 and 0.5% have typically
been used for CR studies in yeast (Jiang et al., 2000; Lin et al.,
2000; Kaeberlein et al., 2004), with greater replicative lifespan
benefits observed for the lower concentrations (Jiang et al.,2000; Kaeberlein et al., 2004). Increasing the glucose con-
centration up to 20%, which elevates the osmolarity of the
culture medium (~1 M glucose), also extended the RLS in a SIR2-and NPT1-dependent manner, suggesting that CR and high
osmolarity extend RLS through similar mechanisms (Kaeberlein
et al., 2002). We therefore tested whether CLS was similarly
regulated by variations in glucose concentration and osmotic
stress. The CLS of BY4741 was tested in SC medium containing
initial glucose concentrations ranging from 0.05 to 20%
(Fig. 5A). As expected, concentrations below 2% extended
lifespan, with 0.1 and 0.05% concentrations having greater
effects than 0.5% (Fig. 5A; extended time course not shown).
However, concentrations above 2% caused a progressive
shortening of lifespan that leveled out at 10–20%. This was in
contrast to the extended RLS that was reported at high con-
centrations due to the high osmolarity (Kaeberlein et al., 2002).
To more directly test whether high osmolarity affected CLS,
we measured the lifespan of BY4741 when the initial glucose
concentration was kept at 2%, and sorbitol was incrementally
increased to a concentration of 2 M (Fig. 5B, top panels). Impor-
tantly, sorbitol is not utilized by S. cerevisiae as a carbon source,
so its concentration will remain steady throughout the experi-
ment. The lowest concentration of sorbitol tested (0.1 M) had
little effect on lifespan (compare the 0.1 M culture in Fig. 5B to
Fig. 3 HST-independent lifespan extension by CR. (A) Nicotinamide (NAM) attenuates CLS. SC media was supplemented with 1 mM or 5 mM NAM. (B) Effects of deleting HST1 (SY163), HST2 (SY164), HST3 (SY165) or HST4 (SY166) on CLS when cells are grown in SC media containing 2% (NR) or 0.5% (CR) glucose. The WT strain is BY4741.
Calorie restriction extends yeast chronological lifespan, D. L. Smith et al.
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654
the 2% glucose culture in Fig. 5A). However, intermediate
concentrations of 0.5 M and 1 M sorbitol did extend CLS in the
context of a 2% glucose culture, although a further increase
to 2 M diminished the benefit. To test whether CR and high
sorbitol osmolarity extended CLS through the same pathway,
the various sorbitol concentrations were combined with CR
(0.5% glucose) and the CLS of BY4741 assayed (Fig. 5B, bottom
panels). Even though 0.5 or 1 M sorbitol extended lifespan
in the context of 2% glucose, combining these sorbitol con-
centrations with 0.5% glucose produced even longer lifespans,
strongly suggesting that CR and osmotic stress affect CLS
through separate pathways.
To determine if other types of cellular stress similarly extended
the CLS under non-CR conditions, we tested the effects of
temperature on the CLS of BY4741. Yeast cultures are normally
grown at 30 °C, so we compared the lifespan of cultures grown
at 30 °C to those grown at 23 °C and 37 °C. As shown in
Fig. 5C, 2% glucose cultures incubated at a reduced temperature
(23 °C) resulted in an extension of CLS compared to 30 °C,
whereas an elevated temperature (37 °C) resulted in a decrease
of CLS. Reducing the glucose concentration to 0.5% extended
the CLS at each temperature, with the combination of 23 °Cand CR extending lifespan more robustly than either condition
alone (Fig. 5C). The effect of CR at 37 °C was modest. From
this data we can conclude that low temperature extends CLS
and is additive with the CR effect.
