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INTRODUCTION |
Saccharomyces cerevisiae is a unicellular model for
studying the molecular mechanisms of aging. Mother yeast cells
undergo replicative senescence, with different strains having
characteristic mean and maximum life spans (1). Senescence is
associated with a number of cellular and molecular phenotypes.
Progressive sterility arises from the loss of silencing at
HM loci (2). This loss of silencing is accompanied by a
redistribution of Sir3p, a component of the Sir-silencing complex, from
HM loci and telomeres to the nucleolus (3). Homologous
recombination at rDNA loci liberates an extrachromosomal rDNA circle
(ERC)1 that contains an
autonomously replicating sequence (4). With each round of cell
division, replicated ERCs segregate to the mother rather than to
daughter, leading to an exponential increase in cellular ERC
concentrations over successive generations.
ERC formation is thought to be a mediator as well as a marker of aging.
Exponential increases in ERCs likely leads to titration of critical DNA
repair/replication and transcription factors (5). Fob1p specifically
acts at the rDNA locus, where it supports opening of the replication
bubble (6) and binds to HOT1 sites, thereby promoting
homologous recombination. fob1
alleles block rDNA
formation and extend life span
20% beyond wild type (wt) (4).
Mutations, such as sir2
, that reduce rDNA silencing are
associated with increased rates of recombination and accelerated aging
(7, 8). The importance of the regulation of silencing in aging is also
emphasized by the findings that Sir2p is a
NAD+-dependent histone deacetylase (9) and that
NAD+ production leads to Sir2p-dependent life
span extension (10). To catalyze deacetylation, Sir2p converts
NAD+ to nicotinamide and O-acetyl-ADP-ribose
(11, 12). Yeast grown on medium containing nicotinamide, a
noncompetitive Sir2p inhibitor, display Sir2p-dependent
rDNA desilencing and a shortened life span (13).
The effects of NAD+ on Sir2p provide a potential link
between aging and cellular glucose/energy metabolism. They also raise the question of how aging affects upstream elements in glucose-sensing pathways and whether such pathways affect replicative life span.
S. cerevisiae has evolved elaborate and elegant mechanisms
for sensing environmental glucose, its principal nutrient source. A
major glucose-sensing pathway involves Snf1, the homolog of mammalian
AMP-activated protein kinase (AMPK) that is involved in regulating
cellular stress responses (14). Snf1 is a heterotrimeric complex
composed of
,
, and
subunits (15, 16). The complex contains
one of three
subunits, Sip1p, Sip2p, or Gal83p, that binds to the
catalytic
subunit, Snf1p (a serine/threonine kinase) (17). The
subunit, Snf4p (18), binds to an autoinhibitory domain of Snf1p,
releasing its catalytic domain. Phosphorylation of Thr210
in the Snf1p C-terminal activation loop also promotes kinase activity
(19), although the Snf1p kinase kinase has yet to be identified.
The Snf1 complex responds to glucose starvation by catalyzing
phosphorylation of a number of target proteins, including
transcriptional regulators of genes involved in alternative carbon
source utilization, gluconeogenesis, and respiration (20). For example,
when glucose is limiting, Mig1p is hyperphosphorylated by Snf1p causing
it to translocate from the nucleus to the cytoplasm and derepress gene
expression (21).
We have reported that forced expression of SNF1, or loss of
Sip2p (sip2
), produces an accelerated aging phenotype
(i.e. a shortened replicative life span accompanied by
progressive sterility, redistribution of Sir3p from telomeres and
HM loci to the nucleolus, and increased ERC accumulation)
(22). The effect of Sip2p on aging is unique among Snf1
-subunits:
gal83
has no effect on life span and sip1
produces a modest (<20%) reduction in life span but without the other
manifestations of accelerated aging. Loss of the
subunit
(snf4
) produces a 20% increase in life span (22).
Together, these results implied that increased Snf1 activity promotes
rapid aging and that Sip2p functions to repress Snf1p activity. Two
additional observations supported this hypothesis: (i) Mig1p undergoes
progressive redistribution from the nucleus to the cytoplasm as wt
cells and sip2
cells age, and (ii) the rapid-aging
phenotype produced by sip2
is completely rescued by
snf4
(22). However, our studies indicated that the
effects of Snf1 activation on life span must extend beyond its
phosphorylation of Mig1p since mig1
cells did not undergo
rapid aging (22).
