Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of...

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


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.


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


et al.


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.


et al.


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.


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.



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.



655

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.



656

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.



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



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,



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




SY386† MATa his3∆1 leu2∆0 met15∆0 ura3∆0 emi1∆::kanMX4 S2

*Brachmann et al. (1998).

†Winzeler et al. (1999)



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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