Deletions of HXK2 or GPA2 are not effective genetic mimics of CR for CLS
HXK2 encodes one of three hexokinases responsible for
converting glucose into glucose-6-phosphate, thus introducing
glucose into the glycolysis pathway. Mutations in this gene are
believed to reduce glucose utilization, producing a genetic
mimic of CR (Lin et al., 2000). Another gene that has been used
as a genetic mimic of CR is GPA2 (Lin et al., 2000), which
encodes a heterotrimeric G-protein subunit that in combination
with Gpr1p responds to glucose levels. Deletion of HXK2 or
GPA2 has been shown to extend the RLS by 30–40% when cells
are grown on a normal glucose concentration of 2% (Lin et al.,2000). As CLS was sensitive to changes in glucose concentration
(Fig. 5A), we were interested in whether these genetic mimics
would extend CLS. Surprisingly, our results showed that hxk2∆and gpa2∆ mutations did not produce any beneficial effects on
CLS when cells were grown in 2% glucose (Fig. 6A). Additionally,
glucose restriction at the 0.5% concentration extended the CLS
of both mutants. These mutants are therefore not efficient CR
mimics in the context of the chronological aging system.
Effects of alternative carbon sources on CLS and CR
As glucose restriction extended the CLS even in hxk2∆ and
gpa2∆ mutants, we theorized that altering the concentration
of other carbon sources may extend lifespan as would be
expected from true general CR. To test this hypothesis, we
measured the CLS of BY4741 grown on a variety of alternative
monosaccharide carbon sources. Yeast can utilize a variety of
carbon sources, the most common being dextrose (glucose).
However, multiple reports in the literature suggested that
glucose can be replaced by fructose, galactose or other mono-
saccharides at equal concentrations (Carlson, 1987). We tested
each sugar at the normal concentration of 2% and a restricted
concentration of 0.5%. As shown in Fig. 6B, we found that CLS
for fructose and galactose tested at 2% was consistently similar
to that of glucose at the 2% concentration (~3 weeks). We also
observed that restricting the fructose or galactose concentration
to 0.5% extended CLS to the same degree or in a similar way
as glucose restriction. The effect of CR is therefore not limited
Fig. 4 Deletion of BNA1 extends CLS. CR growth conditions extend lifespan of mutants defective in de novo NAD+ synthesis (bna1∆-SY8) or NAD+ salvage (npt1∆-SY16 or pnc1∆-SY10). The WT strain was BY4741.
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to glucose, suggesting that mechanisms in addition to the
sensing of exogenous glucose contribute to lifespan regulation.
Yeast can also utilize more complex sugars, both di- and
trisaccharides (Carlson, 1987). We therefore expanded our
investigation to include the disaccharides sucrose, maltose
and trehalose, and the trisaccharide raffinose. Of these more
complex sugars tested, only sucrose exhibited a CLS similar to
glucose at the NR 2% concentration (Fig. 7A). This is reasonable,
as sucrose is composed of one glucose and one fructose moiety,
and each of these monosaccharides alone produces no distinct
difference in CLS (Fig. 6B). The other di- and trisaccharides
greatly extended the CLS of BY4741 at the NR 2% concentration
when compared to 2% glucose or sucrose (Fig. 7A). Yet, the
structures of each of these complex sugars are combinations
of glucose, fructose or galactose. Even though each of these
polysaccharides extended CLS at the NR concentration, reduc-
ing them to 0.5% further extended lifespan, with the exception
of raffinose, and possibly maltose (Fig. 7A). Earlier reports
demonstrated that the breakdown of complex sugars occurs
extracellularly, resulting in the subsequent uptake of the more
simple monosaccharides (Carlson & Botstein, 1982; Carlson, 1987;
Jules et al., 2004). It was therefore possible that the observed
lifespan extension for the polysaccharides was attributable to
relatively poor utilization of the complex sugars caused by
limited uptake. This idea was supported by the delayed initial
growth of the cultures from days 1–7 for maltose and days
1–5 for trehalose compared to the sucrose culture (Fig. 7A). To
test if the CLS benefits of the complex sugars at the NR
concentrations were indeed due to limited uptake, a composite
mixture of glucose, fructose and galactose, the individual
components of raffinose, was added to the SC medium at
concentrations equal to a 2% raffinose control. As shown in
Supplementary Fig. S1, the splitting of raffinose into its easily
utilized monosaccharide components caused a loss of the
beneficial effect on lifespan and restricting the individual sugars
in combination now extended lifespan, albeit not as dramatically.
Taken together, these results suggest that complex sugars may
mimic CR because their requirement for breakdown limits the
uptake of their individual components.