Follow-up DNA microarray analysis revealed that a number of
Mig1p-repressed genes were derepressed in generation 7-8 wt and sip2
cells compared with their generation 0-1
counterparts (23). The analysis also identified a number of other genes
with age-associated changes in their expression that are not regulated
by Mig1p but are involved in various aspects of cellular energy
metabolism. Direct biochemical analysis of cellular metabolism
established that aging in wt cells is accompanied by a shift away from
glycolysis and toward gluconeogenesis and energy storage (23). This
shift is deferred in snf4
cells and accelerated in
isogenic sip2
strains leading to a proposal that the
shift toward glucose and energy storage may be a mediator as well as a
marker of aging (23).
Studies of the promoter region of INO1 (inositol-1-phosphate
synthase) showed that Snf1 phosphorylates histone H3 at
Ser10, which facilitates acetylation at Lys14
by Gcn5p (histone acetyltransferase) (24). These findings in young
cells raise the possibility that Snf1 could promote aging by modifying
chromatin structure at specific genomic loci. Therefore, in the current
study, we have explored the mechanisms by which sip2
operates through Snf1 to regulate aging and chromatin structure. We
show that covalent addition of myristate, a 14 carbon-saturated fatty
acid, to the N-terminal Gly residue of Sip2p is essential for its
localization to the plasma membrane and its contribution to a normal
generational life span. As yeast cells age, Snf4p is redistributed from
the plasma membrane to the nucleus. This redistribution, which is
promoted by sip2
, is accompanied by increased cellular
Snf1p histone H3 kinase activity, desilencing, and increased
recombination at rDNA loci with increased ERC formation. The
rapid-aging phenotype of sip2
cells is completely rescued by removing Fob1p, an essential mediator of ERC formation. Together, our results suggest that the glucose-sensing Snf1 pathway regulates aging by affecting chromatin structure and genomic stability.
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EXPERIMENTAL PROCEDURES |
Strains--
The isogenic strains YB332 (wt;
S288CMATaNMT1ura3-52his3
200ade2-101lys2-801leu2-3,112),
YB810 (sip2::HIS3), and YB614
(snf4::HIS3) are described in Ref. 22.
Additional strains were constructed using standard methods (25) and are
listed in Table I.
Life Span Determination--
Micromanipulation assays (26) were
used to define the number of divisions that individual virgin mother
yeast cells undergo on YPD (1% yeast extract, 2% peptone, 2%
dextrose) agar plates at 24 °C (n =
40
mothers/genotype/experiment; n = two to three independent experiments/strain). Wt cells were used as reference controls in each experiment. The statistical significance of observed differences in life spans was evaluated using the nonparametric Wilcoxen signed rank test.
Isolation of Young and Older Yeast Cell Populations--
When
the surface proteins of a mother yeast cell are labeled with biotin,
the daughter does not inherit the biotinylated proteins because its
surface is generated de novo at the budding site (2). This
allows mothers to be isolated from their progeny by
streptavidin-magnetic bead sorting. The protocol used to obtain sorted
populations of generation 0-1 and generation 7-8 or 9-10 cells is
described in Ref. 2. Note that generation 0-1 refers to the population
of cells that remains after old cells are removed by sorting; on average, 50% of this population will be composed of newly formed daughters, while 25% are mothers that have undergone a single cell division.
Defining the Intracellular Localization of Snf1, Snf4, and
Sip2--
Isogenic wt and sip2
cells were transformed
with one of the following four CEN plasmids: pOV84
(Snf1p-green fluorescent protein (GFP fusion)), pOV76 (Snf4p-GFP), pRT9
(Sip2p-GFP) (27), (gifts from Marian Carlson, Columbia Univ.), or
pBB507 (Sip2p (Gly1
Ala)-GFP). Generation 0-1 and 7-8 cells were
recovered by magnetic bead sorting from cultures grown in synthetic
medium containing 2% glucose, suspended in a solution of 8 mM potassium phosphate buffer, pH 7.0, 4 mM
MgSO4, 26 mM ammonium sulfate, and 2% glucose, incubated for 5 min at 24 °C, washed twice in the same solution, and
then examined under a Zeiss Axioplan 2 microscope (n = two to three independent experiments).
Snf1p Histone H3 Kinase Assay--
Snf1p contains an N-terminal
domain with 13 histidine residues
(His18-His30), facilitating its purification
from cell lysates by Ni-NTA-agarose affinity chromatography
(20). Generation 0-1 and 7-8 isogenic wt and sip2
cells
(2-5 × 107; defined by hemocytometer counts) were
recovered from a mid-log phase YPD culture by magnetic bead sorting,
frozen in liquid N2, freeze-dried, and then incubated for 5 min at 1 °C in 1 ml of extraction buffer (20 mM
potassium phosphate buffer, pH 7.4, 0.02% bovine serum albumin, 0.5 mM EDTA, 5 mM
-mercaptoethanol, 25% glycerol, 0.5% Triton X-100, and 50 mM potassium
fluoride). The resulting cell lysate was added directly to 1 ml of
Ni-NTA-agarose (Qiagen) pre-equilibrated with 5 ml of buffer A (0.3 M NaCl, 75 mM sodium phosphate buffer, pH 7.0, and 50 mM potassium fluoride). The mixture was incubated
for 60 min at 4 °C. The resin was then washed with buffer A (three
cycles, 5 ml/cycle) and poured into a column, and Snf1 activity was
eluted with 1 ml of buffer A supplemented with 50 mM
imidazole. Eluted proteins were concentrated to 0.1-0.2 µg/ml
(Centricon 30 filter column).
