Download Influence of genotype and nutrition on survival and metabolism of starving yeast.

Influence of genotype and nutrition on survival
and metabolism of starving yeast
Viktor M. Boer, Sasan Amini, and David Botstein*
Lewis–Sigler Institute for Integrative Genomics and Department of Molecular Biology, Princeton University, Princeton, NJ 08544
Contributed by David Botstein, March 14, 2008 (sent for review February 8, 2008)
Starvation of yeast cultures limited by auxotrophic requirements
results in glucose wasting and failure to achieve prompt cell-cycle
arrest when compared with starvation for basic natural nutrients
like phosphate or sulfate. We measured the survival of yeast
auxotrophs upon starvation for different nutrients and found
substantial differences: When deprived of leucine or uracil, viability declined exponentially with a half-life of <2 days, whereas
when the same strains were deprived of phosphate or sulfate, the
half-life was ⬇10 days. The survival rates of nongrowing auxotrophs deprived of uracil or leucine depended on the carbon source
available during starvation, but were independent of the carbon
source during prior growth. We performed an enrichment procedure for mutants that suppress lethality during auxotrophy starvation. We repeatedly found loss-of-function mutations in a number of functionally related genes. Mutations in PPM1, which
methylates protein phosphatase 2A, and target of rapamycin
(TOR1) were characterized further. Deletion of PPM1 almost completely suppressed the rapid lethality and substantially suppressed
glucose wasting during starvation for leucine or uracil. Suppression
by a deletion of TOR1 was less complete. We suggest that, similar
to the Warburg effect observed in tumor cells, starving yeast
auxotrophs wastes glucose as a consequence of the failure of
conserved growth control pathways. Furthermore, we suggest
that our results on condition-dependent chronological lifespan
have important implications for the interpretation and design of
studies on chronological aging.
auxotrophy starvation 兩 chronological aging 兩 Warburg effect
T
o grow and survive, all cells must sense and respond to the
availability of nutrients in their environment. In budding
yeast (Saccharomyces cerevisiae), the means by which cells sense
and respond to the availability of sources of nitrogen, phosphate,
sulfate, and fermentable sugars are well studied. The nutrient
sensor and assimilation pathways are known to dynamically
modify activities of diverse cellular functions, including nutrient
utilization, ribosome biosynthesis, and stress responses (1–4).
The onset of cell division is directly dependent on the output of
these pathways: When inadequate carbon, nitrogen, sulfur, or
phosphorus is detected in its environment, it arrests at the start
(G0/G1) of the cell cycle (5–7). Only under favorable conditions
will progression through the cell division cycle proceed (8).
The failure to commit to the cell division cycle, and entering
instead a resting or quiescent state, has always been assumed to
be a survival strategy in the face of inadequate nutrition (6). This
assumption is entirely consistent with findings that pathways that
stimulate growth and division in nutrient-rich conditions, like
target of rapamycin (TOR) and protein kinase A (PKA), repress
entry into a resting state. Moreover, pathways that activate entry
into a resting state, such as protein kinase C (PKC) and Snf1, are
repressed by glucose, arguably the strongest activator of growth
(4, 9). Thus, it came as no surprise that mutations in genes
involved in these signaling pathways and their downstream
targets have been found to affect long-term viability (9–12).
In a comparison with several nutrients, Saldanha et al. (13)
observed that cells respond differently to starvation for a
nutrient made necessary by mutation (leucine in a leucine
6930 – 6935 兩 PNAS 兩 May 13, 2008 兩 vol. 105 兩 no. 19
auxotroph or uracil in a uracil auxotroph), compared with a
‘‘natural’’ starvation for phosphate or sulfate. Notably, in the
latter cases, nearly all of the cells in the culture appeared to have
arrested in G0/G1 (unbudded), whereas there was no similarly
rapid, coordinated arrest upon starvation for leucine and uracil
in the respective auxotrophs. In contrast, when cultures of
exponentially growing cells in nutrient-limited chemostats were
compared with the same limiting nutrients (2), no difference in
the budding index between the natural and auxotrophic limitations was observed. Brauer et al. (2), however, observed that
chemostat cultures limited for uracil or leucine differed from
those limited by phosphate, sulfate, or ammonium in another
way: The latter conserved much of the excess glucose available
to them, whereas the former completely fermented the excess
glucose to ethanol.