Each of the sugars tested above has the potential to be
fermented to ethanol or utilized in respiratory metabolism to
produce adenosine 5’-triphosphate (ATP). CR by glucose limitation
Fig. 5 Effects of glucose concentration, osmotic stress and temperature on CLS. (A) Titration of initial glucose concentrations in the BY4741 culture greatly alters lifespan. (B) Hyperosmotic stress with sorbitol extends lifespan at NR (2%) and CR (0.5%) glucose concentrations. (C) CR extends lifespan independently of temperature variation effects.
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has been proposed to shift this balance towards respiration (Lin
et al., 2002), altering the energy production and metabolic state
of the cells. Using nonfermentable carbon sources such as
glycerol or ethanol provides a means to directly test the effect
of a respiration-only metabolic condition on CLS. Growing
BY4741 in each of these nonfermentable carbon sources at
an NR concentration resulted in a dramatic extension of CLS
compared to 2% glucose (Fig. 7B). As with raffinose, restriction
Fig. 6 Calorie restriction effects on CLS are not limited to glucose as a carbon source. (A) Genetic mimics of CR (gpa2∆-DSY312 or hxk2∆-DSY314) do not extend CLS when cells are grown in the NR (2%) glucose concentration, and do not block CR (0.5% glucose)-mediated extension of CLS. (B) The monosaccharides, fructose and galactose, both extend CLS when restricted from 2% to the 0.5% concentration.
Fig. 7 The effects of restricting complex sugars and nonfermentable carbon sources on CLS. (A) Di- and polysaccharides extend CLS at NR (2%) concentrations. CR carbon source concentrations were 0.5%. (B) Nonfermentable carbon sources extend CLS at NR concentrations (3% glycerol, 2% ethanol or 3% glycerol/1% ethanol when in combination). Restricting the nonfermentable carbon sources does not further extend lifespan. BY4741 was used for these experiments.
Calorie restriction extends yeast chronological lifespan, D. L. Smith et al.
© 2007 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
657
of these carbon sources did not further extend the CLS (Fig. 7B).
This data is consistent with a model previously proposed for RLS
(Lin et al., 2002), in which some of the lifespan benefits caused
by CR in yeast cells is due to a shift in the balance of metabolism
from fermentation towards aerobic respiration.
To determine if the longer lifespan induced by the more
complex sugars correlated with a shift towards respiration, we
spotted fivefold serial dilutions of WT (BY4741), sir2∆ (DSY233)
and emi1∆ (SY386) strains on SC agar plates containing the
various carbon sources. Where indicated, the plates were supple-
mented with the mitochondrial respiration inhibitor, antimycin
A. The emi1∆ mutant was randomly chosen as a strain from
the yeast gene knockout collection with a respiration defect.
We predicted that growth conditions promoting a shift towards
respiration would cause a growth defect in the presence of
antimycin A. Consistent with this hypothesis, antimycin A
prevented growth of each strain on the NR, nonfermentable
combination of glycerol and ethanol (Supplementary Fig. S2A).
As expected, the emi1∆ mutant grew very poorly on glycerol/
ethanol media even without antimycin A. NR maltose and
trehalose produced the same result, strongly suggesting that
these poorly utilized disaccharides cause a drastic shift towards
respiration (Supplementary Fig. S2A). NR raffinose (2%), which
is utilized much better than maltose or trehalose (Fig. 7A),
caused a slow growth phenotype for the WT and sir2∆ strains
on antimycin A compared to without antimycin (Supplementary
Fig. S2A). This effect became more pronounced as the raffinose
concentration was reduced to 0.5 or 0.2%. Importantly, the
antimycin A had little effect on growth of the emi1∆ respiration-
defective mutant even at the lower raffinose concentrations
(Supplementary Fig. S2A), implying that the antimycin effect
on the WT and sir2∆ mutants was due to the shift towards
respiration. Antimycin A had little effect on colony size or
robustness of growth for glucose, fructose or sucrose at the
NR 2% concentration (Supplementary Fig. S2B). However, WT
and sir2∆ growth defects were observed for the restricted
concentrations (Supplementary Fig. S2B). Taken together, these
results show a strong correlation between CR and the shift
towards respiration.