The scheme for measuring histone H3 kinase activity in the
50-mM imidazole fraction is outlined in Fig. 1A.
A 0.5-µl aliquot of the 50-mM imidazole-eluted fraction
(containing 50-100 ng of protein) was added to 0.5 µl of kinase
reagent (50 mM imidazole-HCl, pH 6.7, 0.04% bovine serum
albumin, 4 mM MgCl2, 100 mM
potassium fluoride, 0.5 mM phosphoenolpyruvate (Sigma), 0.5 mM ATP, 40 µg/ml rabbit muscle pyruvate kinase (specific
activity = 200 units/mg; Roche Molecular Biochemicals))
containing 0.5 mM of a previously published histone H3
peptide substrate for Snf1p (24) (ARTKQTARKSTGGKAPRKQLASKAARC; Biomolecules Midwest, Waterloo, IL). The 1-µl mixture was incubated for 60 min at 20 °C. The reaction was stopped with 0.5 µl of 0.2 M HCl (10 min, 20 °C), and a second reaction initiated
by adding 0.5 µl of a solution containing 200 mM
imidazole-HCl, pH 7.0, 80 mM NaOH, 0.08% bovine serum
albumin, 20 mM EDTA, 0.2 mM NADH, 8 µg/ml
beef heart lactate dehydrogenase (specific activity = 500 units/mg; Sigma). The mixture was incubated for an additional 10 min at
20 °C. 0.5 µl of 0.4 M HCl was then introduced to
destroy any excess NADH.
To detect the NAD+ product generated from the Snf1p histone
H3 kinase reaction (Fig. 1A),
the product had to be amplified through a series of coupled reactions
(for details of this well established analytic method, see Refs. 23 and
28). Five thousand cycles of amplification were achieved by adding 0.5 µl of the reaction mixture to 0.1 ml of NAD cycling reagent
(100 mM Tris-HCl, pH 8.1, 2 mM oxaloacetic
acid, 2 mM
-mercaptoethanol, 0.3 M ethanol, 0.02% bovine serum albumin, 12.5 µg/ml alcohol dehydrogenase
(specific activity = 300 units/mg; Sigma), 1.2 µg/ml malic
dehydrogenase (specific activity = 3000 units/mg; Sigma)) and
incubating the solution for 60 min at 24 °C. The reaction was
terminated (100 °C; 5 min), cooled to room temperature, and 1 ml of
malate indicator reagent (50 mM amino-methylpropanol (pH
9.9), 5 mM L-glutamate (pH 9.9), 0.2 mM NAD+, 5 µg/ml malic dehydrogenase (3000 units/mg; Sigma), and 2 µg/ml glutamate oxaloacetate transaminase
(200 units/mg; Roche Molecular Biochemicals)) was added to convert the
cycled product to NADH (23).

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Fig. 1.
Snf1p kinase assay. A,
schematic outline of assay. B, dependence of the assay on
time and amount of input Snf1p, recovered by Ni-NTA-agarose
chromatography from generation 0-1 wt cell lysates. Two 26-residue
peptides were assayed in parallel: one contained the Ser10
phosphorylation site of histone H3
(1ARTKQTARKSTGGKAPRKQLASKAARC); the other was a
prephosphorylated synthetic derivative
(1ARTKQTARKpSTGGKAPRKQLASKAARC) that was used to define
specificity of the Snf1p kinase reaction for Ser10. All
assays were done in duplicate. The results shown are representative of
two independent experiments using different Snf1p preparations.
C, double reciprocal plot determination of the
Km of the histone H3 peptide substrate.
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Several types of reference standards and controls were run in parallel
with the experimental reaction. NAD+ standards were always
added at the cycling step into cycling reagent alone. Known amounts of
malate was included as another reference control. A minus peptide
reaction, and a reaction containing a synthetic phosphorylated histone
H3 peptide (1ARTKQTARKpSTGGKAPRKQLASKAARC;
Biomolecules Midwest) were used to confirm Ser10-specific
phosphorylation. (Note that we were unable to detect Snf1p-catalyzed
phosphorylation of the H3 peptide using unfractionated whole
cell lysates due to the high background signal, as defined with the
histone H3 peptide and its phospho-Ser10 derivative).