Both the lack of a uniform arrest during starvation of auxotrophs and the wasting of excess glucose suggest a failure of
regulation. Specifically, they raise the possibility that one or
more signals given during starvation for the natural nutrients,
leading to cell-cycle arrest and cessation of glucose fermentation, are lacking when a nutrient made necessary only by a
mutation runs out. It seemed likely that this failure of regulation
might have consequences for the viability of cells that were
starved of auxotrophic nutrients.
Here we show that starvation does indeed have differential
effects on viability depending on the nature of the missing
nutrient. Several auxotrophic strains we tested die exponentially
when deprived of their cognate nutrients, whereas the same
strains survive when deprived of phosphate or sulfate; the degree
of the difference depends on the carbon source. We selected
mutants that survive auxotrophic starvation for leucine and
repeatedly found null mutations in PPM1 and TOR1. Our data
further point to a major involvement of protein phosphatase 2A
(PPA2) as a central component in the response upon auxotrophy
starvation.
Results
We determined the viability of yeast cells in dilute, nonsaturating
media, where only a single nutrient limits growth. The survival
of cells starved for phosphate, sulfate, uracil, or leucine was
tested in triplicate in a single strain (DBY8863) doubly auxotrophic for leucine and uracil. In each case, this strain was
growing in the appropriate nutrient-limited medium before
being completely deprived of the nutrient by dilution, as described in Materials and Methods. Fig. 1 shows that the cells
Author contributions: V.M.B. and D.B. designed research; V.M.B. and S.A. performed
research; V.M.B., S.A., and D.B. contributed new reagents/analytic tools; V.M.B. and D.B.
analyzed data; and V.M.B. and D.B. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
*To whom correspondence should be addressed at: Carl Icahn Laboratory, Lewis–Sigler
Institute, Princeton University, Washington Road, Princeton, NJ 08544. E-mail: botstein@
genomics.princeton.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0802601105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0802601105
10
10 0
10
6
5
PHO
SUL
10 -1
10 -2
GLU to GAL
GAL to GAL
3
10
2
10
GAL to GLU
E&G to GLU
GLU to GLU
LEU
1
URA
10
10
10
-4
0
5
10
15
time (days)
20
25
30
Fig. 1. Survival of strain DB8863, measured as colony-forming units, during
starvation for phosphate (triangles), sulfate (diamonds), leucine (circles), and
uracil (squares). The number of viable cells was normalized to 100% and
measured at the onset of starvation (day 0). The average values and SD for
three independent replicate experiments are shown. Survival of prototrophs
in limiting phosphate or sulfate is identical to that of auxotrophs and is
unaffected by added uracil or leucine (data not shown).
survive for weeks when starved of phosphate or sulfate (viability
declines exponentially with a half-life of ⬇10 days), whereas the
cells starved of uracil or leucine die exponentially at a much
higher rate (viability declines with a half-life of 0.8 or 1.9 days,
respectively).
This limitation-dependent difference in survival extends previous observations of other significant differences in the physiological responses to starvation for sulfate or phosphate, on the
one hand, and to leucine or uracil, on the other hand. Brauer et
al. (2) found that leucine or uracil limitation in chemostat
cultures results in an excess glucose consumption, compared
with phosphate or sulfate limitation. Saldanha et al. (13) found
a relative failure to arrest the cell cycle promptly during leucine
or uracil starvation, compared with the prompt arrest at G0/G1
that is found during phosphate or sulfate starvation. Supporting
information (SI) Fig. S1 shows two typical experiments showing
the excess consumption of glucose by starving auxotrophs. These
phenomena do not appear to depend on the yeast strain background: We have observed them in S288c-, CEN.PK-, and
W303-derived strains.
Because of the potential connection between survival and
glucose consumption, we tested survival during leucine starvation in media containing either galactose or a combination of
ethanol and glycerol instead of glucose as carbon sources. Fig. 2
shows that the nature of the carbon source has a significant
impact on the rate of loss of viability during leucine starvation.