Discussion
S. cerevisiae has proven to be an outstanding model for
studying basic cellular processes. The CLS of this yeast species is
known to be sensitive to oxidative damage, which accumulates
in both aging yeast (Reverter-Branchat et al., 2004) and aging
postmitotic tissues of higher organisms, including the nervous
system (Chen et al., 2005). The yeast CLS system has therefore
been billed as a model for the aging of these types of post-
mitotic tissues (MacLean et al., 2001; Chen et al., 2005). Even
though CLS measures longevity of nondividing cells, previous
studies have found similarities with replicatively aging cells,
including sensitivity to oxidative stress and negative regulation
by the Sch9p kinase pathway (Fabrizio et al., 2004). Furthermore,
old cells in both systems die through an apoptosis-like pathway
(Laun et al., 2001; Herker et al., 2004). There are also significant
differences between the two types of yeast aging, including
opposite regulation by Ras2p (Sun et al., 1994; Longo, 2004)
and now Sir2p (this study and Fabrizio et al., 2005).
There are several ways in which CLS experiments can be
carried out. Our laboratory grows the strains in 10-mL SC cultures
slowly rotating on a tilted wheel. Once the cells reach stationary
phase, they are left in the expired media. Some laboratories
transfer the stationary phase cells to sterile water to measure
CLS, which greatly extends the lifespan (Fabrizio & Longo,
2003). In fact, the transfer to water has been used as an extreme
form of CR (Fabrizio et al., 2005). Other laboratories grow
strains in glycerol to maximize the lifespan (Piper et al., 2006),
which, as we have also determined, extends lifespan in this
study. The spot test method of determining cell viability in the
aging cultures has advantages and disadvantages. The big
advantage is the simplicity and the ability to monitor the lifespan
of a large number of individual mutants or growth conditions
at the same time. The assay is also as consistent as the traditional
colony counting method in determining maximum lifespans. A
disadvantage is in quantitation of relatively small lifespan effects
where calculation of a mean lifespan would be needed. Large
effects like that of CR are within the spot test assay’s capacity.
We were initially surprised that CR extended yeast CLS
because the glucose in these cultures is rapidly utilized before
they reach stationary phase. However, the longevity phenotype
is not noticed until almost 1 week later, when the glucose is
depleted. This result strongly suggests that cellular changes
occur during the early growth phase of the culture that impact
on cell viability later in the experiment. In contrast, calorie-
restricted cells in replicative aging experiments grow on agar
plates with a reduced glucose concentration that remains
relatively constant throughout the experiment, a difference
that provides a plausible explanation for why gpa2∆ and
hxk2∆ mutations extend RLS, but not CLS. For the CLS system,
any effect on glucose metabolism or sensing would be limited
to the initial growth phase of the culture, and not to changes
occurring later in the experiment.
Even though the carbon source concentration is reduced by
75% when cells are grown under the CR condition, the resulting
culture density, as measured by spectrophotometry, is not
reduced by 75% during the CLS experiments. This suggests that
stored carbon sources such as glycogen and trehalose, and
perhaps amino acids, are utilized at an elevated rate during CR.
Support for this hypothesis comes from previous studies in which
gluconeogenic genes were up-regulated in calorie-restricted
mice and replicatively old yeast cells (Lee et al., 1999; Lin et al.,2002). Gluconeogenesis is also induced in yeast cells when they
enter the diauxic shift and stationary phase (DeRisi et al., 1997).
CR may enhance this transition to gluconeogenesis.
One of the main conclusions of this study is that SIR2 is not
required for the extension of lifespan triggered by CR in the
BY4741 strain background when cultured using the conditions
in our study. Previous work from the Longo laboratory
showed that deleting SIR2 from BY4741 caused a slightly
Calorie restriction extends yeast chronological lifespan, D. L. Smith et al.