ERC Analysis--
DNA was prepared from sorted generation 0-1
and 7-8 cells according to Sinclair and Guarente (5) with one
exception: no zymolyase was used in the first step; instead, 0.5 ml of
sorbitol solution (0.9 M sorbitol, 0.1 M Tris,
pH 8.0, 0.1 M EDTA) was added to freeze-dried cell pellets.
Purified DNA was fractionated by electrophoresis through 0.7% agarose
containing TAE buffer (40 mM Tris acetate, 1 mM
EDTA, pH 7.5). Following capillary transfer to GeneScreen Plus
(Invitrogen), blots were probed with a 32P-labeled, 2.8-kB
EcoRI fragment containing rDNA sequences from pNL47 (5).
Recombination and Silencing Assays--
Assays for recombination
and silencing used PSY316a strains containing ADE2
integrated into RDN1 or telomeric loci (Table I). To measure
recombination rates, cells with a 2µ plasmid containing SNF1 under the control of its own promoter (21), or the
empty YEp24 vector, were spread onto YPD/agar plates and incubated for 30 °C for 3 days. Recombination frequency was defined as the
number of half-sectored red/white colonies divided by the total number of colonies (n = >104 colony
scored/strain; n = four independent experiments). To
measure silencing, cells were grown on synthetic complete medium.
Individual colonies were picked and transferred to 1 ml of PBS. Cells
were serially diluted in PBS and plated onto synthetic medium minus adenine. Plates were incubated for 20 h at 30 °C, and the
number of viable colonies scored per dilution.
Microanalytic Biochemical Assays--
Levels of fructose
1,6-bisphosphatase, glycogen, NAD+, ATP, and AMP were
assayed in young and old isogenic wt, sip2
,
fob1
, and sip2
/fob1
cells using pyridine
nucleotide-based enzyme-cycling methods. These previously described,
sensitive methods permit analysis of enzymes and metabolites in a
single sample of 106 sorted cells (23, 28, 29).
Gene Expression Profiles of Young and Old Cells--
Details of
this analysis are provided in an earlier publication (23). Briefly,
RNAs were prepared from sorted generation 0-1 and 7-8 wt and
sip2
cells (5 × 107 cells/sort;
n = three independent sorts from separate cultures). Equivalent amounts of RNA from generation-matched cells from each sort
of a given strain were pooled. Two cRNA targets were independently prepared from each RNA pool, and each cRNA used to interrogate a high
density oligonucleotide-based Ye98 GeneChip (23). Pairwise comparisons
of expression levels in old versus young cells were performed for both strains using proprietary GeneChip software (Affymetrix).
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RESULTS |
Age-associated Changes in the Intracellular Distribution of
Components of the Snf1p Kinase Complex--
We began our analysis of
the mechanisms by which Snf1 affects aging by characterizing the
intracellular distributions of its Snf1p
subunit, Sip2p
subunit, and Snf4p
subunit in generation 0-1 and 9-10 wt cells
that had been grown in synthetic medium containing 2% glucose. Snf1p,
Snf4p, and Sip2p with C-terminal GFP reporters were all expressed from
low copy CEN plasmids under the control of their own
promoters. Previous studies have shown that each of these constructs
fully rescues the growth phenotypes produced by the null alleles of the
corresponding gene in log phase cells (27). This rescue suggests that
the GFP fusions accurately represent the trafficking patterns of the
wild type proteins.
The majority of Snf1p-GFP is present in the cytoplasm of young
(generation 0-1) cells, with a small fraction appearing in the
nucleus. By generation 9-10, there is a modest shift in the protein to
the nucleus (Fig. 2A).
Sip2p-GFP is largely associated with the plasma membrane in young
cells. As cells age, there is increased representation in the cytoplasm
of a subset of cells (Fig. 2B). Like Sip2p-GFP, Snf4p-GFP is
plasma membrane-bound in young cells, although there is also
cytoplasmic localization. With age, Snf4p-GFP shifts away from the
plasma membrane to the cytoplasm and nucleus (Fig. 2C).

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Fig. 2.
