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Regulation of Longevity and Stress Resistance by Sch9 in Yeast

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The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling in mammals and functions in a pathway that regulates longevity and stress resistance in Caenorhabditis elegans. We screened for long-lived mutants in nondividing yeastSaccharomyces cerevisiae and identified mutations in adenylate cyclase and SCH9, which is homologous to Akt/PKB, that increase resistance to oxidants and extend life-span by up to threefold. Stress-resistance transcription factors Msn2/Msn4 and protein kinase Rim15 were required for this life-span extension. These results indicate that longevity is associated with increased investment in maintenance and show that highly conserved genes play similar roles in life-span regulation in S. cerevisiae and higher eukaryotes.
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DOI: 10.1126/science.1059497
, 288 (2001); 292Science
et al.Paola Fabrizio,
Sch9 in Yeast
Regulation of Longevity and Stress Resistance by
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Regulation of Longevity and
Stress Resistance by Sch9 in
Yeast
Paola Fabrizio,
1
Fabiola Pozza,
1
Scott D. Pletcher,
2
Christi M. Gendron,
2
Valter D. Longo
1
*
The protein kinase Akt/protein kinase B (PKB) is implicated in insulin signaling
in mammals and functions in a pathway that regulates longevity and stress
resistance in Caenorhabditis elegans. We screened for long-lived mutants in
nondividing yeast Saccharomyces cerevisiae and identified mutations in ade-
nylate cyclase and SCH9, which is homologous to Akt/PKB, that increase re-
sistance to oxidants and extend life-span by up to threefold. Stress-resistance
transcription factors Msn2/Msn4 and protein kinase Rim15 were required for
this life-span extension. These results indicate that longevity is associated with
increased investment in maintenance and show that highly conserved genes
play similar roles in life-span regulation in S. cerevisiae and higher eukaryotes.
Mutations that extend life-span in C. elegans,
Drosophila melanogaster, and mice are asso-
ciated with increased resistance to oxidative
stress (1, 2). However, the mechanisms that
regulate aging in these multicellular organ-
isms are poorly understood. As in higher
eukaryotes, the unicellular yeast Saccharo-
myces cerevisiae undergoes an age-depen-
dent increase in cell dysfunction and mortal-
ity rates (3, 4 ). Aging in yeast is associated
with an enlargement of the cell and a slowing
in the budding rate, and is commonly mea-
sured by counting the number of buds gener-
ated by a single mother cell (replicative life-
span) (5, 6 ). The replicative life-span of yeast
is regulated by the Sir2 protein, which medi-
ates chromatin silencing in a nicotinamide
adenine dinucleotide– dependent manner (6,
7). However, yeast can also age chronologi-
cally as a population of nondividing cells (2,
4, 6). Saccharomyces cerevisiae grown in
complete glucose medium [synthetic com-
plete (SC) medium] stop dividing after 24 to
48 hours and survive for 5 to 7 days while
maintaining high metabolic rates (2, 8, 9), a
situation more akin to their experience in
nature where they are likely to survive as
nondividing populations exposed to scarce
nutrients. For these reasons, and to avoid
extended growth and entry into the hypo-
metabolic stationary phase induced by incu-
bation in the nutrient-richer yeast extract/
peptone/dextrose (YPD) medium (10), our
studies were performed exclusively in SC
medium. The survival of nondividing yeast is
shortened by null mutations in either or both
superoxide dismutases (SODs) (2, 11, 12)
and is modestly extended by overexpressing
the antiapoptotic protein Bcl-2 (8).