© 2007 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
658
shorter lifespan during the early part of the growth curve when
grown in SDC minimal medium with an initial 2% glucose
concentration, and slightly longer later on in the curve (Fabrizio
et al., 2005). The subtle differences between our sir2∆ results
and the Longo laboratory’s sir2∆ results could potentially be due
to the differences between the synthetic medium composition
and the long-term culturing conditions. It is also possible that
the minor extension in CLS that we observe with the sir2∆mutant is related to the effect of deleting SIR2 on CLS during
extreme CR conditions in water, which greatly extends CLS
(Fabrizio et al., 2005). A lack of SIR2 causes uptake and catab-
olism of ethanol and up-regulation of many stress-resistance
genes, consistent with improved viability over time (Fabrizio
et al., 2005). Ethanol was previously shown to shorten the
lifespan of BY4741 when added to synthetic media, but we
observed lifespan extension when ethanol was the sole carbon
source (Fig. 7B). Perhaps ethanol is only toxic when cells are
preferentially utilizing glucose.
Unlike SIR2, the HST1 and HST3 genes appear to function in
chronological longevity when cells are grown in SC media, with
each deletion mutant having a shorter lifespan than BY4741
(Fig. 3A). However, the HSTs are still not required for the CR-
mediated lifespan extension. At this time we cannot rule out
the possibility that there is redundancy between SIR2 and the
HSTs, and that cells lacking specific combinations of the NAD+-
dependent protein deacetylases would be nonresponsive to CR.
We do not favor this idea because redundancy between SIR2and the HSTs (specifically HST2) in replicative lifespan has cen-
tered on the suppression of rDNA recombination (Lamming
et al., 2005), which does not affect CLS. Our data therefore lend
support to a model in which yeast SIR2 positively functions in
longevity through the suppression of rDNA recombination and
ERC formation. For replicative aging, the cells are highly sen-
sitive to ERC accumulation and are therefore reliant on SIR2for longevity. For chronological aging, ERCs do not appear to
influence lifespan, and SIR2 is therefore dispensable. This SIR2-
independent CR pathway that occurs for yeast CLS could be
related to a putative ERC/SIR2/HST–independent longevity
pathway that has been proposed for the RLS system (Kaeberlein
et al., 2004). While the role of SIR2 in mediating CR in RLS
remains controversial, it is now becoming clear that SIR2 does
not mediate the CR effects on yeast CLS (this study and Agarwal
et al., 2005; Fabrizio et al., 2005).
What is the mechanism of CR-mediated lifespan extension?
The benefits of CR on lifespan and health are likely due to
changes in multiple cellular processes and pathways, but there
may be specific aspects that are more critical than others. Our
results illustrate that CR by glucose limitation is not exclusively
working through a stress-induced pathway and that CR can be
protective against harmful cellular stress. Hyperosmotic stress
(1 M sorbitol) has previously been shown to extend yeast RLS,
but combined with CR (0.5% glucose), did not result in any
additional RLS lifespan benefit (Kaeberlein et al., 2002), indicating
that the work through the same pathway that was postulated
to lead to Sir2p activation. In contrast, even though 1 M sorbitol
extended CLS, reduction of the glucose concentration to
0.5% extended lifespan further (Fig. 5B), consistent with the
two processes working through independent pathways.
Interestingly, CR was even able to suppress the short lifespan
caused by elevated temperature (Fig. 5C) and oxidative stress
(Agarwal et al., 2005) consistent with the disease-protective
effects of CR known to occur in higher eukaryotes.
The extended lifespan caused by many of the alternative
carbon sources even at NR concentrations provides another
possible clue. The relatively inefficient utilization of sugars such
as maltose and trehalose could be a form of CR that becomes
more pronounced when their concentration in the starting
growth medium is further reduced. Of particular interest are the
nonfermentables and raffinose, each of which extended lifespan
at normal concentrations, but reducing their concentrations by
66.7 or 75%, respectively, provided no further benefit. Growth
on these three carbon sources can therefore be considered as
mimicing CR effects on CLS. Growth in these carbon sources
may induce changes in gene expression and/or metabolism that
partially overlap with those that occur under typical glucose CR
growth conditions. One such change that occurs with glycerol
or ethanol is a switch from fermentation to respiration. This shift
occurs in replicatively aging yeast cells upon glucose CR (Lin
et al., 2002). In addition, CR induces mitochondrial biogenesis
in mammalian cells (Nisoli et al., 2005; Lopez-Lluch et al., 2006).