Localization of Snf1 complex subunits
in young and aged cells. A-D, Wt cells
containing a CEN plasmid encoding either Snf1p-GFP,
Sip2p-GFP, Snf4p-GFP, or a N-myristoylation-defective Sip2p
(Gly1 Ala) mutant were grown in synthetic medium
containing 2% glucose, and generation 0-1 and/or 9-10 cells isolated
by magnetic bead sorting. Note that with increasing age, Snf4p is
redistributed from the plasma membrane to the nucleus. Loss of the
myristoyl moiety from Sip2p abrogates its ability to affiliate with the
plasma membrane. E, studies of sip2 cells
showing the effect of loss of myristoylSip2p on Snf4p localization in
generation 0-1 cells. Comparison with C reveals that Sip2p
is a key contributor to the plasma membrane association of Snf4p.
Bars, 2.5 µm.
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Our previous in vitro studies had established that Sip2p is
substrate for myristoyl-CoA:protein
N-myristoyltransferase (Nmt1p) (30). Nmt1p catalyzes the
transfer of tetradecanoate (myristate; C14:0) from CoA to the
N-terminal Gly1 residues of ~70 yeast proteins (30). This
modification occurs co-translationally and appears to be irreversible.
N-Myristoylation is known to promote protein-membrane and
protein-protein interactions that typically involve components of
signal transduction cascades (e.g. kinases, kinase
substrates, protein phosphatases, and
subunits of heterotrimeric G
proteins; reviewed in Ref. 31).
We examined the contribution of N-myristoylation to the
plasma membrane targeting of Sip2p. Substituting the Gly1
of Nmt1p substrates with Ala is sufficient to completely block their
N-myristoylation (31). Therefore, we mutated
Gly1 of Sip2-GFP to Ala, yielding Sip2G1A-GFP. Studies of
generation 0-1 wt cells containing a sip2G1A-GFP CEN
episome revealed that loss of the myristoyl moiety changed the protein
distribution from the plasma membrane to the cytoplasm and nucleus
(Fig. 2D).
Comparison of isogenic generation 0-1 wt and sip2
cells
containing SNF4-GFP or SNF1-GFP CEN episomes
disclosed that in the absence of Sip2p, Snf4p was redistributed from
the plasma membrane to the cytoplasm and nucleus (Fig. 2E).
The distribution of Snf1p was not affected; it remained principally
cytoplasmic with a small fraction in the nucleus (data not shown).
Based on these findings, we conclude that N-myristoylation
of Sip2p is essential for its plasma membrane localization, as well as
the plasma membrane localization of Snf4p, and that loss of Sip2p from
the plasma membrane allowed Snf4p to enter the nucleus.
We next assessed the impact of N-myristoylation of Sip2p on
cellular life span. To do so, CEN episomes containing
SIP2 or sip2-G1A were introduced into
sip2
cells. Wt cells as well as sip2
cells
with an empty CEN episome served as controls. The SIP2 episome completely rescued the shortened generational
life span of sip2
cells, while sip2G1A, or the
vector alone, had no effect (Fig. 3).
Thus, N-myristoylation of Sip2p is required for a normal
cellular life span.

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Fig. 3.
Genetic evidence that
N-myristoylation of Sip2p is essential for a normal
cellular life span. Life spans of sip2 cells
containing SIP2 or sip2G1A CEN episomes or the
empty vector.
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Snf1p-directed Histone H3 Kinase Activity Increases in Aging Wt
Cells--
To further define the effects of the observed
age-associated redistribution of Snf1 complex components to the
nucleus, we developed a sensitive in vitro assay to measure
Snf1p histone H3 kinase activity in small numbers (~3 × 104) of sorted generation 0-1 and 7-8 wt and
sip2
cells. Snf1p can catalyze phosphorylation of
Ser10 in a 26-amino acid peptide acceptor representing
Ala1-Arg26 of S. cerevisiae histone
H3 (ARTKQTARKSTGGKAPRKQLASKAARC; Ref. 24). Our assay was linear with
time, and with H3 peptide substrate and input protein concentration
(Fig. 1B). The specificity of the Snf1 kinase for
Ser10 was confirmed in control reactions that either
contained no peptide, or the preformed phosphopeptide
(1ARTKQTARKpSTGGKAPRKQLASKAARC; e.g. Fig.
1B) The Km for the H3 peptide (163 µM; Fig. 1C) is significantly better than the
Km of a peptide that contains the phosphorylation site of another known Snf1p target, acetyl-CoA carboxylase
(HMRSAMSGLHLVKRR; Km = 1000 µM).
The in vitro assay disclosed that Snf1 histone H3 kinase
activity was 2-fold higher in generation 7-8 compared generation 0-1
wt cells (p < 0.05; Fig.
4A). Moreover, the activity in
young (generation 0-1) sip2
cells was up to 3-fold
higher than young wt cells. Levels did not increase significantly as
sip2
cells aged to generation 7-8 (Fig.
4A).

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Fig. 4.