In galactose, the viability declines (half-life of 2.8 days) significantly more slowly than in glucose (half-life of 1.9 days). In
ethanol/glycerol, viability during leucine starvation is even
higher and declines at about the same rate as that found for
phosphate starvation in glucose (11 days).
A series of experiments determined that these differences
depend only on the carbon source in which the cells are starving
not on their prior growth conditions. When cells were pregrown
on leucine-limited glucose media and transferred to leucinelimited media with galactose, they displayed the same death rates
as when the cells were both growing and starving in galactose
medium. The same holds true for the transfer to ethanol/glycerol
media and the reciprocal switch: Death rates in leucine-limited
glucose medium were independent of the carbon source during
prior growth.
To investigate the mechanisms of sensitivity to auxotrophy
starvation, we performed a genetic screen for mutants with
Boer et al.
0
10
0
5
10
15
time (days)
20
25
30
Fig. 2. Survival of strain DB8863, measured as colony-forming units, during
leucine starvation in media containing as carbon source ethanol and glycerol
(triangles), galactose (squares), or glucose (circles). Cells were pregrown on
leucine-limited media containing, as a carbon source, glucose (black symbols),
galactose (gray), or ethanol and glycerol (white).
enhanced survival upon leucine starvation (see Materials and
Methods). Briefly, this process involved allowing independent
cultures to starve for leucine until viability declined ⬇200,000fold, regrowing cultures in limiting phosphate and excess leucine,
and repeating the starvation for leucine. After five to seven
rounds of starvation and recovery, single colonies were isolated
and tested for viability in media lacking leucine. These viability
assays were performed, as in Fig. 1, at low cell density (⬇3 ⫻ 105
per ml) to avoid acidification of the medium or depletion of
nutrients other than leucine.
Initially, two independent mutants with enhanced viability
during leucine starvation were isolated (strains DBY11202 and
DBY11203). Complementation tests revealed that these mutants
do not complement each other, implicating a single locus. One
of the mutants, DBY11202, was used for both array-assisted bulk
segregant analysis (14) and mutation detection by using overlapping tiling arrays of the entire yeast genome (15). The bulk
segregant analysis identified one linkage peak on chromosome
IV (Fig. 3). Within that region, tiling arrays analysis predicted
one polymorphism within the gene PPM1 (Table 1). The PPM1
gene was sequenced in DBY11202 and DBY11203, and in both
Fig. 3. Bulk segregant analysis on tiling arrays reveals a single locus on
chromosome IV. The difference in signal intensity (resistant pool–sensitive
pool) is shown for chromosome IV and the whole genome (Inset).
PNAS 兩 May 13, 2008 兩 vol. 105 兩 no. 19 兩 6931
CELL BIOLOGY
10
-3
GLU to E&G
E&G to E&G
4
viable cells/ml
fraction of viable cells
10 1
Table 1. Viability during nutrient starvation of wild-type
and mutant strains
6
10
Strain
Limitation
Carbon source
Half-life, days
DBY8863
Phosphate
Sulfate
Leucine
Uracil
Leucine
Leucine
Leucine
Uracil
Leucine
Uracil
Leucine
Uracil
Glucose
Glucose
Glucose
Glucose
Galactose
Ethanol plus glycerol
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
11
9.4
1.9
0.8
2.8
11
2.0
0.7
8.6
2.6
2.4
1.0
ppm1⌬0
tor1⌬0
4
viable cells/ml
BY4742
ppm1∆0
5
10
10
3
10
2
10
1
10
WT
0
10
0
strains, we found frameshift mutations introducing premature
STOP codons (a single-bp deletion at bp 1,333,843 introducing
a STOP codon at L49 in DBY11202 and a 71-bp deletion starting
at bp 1,333,461 introducing a STOP codon at L146 in
DBY11203).
Another 12 independent mutants were isolated, and all were
resequenced by using tiling arrays and Sanger sequencing (15).