To understand the molecular mechanism
that regulates yeast longevity, we transposon-
mutagenized yeast cells and isolated long-lived
mutants (13). Because of the association be-
tween stress resistance and longevity in higher
eukaryotes, we screened for mutants that sur-
vived both a 1-hour heat stress at 52°C and a
9-day treatment with the superoxide-generating
agent paraquat (1 mM). From 2 billion cells
screened, we isolated 4000 thermotolerant col-
onies and 40 paraquat-resistant colonies carry-
ing transposons. From the 4040 stress-resistant
mutants, we isolated nine that were able to
survive to day 9, when most of the wild-type
cells are dead. The only two long-lived mutants
isolated independently in both the paraquat and
heat shock selections, designated Tn3-5 and
Tn3-24, were also the longest lived (Fig. 1A),
suggesting that resistance to multiple stresses is
associated with increased longevity. Allele res-
cue of the mutants revealed that transposons
had integrated in the promoter region of the
Sch9 protein kinase gene (sch9::mTn) (Tn3-5)
(33 base pairs upstream of the start codon) and
in the NH
2
-terminal regulatory region of ade-
nylate cyclase (cyr1::mTn) (Tn3-24) (between
codon 208 and 209). The mean life-spans of
sch9::mTn and cyr1::mTn were extended by 30
and 90%, respectively. Transformation of
Tn3-5 cells with wild-type SCH9, and of Tn3-
24 cells with CYR1, abolished the survival ex-
tension, strongly suggesting that the decreased
expression or activity of Sch9 and Cyr1 extends
survival (not shown).
To investigate further the role of SCH9 in
chronological survival, we deleted the SCH9
gene (14). The sch9 mutants grew slowly, but
survived three times longer than wild-type cells
(Fig. 1B). To determine whether the protein
kinase activity of Sch9 accelerates mortality in
nondividing yeast, we transformed mutants
with either wild-type SCH9 or with forms of
SCH9 bearing kinase-inactivating mutations:
sch9
K441A
and sch9
D556R
(15). Transformation
of sch9 with wild-type SCH9 reversed the
life-span extension, whereas transformation
1
Division of Biogerontology, Andrus Gerontology
Center, and Department of Biological Sciences, Uni-
versity of Southern California, Los Angeles, CA
90089 0191, USA.
2
Max Planck Institute for Demo-
graphic Research, Rostock, 18057 Germany.
*To whom correspondence should be addressed. E-
mail: vlongo@usc.edu
Fig. 1. Mutations in CYR1 and in SCH9
increase chronological life-span of S.
cerevisiae. Survival of (A) the wild
type (DBY746), and transposon-mu-
tagenized cyr1::mTn (Tn3-24) and
sch9::mTn (Tn3-5); (B) the wild type
and sch9;(C) sch9 transformed
with vector alone wild-type SCH9
or with a mutated sch9 encoding
for a catalytically inactive proteins
(Sch9
K441A
, Sch9
D556R
). Cell viability
was measured every 2 days starting at
day3(14). Experiments were repeat-
ed between three and seven times
with two or more samples per exper-
iment with similar results. The aver-
age of all experiments is shown. The
mean life-span increase in cyr1::mTn
(90%), sch9::mTn (30%), and sch9
(300%) is significant [P 0.05, anal-
ysis of variance (ANOVA)].
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with the genes encoding for the inactive
Sch9
K441A
or Sch9
D556R
kinases did not (Fig.
1C).
Both Sch9 and Cyr1 function in path-
ways that mediate glucose-dependent sig-
naling, stimulate growth and glycolysis,
and decrease stress resistance, glycogen ac-
cumulation, and gluconeogenesis (16). The
COOH-terminal region of Sch9 is highly
homologous to the AGC family of serine/
threonine kinases, which includes Akt/
PKB, whereas the NH
2
-terminal region
contains a C2 phospholipid and calcium-
binding motif. The 327–amino acid serine/
threonine kinase domain of yeast Sch9 is,
respectively, 47 and 45% identical to that
of C. elegans AKT-2 and AKT-1, which
function downstream of the insulin-recep-
tor homolog DAF-2 in a longevity/diapause
regulatory pathway (14, 17, 18). In this
domain conserved from yeast to mammals,
Sch9 is also 49% identical to human AKT-
1/AKT-2/PKB, which are implicated in bi-
ological functions including insulin signal-
ing, the translocation of glucose transport-
er, apoptosis, and cellular proliferation
(19).
The CYR1 gene encodes for adenylate
cyclase, which stimulates cyclic adenosine
monophosphate (cAMP)–dependent protein
kinase (PKA) activity required for cell cycle
progression and growth. The catalytic sub-
units of PKA are also 35 to 42% identical to
C. elegans and human AKT-1/AKT-2, al-
though PKA belongs to a different family of
serine/threonine kinase. The inactivation of
the Ras/cAMP/PKA pathway in S. cerevisiae
increases resistance to thermal stress, in part,
by activating transcription factors Msn2 and
Msn4, which induce the expression of genes
encoding for several heat shock proteins,
catalase (CTT1), and the DNA damage induc-
ible gene DDR2 (14, 16). MnSOD also ap-
pears to be regulated in a similar manner (20).