Effects of aging on Snf1-catalyzed
Ser10 phosphorylation of histone H3. A,
histone H3 kinase assay of Snf1p recovered from generation 0-1 and
7-8 wt and sip2 cell lysates. n, number of
determinations on independently sorted cell populations. Mean
values ± S.D. are plotted. An asterisk indicates that
kinase activity is significantly different (p < 0.05, Students' t test) compared with generation 0-1 wt cells.
B, ATP and AMP concentrations in generation 0-1 and 7-8
cells. Mean values ± S.E. are plotted (n = three
to six independent determinations, each in duplicate).
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Like its mammalian homolog AMPK, Snf1 may be activated by the low
ATP/AMP ratios that result from impaired glycolytic activity during
starvation (20). However, the difference in histone H3 kinase levels
observed between wt and sip2
cells was not
attributable to differences in their ATP:AMP ratios (Fig.
4B).
These results establish that the redistribution of Snf1 complex
components to the nucleus in aging wt cells and the shift in
Snf1p/Snf4p to the nucleus in young cells lacking plasma
membrane-associated N-myristoylated Sip2p are associated
with increased cellular levels of histone H3 kinase activity. Together
with the Snf1 subunit localization data presented above, our findings
indicate that (i) the Sip2p
subunit functions as a negative
regulator of nuclear Snf1 activity in young cells by sequestering its
activating Snf4p
subunit at the plasma membrane and (ii) this
inhibition is overcome in aging cells through a shuttling of Sip2p from
the plasma membrane to the cytoplasm and a concomitant rise in nuclear
Snf4p/ Snf1p.
Snf1p Promotes Recombination and Desilencing--
We next examined
whether the observed in vitro changes in Snf1p-catalyzed
histone H3 kinase activity were accompanied by in vivo
alterations in chromatin structure at rDNA loci. Fob1p regulates recombination at rDNA repeats (6), and fob1
produces
decreased ERC formation and increased replicative life span in wt
strains (4). Therefore, we introduced a fob1
allele into
sip2
cells. Removal of Fob1p not only rescued the
shortened generational life span of sip2
cells, but
extended it beyond wt to levels observed with the isogenic
fob1
strain (125% of wt; Fig.
5A). ERC levels in generation
7-8 sip2
fob1
cells were also equivalent to that of
fob1
alone (Fig. 5B). Finally, the life span
of the snf4
fob1
strain did not extend beyond
fob1
alone (data not shown). These findings indicate the
Snf1 pathway is a regulator of ERC formation and that recombination at
rDNA loci is an essential component of the rapid-aging phenotype
observed in sip2
cells.

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Fig. 5.
Deletion of FOB1 rescues
the rapid-aging phenotype of sip2 cells.
A, life spans of the isogenic wt, sip2 ,
fob1 , and sip2 fob1 strains.
B, Southern blot analysis of ERC levels in sorted generation
0-1 and 7-8 cells. Blots were probed with a radiolabeled rDNA
probe.
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Additional in vivo proof that Snf1p itself is a positive
regulator of recombination at rDNA loci came from an independent assay
of recombination in generation 0-1 wt cells with a high copy 2µ
plasmid containing SNF1 under the control of its own
promoter. Recombination was monitored by scoring loss of an integrated
ADE2 reporter from the rDNA locus (10). Recombination causes
ADE2 to become a part of the ERC. Since ERCs only segregate
to the mother, all daughter cells will appear red on YPD medium due to their adenine auxotrophy. Loss of the ADE2 marker after the
first cell division results in a "half-sectored" colony,
i.e. one that is half-red and half-white. Based on this
assay, wt cells with the SNF1-containing episome have a
statistically significant (p < 0.05) 4-fold higher
rate of recombination at the rDNA locus compared to cells with the
empty 2µ vector (Fig.
6A).

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Fig. 6.
Forced expression of SNF1 causes desilencing
and increased recombination at the rDNA locus. A, rDNA
recombination assay. The rDNA:ADE2 strain was plated on
YPD/agar at a dilution that allowed individual colonies to be scored
for red/white sectoring. Recombination frequency was defined as the
ratio of half-sectored red colonies to total colonies. B, a
high-copy SNF1-containing episome or the empty 2µ vector
was introduced into PSY316a cells with ADE2 placed at a rDNA
locus. Cells were serially diluted and spotted onto synthetic medium
with or without adenine.
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Further evidence that Snf1p affects chromatin structure at the rDNA
locus came from a silencing assay. This assay is based on the finding
that loss of silencing at rDNA containing an ADE2 marker
results in increased expression of the integrated gene (7) and faster
growth on synthetic complete medium lacking adenine. Forced expression
of Snf1p promoted growth of wt cells on synthetic complete medium minus
adenine, while control experiments showed that there was no difference
in growth on medium with adenine (Fig. 6B). A similar result
was obtained with a strain that contained ADE2 integrated
into a telomeric region (data not shown).