However, the link between CR and respiration in yeast RLS has
come under scrutiny because CR-mediated extension of RLS was
recently shown to still work in rho0 petite yeast strains that are
respiration defective (Kaeberlein et al., 2005). Antimycin A
sensitivity results from our current study demonstrate a strong
correlation between the effectiveness of a particular carbon
source for CLS extension and the utilization of the carbon source
in respiration. This could be related to the fact that proper entry
into stationary phase requires efficient mitochondrial function
and respiration, similar to the effect of pregrowing cultures in
glycerol for long-term CLS determination in water (MacLean
et al., 2001). Improper stationary phase entry would impair
long-term cell survival. Further work in the CLS system will help
elucidate additional CR mechanisms. Together, the data in this
study underscore the utility of the yeast CLS system for dissecting
the general mechanism(s) of CR-mediated lifespan extension
and cellular stress resistance.
Experimental procedures
Strains and growth media
All strains used in this study were derived from the FY2 back-
ground, which is a direct descendent of S288C (Brachmann
et al., 1998), and are listed in Table 1. Gene deletions were
created using polymerase chain reaction (PCR)-mediated one-
step gene replacement with the dominant drug resistance gene,
Calorie restriction extends yeast chronological lifespan, D. L. Smith et al.
© 2007 The AuthorsJournal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
659
kanMX4. The strains were obtained either from the haploid
Yeast Knockout collection from Invitrogen (Carlsbad, CA, USA),
or from sporulation of strains from the heterozygous diploid
collection (Winzeler et al., 1999). Double mutant combinations
were generated by genetic crossing and tetrad dissections.
Within each experiment the strains are congenic except for the
deletion mutations in question. To test the effect of increased
SIR2 dosage, an empty LEU2 vector [pASC405; derivative of
pRS405 (Christianson et al., 1992)] or a similar plasmid con-
taining the SIR2 gene [pSB760 (Buck et al., 2002)] were digested
with BstEII, which cuts within the LEU2 gene, and integrated
into the leu2∆1 locus of SY108 (see Table 1 for genotype),
producing DSY253 and DSY273, respectively. To test for rDNA
silencing, the same BstEII-digested plasmids were integrated
into the leu2∆1 locus of JS201, which contains a Ty1-mURA3silencing reporter (Smith & Boeke, 1997) within the NTS2
sequence of a single rDNA repeat.
All chronological aging assays were performed with SC media
[1.5 g L–1 yeast nitrogen base w/o amino acids and ammonium
sulfate (Difco); 5 g L–1 ammonium sulfate; 2 g L–1 SC dropout
mix lacking adenine, histidine, leucine, tryptophan and uracil
(Burke et al., 2000)]. All other amino acids, inositol and para-
aminobenzoic acid (PABA) are included in the dropout mix
(Burke et al., 2000). We then supplement the final media
with adenine (0.1 mM), histidine (0.3 mM), leucine (1.66 mM),
tryptophan (0.8 mM) and uracil (0.2 mM) to complement the
components missing from the SC-5 dropout mix. See Burke
et al. (2000) for the concentrations of other amino acids and
dropout mix components. Carbon sources were added to the
concentrations indicated in the results section. For glucose,
fructose, galactose, sucrose, trehalose, maltose and raffinose,
2% (w/v) was used as the NR concentration and 0.5% was used
as the calorie-restricted concentration. Glycerol and ethanol were
used at 3% (NR) and 1% (CR) concentrations. SC agar medium
was supplemented with 1 µg mL–1 antimycin A where indicated,
along with the indicated concentration of carbon source variety.