To determine whether MSN2/MSN4 mediate
survival extension, we deleted both genes in
the cyr1::mTn mutants. The absence of both
transcription factors abolished the life-span
extension conferred by cyr1::mTn, but did
not affect the survival of wild-type cells (Fig.
2A). By contrast, the deletion of MSN2/
MSN4 did not reverse the survival extension
in sch9 cells (Fig. 2B).
The protein kinase Rim15 regulates genes
containing a PDS ( postdiauxic shift) element
T(T/A)AG
3
AT involved in the induction of
thermotolerance and starvation resistance by
a Msn2/Msn4-independent mechanism (21).
To test the role of Rim15 in survival, we
generated sch9 rim15 mutants. The life-
span of the double mutant was decreased
compared to sch9 (Fig. 2B). The deletion of
RIM15 also abolished the life-span extension
in cyr1::mTn cells (Fig. 2A). However, it is
difficult to establish whether Rim15 mediates
the survival extension in these mutants, be-
cause rim15 single mutants are short-lived
(Fig. 2A).
To test whether the long-lived strains
were stress-resistant, we exposed the mutants
to hydrogen peroxide, menadione, or heat.
All mutants were resistant to a 1-hour heat
shock treatment at 55°C (Fig. 3A). Similarly,
3- to 5-day-old mutants were resistant to a
30-min treatment with 100 mM hydrogen
peroxide (Fig. 3B) or with the superoxide/
H
2
O
2
-generating agent menadione (20 M)
(Fig. 3C).
In yeast sod2 mutants, superoxide specif-
ically inactivates aconitase and other [4Fe-4S]
cluster enzymes and causes the loss of mito-
chondrial function and cell death (11, 12). To
investigate further the role of superoxide toxic-
ity in aging, we monitored the activity and
reactivation of mitochondrial aconitase, which
can also serve as an indirect measure of super-
oxide concentration (22). In agreement with the
pattern of resistance to superoxide toxicity (Fig.
3C), aconitase specific activity decreased by
50% in wild-type cells, and by 30% in
cyr1::mTn mutants, but did not decrease in
sch9::mTn and sch9 mutants at day 7 com-
pared to day 3 (14 ). The percent reactivation of
aconitase was lowest in the long-lived sch9
mutants and highest in wild-type cells (Fig. 4A)
and correlated with death rates (Fig. 4B), sug-
gesting that cyr1 and sch9 mutants increase
survival, in part, by preventing superoxide tox-
icity. However, the overexpression of both
SOD1 and SOD2 only increases survival by
30% (9), indicating that additional systems, reg-
ulated by Msn2, Msn4, and Rim15, are respon-
sible for the major portion of chronological
life-span extension in cyr1::mTn and sch9
mutants.
There are many phenotypic similarities
between long-lived mutants in S. cerevisiae,
C. elegans, Drosophila, and mice (1, 2). Cae-
Fig. 2. Transcription factors Msn2,
Msn4, and protein kinase Rim15 are
required for the chronological life-
span extension of cyr1::mTn and
sch9 mutants. (A) Survival of the
wild type and cyr1::mTn mutants lack-
ing either the stress-resistance genes
MSN2/MSN4 or RIM15.(B) Survival of
the wild type and sch9 mutants
lacking either MSN2/MSN4 or RIM15.
Experiments were repeated between
three and seven times with two or
more samples per experiment with
similar results. The average of all ex-
periments is shown.