Results obtained from a GeneChip analysis of generation 0-1 and 7-8
isogenic wt and sip2
cells provided a final set of
observations supporting the notion that age-associated increases in
Snf1p-mediated histone H3 phosphorylation/acetylation is accompanied by
changes in chromatin structure and desilencing at sites other than rDNA loci. Levels of Ino1p mRNA are 23-fold higher in old
versus young wt cells and 14-fold higher in old
versus young sip2
cells (Table II). Previous studies of young cells (32)
indicated that in the absence of glucose, Snf1-mediated phosphorylation
of histone H3 regulates binding of the transcriptional activator Adr1p
to its target gene promoters, leading to induction of ADH2
(ADHII isozyme that catalyzes the first step in ethanol metabolism), ACS1 (acetyl-CoA synthetase), and POT1
(3-oxoacyl-CoA thiolase) expression. Our GeneChip analysis disclosed
that each of these mRNAs rises in aging wt and/or
sip2
cells (Table II).
The Metabolic Shift Observed in sip2
Cells Also Occurs in
sip2
fob1
Cells--
Snf1 promotes glycogen production by
activating Glc7p, a protein phosphatase that activates glycogen
synthase (33). We found that the rapid-aging phenotype of
sip2
cells is associated with a metabolic shift toward
gluconeogenesis and glycogen storage (23). This shift is also a natural
consequence of aging. Wt cells accumulate glycogen as they undergo
replicative senescence; the increase is equivalent to that observed in
sip2
cells that have traversed an equivalent percentage
of their mean generational life span (Fig.
7A).

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Fig. 7.
fob1 rescues the rapid-aging
phenotype of sip2 cells without affecting the age-associated shift
toward glucose storage. A, glycogen concentrations in
generation 0-1 and generation 7-8 wt, sip2 ,
fob1 , and sip2 fob1 cells recovered from
YPD medium by magnetic bead sorting. Generation 18-21 cells were
harvested by micromanipulation directly from YPD/agar plates
(n = 59 cells assayed). B, fructose
1,6-bisphosphatase levels. C, total cellular
NAD+ concentrations. Mean values ± S.E. are plotted
(n = three to six independent determinations for sorted
populations, each in duplicate).
|
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We had speculated that the shift toward gluconeogenesis, glucose
storage, and energy conservation may be a mediator as well as a marker
of aging since it was forestalled in longer-lived snf4
cells. The extended life span of the sip2
fob1
strain
allowed us to explore this hypothesis further.
Populations of sorted generation 0-1 and 7-8 cells were prepared from
isogenic wt, sip2
, fob1
, and
sip2
fob1
strains grown in YPD medium. Well established
microanalytic biochemical assays (23) revealed that introduction of
fob1
does not ameliorate the marked age-associated
increase in fructose 1,6-bisphosphatase and glycogen levels prompted by
sip2
, nor does it significantly change total cellular
NAD+ levels (Fig. 7, A-C). In addition,
pgm1
cells (phosphoglucomutase; converts glucose
6-phosphate to glucose 1-phosphate) have a normal life span (data not
shown). We concluded that the metabolic shift toward gluconeogenesis is
a marker but not necessarily a mediator of aging.
 |
DISCUSSION |
Our studies have revealed a mechanism that links the Snf1
glucose-sensing pathway in S. cerevisiae to modification of
chromatin structure and aging. An age-associated increase in Snf1
histone H3 kinase activity occurs coincident with a shift in its
subunit from the plasma membrane to the cytoplasm and its activating
subunit (Snf4p) from the plasma membrane to the nucleus. These shifts in subunit localization are accompanied by age-associated increases in recombination at rDNA loci (generating ERCs) and augmented
desilencing at rDNA loci, telomeres, as well as sites known in younger
cells to be affected by Snf1p-catalyzed phosphorylation of histone H3.
Several observations established that N-myristoylated Sip2p
is a key regulator of Snf1 in aging cells. Among the three genes
encoding Snf1
subunits (SIP1, SIP2,
GAL83), only sip2
affects aging. The
rapid-aging phenotype of sip2
cells is completely rescued
by blocking recombination at rDNA loci with a fob1
allele. N-Myristoylation of Sip2p is essential for normal
cellular life span. When the plasma membrane association of Sip2p is
abolished by a mutation that blocks its N-myristoylation,
Snf4p is shifted to the nucleus. Together, these findings are
consistent with a model where Sip2p functions as a negative regulator
of nuclear Snf1 activity in young cells by sequestering its activating
subunit at the plasma membrane and where age-associated loss of Sip2p from the plasma membrane to the cytoplasm facilities Snf4p/Snf1p entry into the nucleus and Snf1-catalyzed modification of chromatin structure.
subunits play a key role in mediating the diverse functions of Snf1
in younger cells. Snf1p is not functional in strains that lack all
three subunits (16). Sip2p and Gal83p are required for growth on
glycerol/ethanol, a poor carbon source that mimics low glucose (16).