CLS assays
For the spot test assays, strains that had been patched onto rich
YPD [Bacto yeast extract (10 g L–1), Bacto peptone (20 g L–1),
tryptophan (0.32 g L–1), 2% glucose] plates were inoculated
into 10 mL of SC media in 18-mm glass culture tubes with metal
caps. These starter cultures were grown overnight on a rotating
roller drum (model TC-7; New Brunswick Scientific, Edison, NJ,
USA) such that the tubes were vertically tilted ~10° from
horizontal and rotating at ~50 r.p.m. to maintain the cells in
suspension. The roller drum was positioned within a Fisher
Isotemp Standard Capacity Refrigerated Incubator set at 30 °C,
unless stated otherwise. One hundred microliters of the
overnight culture was then inoculated into a fresh 10-mL SC
culture and incubated on the roller drum for the duration of the
experiment, except for brief times that cultures were removed
for plating of aliquots. Twenty-four hours later was considered
as the first time-point (day 1). At day 1 and every 2–4 days there-
after, 20 µL aliquots were removed from the cultures and serially
Table 1 Yeast strains used in the study
Strain Genotype Figures
BY4741* MATa his3∆1 leu2∆0 met15∆0 ura3∆0 1, 3, 4, 5, 6, 7, S1, S2
DSY233 MATa his3∆1 leu2∆0 met15∆0 ura3∆0 sir2∆::kanMX4 1, S2
DSY253 MATa his3∆200 lys2∆202 trp1∆63 ura3-52 leu2∆1::pASC405 (empty LEU2 vector) 2B
DSY273 MATa his3∆200 lys2∆202 trp1∆63 ura3-52 leu2∆1::pSB760 (LEU2-SIR2 vector) 2B
DSY278 MATα his3∆1 leu2∆0 ura3∆0 2A
DSY281 MATα his3∆1 leu2∆0 ura3∆0 sir2∆::kanMX4 2A
DSY283 MATα his3∆1 leu2∆0 ura3∆0 fob1∆::kanMX4 sir2∆::kanMX4 2A
DSY291 MATα his3∆1 leu2∆0 ura3∆0 fob1∆::kanMX4 2A
DSY312 MATa his3∆1 leu2∆0 met15∆0 ura3∆0 gpa2∆::kanMX4 6A
DSY314 MATa his3∆1 leu2∆0 met15∆0 ura3∆0 hxk2∆::kanMX4 6A
JS201 MATα his3∆200 leu2∆1 lys2∆202 trp1∆63 ura3–52 RDN1(NTS2)::Ty1-mURA3 2C
JS204 JS201 sir2∆::HIS3 2C
JS1121 JS201 leu2∆1::pASC405 (empty LEU2 vector) 2C,D
JS1122 JS201 leu2∆1::pSB760 (LEU2 SIR2 vector) 2C,D
SY8† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 bna1∆::kanMX4 4
SY10† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 pnc1∆::kanMX4 4
SY16† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 npt1∆::kanMX4 4
SY108 MATa his3∆200 lys2∆202 trp1∆63 ura3-52 leu2∆1
SY163† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 hst1∆::kanMX4 3B
SY164† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 hst2∆::kanMX4 3B
SY165† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 hst3∆::kanMX4 3B
SY166† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 hst4∆::kanMX4 3B
SY386† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 emi1∆::kanMX4 S2
*Brachmann et al. (1998).
†Winzeler et al. (1999)
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660
diluted 1 : 10 in sterile water in a 96-well clear flat bottom plate.
Therefore, the first spot in each figure is a tenfold dilution of
the culture. Three microliter of each tenfold serial dilution
was spotted onto YPD plates and incubated for 2–3 days to
determine viability based on the colony growth in each spot.
Images of the plates were collected with a gel/plate imaging
system (Alpha Innotech, Inc., San Leandro, CA, USA). At the
end of each experiment, the time course of TIFF images for each
strain or growth condition was spliced together in a composite
image using Adobe Photoshop software for presentation in
the figures.
For quantitative measurements of CFUs, 20 µL aliquots of the
10 mL cultures were removed at the indicated time-points,
diluted in sterile water, spread onto at least three YPD plates
(maximum ~300 colonies per plate), and allowed to grow
into colonies for 3 days. The colonies were then counted and
the number of CFUs per mL calculated. Over time, more
concentrated dilutions had to be spread onto the YPD plates
to compensate for the cell death that was taking place in the
culture tube. There were no differences between culture growth
protocols for the spot assays and CFU assays. Tubes for each
of the individual experiments in the study were positioned at
the same distance from the center of the rotating wheel to
control for the speed of movement.
rDNA silencing assay
Reporter strains were patched onto YPD agar plates and grown
overnight. The cells were then scraped from the agar with a
wooden applicator stick and resuspended in 1 mL of sterile
water. The optical density of each cell suspension was normal-
ized to 1.0 at 600 nm, and then fivefold serially diluted with
water in a 96-well plate. Five microliters of each dilution was
spotted onto SC agar plates as a nonselective control for cell
growth, or SC media lacking uracil (SC-ura) to measure silencing
of the mURA3 reporter gene. Silencing is indicated by poor
growth on the SC-ura plates. Plates were incubated for 2 days
at 30° prior to photography with the Alpha Innotech gel/plate
imaging system.