Fig. 3. Heat-shock and oxidative stress resis-
tance are increased in long-lived mutants. (A)
Serial dilutions (1:1 to 1:1000, left to right) of
cells removed from day 1 postdiauxic phase
cultures were spotted onto YPD plates and
incubated at 30°C (control) or 55°C (heat-
shocked) for 1 hour. Pictures were taken after a
4-day incubation at 30°C. The experiment was
performed twice with two or more samples per
experiment with similar results. Cells removed
from days 3 or 5 in the postdiauxic phase were
(B) diluted to an OD
600
(optical density at 600
nm) of 1 in expired medium and incubated with
hydrogen peroxide (100 mM) for 30 min or (C)
diluted to an OD
600
of 0.1 in potassium phos-
phate buffer and treated with 20 Mofthe
superoxide/H
2
O
2
-generating agent menadione
for 60 min. Viability was measured by plating
cells onto YPD plates after the treatment. The
experiments were performed twice with similar
results. The average of the two experiments is
shown.
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norhabditis elegans age-1 and daf-2 mutations
extend the life-span in adult organisms by 65 to
100%, by decreasing AKT-1/AKT2 signaling
and activating transcription factor DAF-16 (14,
18, 23). These changes are associated with the
induction of superoxide dismutase (MnSOD),
catalase, and the heat shock proteins HSP70
and HSP90 (14, 17). A role for oxidants in the
aging of C. elegans was confirmed by the ex-
tended survival of wild-type worms treated
with small synthetic SOD/catalase mimetics
(24). Thus, the yeast Gpr1/Cyr1/PKA/Msn2/4-
Sch9/Rim15 and the C. elegans DAF-2/AGE-
1/AKT/DAF16 pathways play similar roles in
regulating longevity and stress resistance (14 ).
Analogously, a Drosophila line with a mutation
in the heterotrimeric guanosine triphosphate–
binding protein (G protein)–coupled receptor
homolog MTH gene displays a 35% increase in
life-span and is resistant to starvation and para-
quat toxicity (25). Furthermore, in flies,
aconitase undergoes age-dependent oxidation
and inactivation (26), and the overexpression
of SOD1 increases survival by up to 40% (27,
28). A mutation in a signal-transduction gene
also increases resistance to stress and length-
ens survival in mammals. A knockout muta-
tion in the signal transduction p66
SHC
gene
increases resistance to paraquat and hydrogen
peroxide and extends survival by 30% in
mice (29).
We propose that yeast Sch9 and PKA and
worm AKT-1/AKT-2 evolved from common
ancestors that regulated metabolism, stress re-
sistance, and longevity in order to overcome
periods of starvation. Analogous mechanisms
triggered by low nutrients may be responsible
for the extended longevity of dietary restricted
rodents (3). The phenotypic similarities of long-
lived mutants ranging from yeast to mice (1, 2),
and the role of the conserved yeast Sch9 and
PKA and mammalian Akt/PKB in glucose me-
tabolism, raise the possibility that the funda-
mental mechanism of aging may be conserved
from yeast to humans.
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1 February 2001; accepted 15 March 2001
Published online 5 April 2001;
101126/science.1059497
Include this information when citing this paper.
Functional Specialization in
Rhesus Monkey Auditory Cortex
Biao Tian, David Reser, Amy Durham, Alexander Kustov,
Josef P. Rauschecker*
Neurons in the lateral belt areas of rhesus monkey auditory cortex prefer complex
sounds to pure tones, but functional specializations of these multiple maps in the
superior temporal region have not been determined. We tested the specificity
of neurons in the lateral belt with species-specific communication calls pre-
sented at different azimuth positions. We found that neurons in the anterior
belt are more selective for the type of call, whereas neurons in the caudal belt
consistently show the greatest spatial selectivity. These results suggest that
cortical processing of auditory spatial and pattern information is performed in
specialized streams rather than one homogeneously distributed system.
Hearing plays a dual role in the identification
of sounds and in their localization. Although
it is undisputed that auditory cortex partici-
pates in the analysis of spectro-temporal pat-
terns for the identification of complex sound
objects, including speech and music, the neu-
ral basis of auditory spatial perception re-
mains a matter of controversy. Brainstem-
structures play a significant role in the pro-
cessing of binaural cues, which contain im-
portant information for sound localization
(1). However, lesions of auditory cortex also
impair auditory spatial analysis (2, 3). With
the recent discovery of multiple cochleotopic
maps in nonprimary auditory cortex of the
rhesus monkey (4, 5), the question arises
whether neurons in some of these areas show
greater specificity for sound source location
than in others. This could indicate the exis-
tence of a specialized cortical stream for the
processing of auditory space, similar to what
Georgetown Institute for Cognitive and Computa-
tional Sciences, Department of Physiology and Bio-
physics, Georgetown University Medical Center,
Washington, DC 20007, USA.