Sip1p appears to be involved in the dephosphorylation of Sip4p, a
transcription factor that binds to carbon source-responsive elements
present in genes encoding gluconeogenic enzymes (16, 34). Gal83p alone
is required for phosphorylation of the same protein (16).
All
subunits have conserved C-terminal domains that mediate
interactions with Snf1p and Snf4p (these domains are termed KIS and
ASC, respectively) (35). The N termini of the three
subunits are
more divergent than their C-terminal domains and contribute to their
distinct intracellular locations and functions. Sip1p is found in the
vacuole when cells are grown in glycerol (27). In contrast, Gal83p
moves from the cytoplasm to the nucleus when cells are shifted from
glucose to glycerol (27). The N-terminal 90 residues of Gal83p are
sufficient to confer nuclear localization (27). Sip2p is the only Snf1
subunit that is N-myristoylated and, as noted above, is
affiliated with the plasma membrane. (The
subunit of the mammalian
Snf1 homolog, AMPK, is also predicted to be a substrate for
N-myristoyltransferase) (14).
The importance of N-myristoylation of Sip2p in modulating
cellular life span is highlighted by two findings. First, the
accelerated aging phenotype of sip2
cells can be fully
rescued by a CEN episome containing SIP2 under
the control of its own promoter but not by an episome containing a
sip2 mutant with a Gly1
Ala substitution
that blocks N-myristoylation. Second, a strain containing a
mutant nmt1 allele that produces global defects in protein
N-myristoylation (nmt1-451D) has a rapid-aging
phenotype (22). The mean life spans of isogenic nmt1-451D
and nmt1-451Dsip2
strains are the same. In addition,
introducing null alleles of genes encoding a number of prominent
N-myristoylproteins into wt cells has no effect on life span
(22). Together, these observations suggest that loss of myristoylSip2p
is the principal contributor to the rapid aging of
nmt1-451D cells.
We know that the trigger to Snf1 activation in aging cells is not a
reduction in intracellular glucose levels; they rise 2- and 8-fold as
wt and sip2
cells go from generation 0-1 to 7-8, respectively (23). Impaired glucose transport does not appear to be the
trigger: transport rates rise modestly in aging cells as judged by the
uptake of 2-deoxyglucose (22). Although one possible trigger could be
an age-associated change in the level of N-myristoylation of
Sip2p, to date we have no evidence to support such a notion. Our
GeneChip analysis revealed that Nmt1p mRNA levels were not
appreciably different in young and old wt and sip2
cells,
nor were there detectable changes in expression of genes that regulate
myristoyl-CoA pool size, e.g. FAA1-4 (fatty acyl-CoA synthetases) and FAS1,FAS2 (fatty acid
synthetases) (data not shown). A sensitive assay for Nmt1p activity
(36) could not be used to directly measure enzyme activity in
unfractionated lysates prepared from young and old cells because of the
high background. However, immunoblot analysis of total cellular
proteins using previously characterized antibodies to Nmt (37) revealed no apparent differences in enzyme concentration between young and old
wt or sip2
cells (data not shown).
It is important to note that for many N-myristoylproteins
myristate is a key but not an exclusive regulator of membrane
association. Protein N-myristoylation promotes weak and
reversible protein-membrane interactions (38). Proteins such as
myristoylated alanine-rich C kinase substrate use myristate
plus electrostatic interactions between positively charged
protein side chains and negatively charged phospholipids to stabilize
their association with the plasma membrane (39). Phosphorylation of
these side chains functions is used to sever the association (39).
Other N-myristoylproteins such as ADP-ribosylation factors
or recoverin use ligand binding to produce a conformational
change that exposes or sequesters their myristoyl chain
("myristoyl-conformational switch") (40, 41). Thus, the
age-associated redistribution of Sip2p from the plasma membrane to the
cytoplasm could reflect changes in its levels of
N-myristoylation or reversible modifications that affect electrostatic interactions with membrane phospholipids and/or a
conformation change triggered by acquisition of a ligand that affects
presentation of its myristoyl group. Distinguishing among these
possibilities should provide further mechanistic details about how
Snf1p is activated in aging cells and how such activation can be overcome.