Protein extraction and Western blotting
JS201, JS204, DSY253 and DSY273 were grown in 5 mL SC
media (2% glucose) to OD600 of 2.0. Cells were harvested by
centrifugation, washed once with ice-cold water, once with
20% TCA, and frozen in liquid nitrogen. The cell pellets were
thawed and resuspended in 0.5 mL 20% TCA. Whole cell
lysates were prepared by shearing with glass beads (0.4 mL glass
beads and 4 × 30 s vortex with 15 s on ice in between). Lysates
were transferred to new microfuge tubes. The beads were
washed two times with 0.5% TCA and these washes were com-
bined with the initial extract. The combined whole cell lysates
were centrifuged at 800 g in an Eppendorf microcentrifuge at
4 °C for 10 min, and pellets resuspended in 1× sample buffer
[50 mM Tris–HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10%
glycerol, 0.0015% bromphenol blue and 5% β-mercaptoethanol].
Samples were heated for 3 min at 100 °C and loaded onto a
10% SDS-polyacrylamide gel electrophoresis (PAGE) gel. After
electrophoresis, the proteins were transferred to Immobilon-P
membranes (Millipore, Beford, MA, USA) and probed with a
1 : 2000 dilution of α-Sir2 antibody (yN-19, Santa Cruz Bio-
technology, Santa Cruz, CA, USA) and a horseradish peroxidase
(HRP)-conjugated secondary antibody. Proteins were detected
using ECL (G.E. Healthcare, Buckinghamshire, England, UK).
A parallel membrane was probed with 1 : 5000 dilution of
α-tubulin antibody (B-5-1-2; Sigma, St. Louis, MO, USA) for a
loading control.
Acknowledgments
We thank Emily Glidden and Min Shin for technical assistance,
and members of the Smith laboratory and Marty Mayo for
helpful discussions. We also thank Dan Burke and Mitch Smith
for assistance with strains. This work was supported by
grants AG022685 and GM075240 to Jeffrey S. Smith from the
National Institutes of Health (NIH) and a pilot project award from
the University of Virginia Institute on Aging. Daniel L. Smith was
supported in part by a Cell and Molecular Biology training grant
from the NIH (GM08136).
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Supplementary material
The following supplementary material is available for this article:
Fig. S1 The individual monosaccharide components of raffinose
do not extend lifespan when used in combination. Chronological
lifespan (CLS) assay in which BY4741 was grown in SC medium
containing 2% glucose, 2% fructose and 2% galactose (non-
restricted; NR) or 0.5% of each sugar (calorie restricted; CR).
Fig. S2 Characterization of the shift from fermentation toward
respiration induced by calorie restriction (CR) or poorly utilized
carbon sources. The strains tested were WT (BY4741), sir2∆(DSY233), and emi1∆ (SY386). Strains were initially grown over-
night as patches on SC plates containing 2% glucose. Cells were
then scraped from the patches, resuspended in water, normalized
for cell number, serially diluted in fivefold increments, and then
spotted (5 µL) onto SC plates containing (A) glycerol/ethanol
(Gly/Eth), maltose (Mal), trehalose (Tre), raffinose (Raff), or (B)
glucose (Glu), fructose (Fru) or sucrose (suc). The carbon source
concentration and the presence or absence of antimycin A
(1 µg mL–1) is indicated for each panel. The Gly/Eth, Mal and Tre
plates were incubated at 30 °C for 5 days, whereas the Glu, Fru,
Suc and Raff plates were incubated for 2 days.
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/
10.1111/j.1474-9726.2007.00326.x
(This link will take you to the article abstract).
Please note: Blackwell Publishing are not responsible for the
content or functionality of any supplementary materials supplied
by the authors. Any queries (other than missing material) should
be directed to the corresponding author for the article.
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