*To whom correspondence should be addressed. E-
mail: rauschej@georgetown.edu
Fig. 4. Mutations in cyr1
and sch9 delay the re-
versible inactivation of
the superoxide-sensitive
enzyme aconitase in the
mitochondria. (A) Mito-
chondrial aconitase per-
cent reactivation after
treatment of whole-cell
extracts of yeast re-
moved from cultures at
day 5 through 7 with
agents (iron and Na
2
S) able to reactivate superoxide inactivated [4Fe-4S] clusters. (B) Death rate
reported as the fraction of cells that lose viability in the 24-hour period following the indicated day.
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... The effect of the deletion of the gene coding for the Ras2 protein on longevity extension and stress resistance allowed the identification of Ras2 as one of the very first longevity regulatory genes in any organism [28]. RAS2 deletion doubles CLS and increases the resistance to heat and oxidative stresses [29,30] (Figure 1). Ras2 codes for one of the two monomeric G-proteins that are capable, when GTP-bound, of stimulating adenylate cyclase, which in turn activates PKA. ...
... Ras2 codes for one of the two monomeric G-proteins that are capable, when GTP-bound, of stimulating adenylate cyclase, which in turn activates PKA. The involvement of PKA as a pro-aging pathway was further confirmed by the observation that mutants with impaired synthesis of cAMP were also long-lived [29]. The Ras pathway appears to have a pro-aging role conserved from yeast to mammals, since in mice maternal uniparental disomy of the Ras exchange factor coding gene RasGRF (which is inactivated during oogenesis) has been associated with increased longevity [30], a hypothesis confirmed by the lifespan extension in mice carrying homozygous deletion of the gene coding for this nucleotide exchange factor [31]. ...
... The role of this pathway in regulating aging has been since confirmed in worms and flies [41,42]. Inhibition of Tor-S6k activity increases both chronological and replicative lifespan [29,35,43] (Figure 1). Decreased Tor signaling increases respiration [44] as well as mitochondrial protein expression [45], and reactive oxygen species are also increased, likely activating an adaptive response that extends CLS. ...
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S. cerevisiae plays a pivotal role as a model system in understanding the biochemistry and molecular biology of mammals including humans. A considerable portion of our knowledge on the genes and pathways involved in cellular growth, resistance to toxic agents, and death has in fact been generated using this model organism. The yeast chronological lifespan (CLS) is a paradigm to study age-dependent damage and longevity. In combination with powerful genetic screening and high throughput technologies, the CLS has allowed the identification of longevity genes and pathways but has also introduced a unicellular “test tube” model system to identify and study macromolecular and cellular damage leading to diseases. In addition, it has played an important role in studying the nutrients and dietary regimens capable of affecting stress resistance and longevity and allowing the characterization of aging regulatory networks. The parallel description of the pro-aging roles of homologs of RAS, S6 kinase, adenylate cyclase, and Tor in yeast and in higher eukaryotes in S. cerevisiae chronological survival studies is valuable to understand human aging and disease. Here we review work on the S. cerevisiae chronological lifespan with a focus on the genes regulating age-dependent macromolecular damage and longevity extension.
... The Workman group showed that H3T11 was phosphorylated by both Sch9 and Cka1, and reported that Δsch9 and Δcka1 as well as H3T11A prolonged CL [20]. Although this seems opposite to our result where the phosphorylated mimic H3T11D showed CLE, we note that the Δsch9 mutant strain will facilitate activated expression of Msn2/4 stress response genes associated with CLE [42], and it is not clear if the CLE observed was due to absence of H3T11 phosphorylation or induction of stress response genes. A phosphorylated mimic H3T11D was not tested [20]. ...
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We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the tran-scriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism.
... It is worth noting that the CLS analysis begins with the entry of cells into the stationary phase. It has been proposed that, during CLS, two main pathways are activated: the Tor/S6K pathway [63] and the Ras/adenylate cyclase/PKA pathway [64]. Interestingly, all of the studied strains showed a longevity comparable to that of the WT. ...
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