The mutations that were found in annotated genes are listed in
Table 2. Mutations in some genes were found more than once
and thus are likely to be causative. In addition to the two
truncating ppm1 mutations, we found three independent truncating mutations in TOR1, one truncating and one missense
mutation in SCH9, one nonsense and one missense mutation in
HRD1, and one nonsense and one missense mutation in HAP1.
Initial tests of the ability of these spontaneous mutant strains to
survive leucine starvation suggested that the ppm1 mutations
had the strongest effect of restoring survival to levels found with
sulfate or phosphate starvation, followed by sch9 and tor1
mutations; the hap1 and hrd1 mutations had very minor effects.
Below, we further characterize the effect of loss of function of
ppm1 and tor1, recognizing that it is likely that many of the other
genes in which we recovered mutations also may be involved in
starvation survival. Specifically, the sch9 mutants seem to confer
significant resistance (half-life on leucine-limited glucose me-
5
10
15
time (days)
20
25
30
Fig. 4. Survival of strain DBY8863 (circles) and isogenic ppm1⌬0 derivative
(squares) during leucine starvation, with mean and SD of six independent
replicates.
dium is ⬎5 days); however, the severe growth defect of the sch9
deletion strain (16) compromises a straightforward comparison.
To confirm more rigorously that the ppm1 and tor1 mutations
and not other bp differences (whether detected or not) in the
strains we recovered are responsible for the enhanced survival,
we tested the survival of constructed ppm1- and tor1-deletion
mutants. Fig. 4 shows survival during leucine starvation in a
ppm1-deletion mutant and the isogenic parent DBY8863. Fig. S2
shows results for a ppm1- and tor1-deletion strain from the
systematic yeast-deletion project (17), tested for survival upon
leucine and uracil starvation; the half-lives also are given in
Table 1. The isogenic parent strain (BY4742) dies at about the
same rate as the closely related strain DBY8663, which was the
parental strain in our selection (2.0 days for leucine and 0.7 day
for uracil). Survival of the ppm1⌬0 mutant is considerably higher
than wild type (half-life of 8.6 days for leucine and 2.6 days for
uracil). Survival of the tor1⌬0 mutant is only slightly higher than
wild type (half-life of 2.4 days for leucine and 1 day for uracil).
These results strongly support a causal role for ppm1-null
mutations in survival upon auxotrophy starvation, which is
Table 2. Mutations found in isolated strains with enhanced survival upon leucine starvation
Gene
Mutation
(chr:bp)
Protein
Parent
Strain
Other mutations in
PPM1
PPM1
TOR1
TOR1
TOR1
SCH9
SCH9
HRD1
HRD1
HAP1
HAP1
KOG1
TPD3
HRD3
4:1333843 A to ⌬
4:1333461 71bp⌬
10:559755 C to ⌬
10:563920*
10:565483 C to G
8:511331 G to A
8:509689 C to AA
15:302637 C to T
15:301850 G to T
12:646559 C to G
12:647879 T to G
8:480255 T to C
1:126722 G to C
12:557609 G to T
L49X
L146X
S116X
S1509X
Y2025X
G657S
N125X
Q134X
C396F
S48X
F488C
S140 P
D615H
G274C
1
1
1
1
1
2
2
1
1
2
2
2
1
1
1
DBY11202
DBY11203
DBY11204
DBY11205
DBY11206
DBY11211
DBY11214
DBY11207
DBY11208
DBY11213
DBY11215
DBY11212
DBY11210
DBY11210
DBY11209
CPR3, GCD1, ACF4, KEX1
ADP1
SSK22
CDC60
AUS1
FLO1, FLO5
FLO1, FLO5
UBR2, 15:1042k to end
Genes contributing to the gene neighborhood (Fig. 6) are given in the first column. Parent 1, FY strain DBY8863,
parent 2, SK1 strain NKY1060. These 14 strains were randomly chosen from 27 independent selections.
*TTACGA to CTTCTTA
6932 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0802601105
Boer et al.
15
OD 600
0.8
WT PHO
ppm1∆0 URA
0.4
10
5
WT URA
0
0
10
20
30
time (h)
40
50
0
Fig. 5.
Growth and glucose consumption during phosphate and uracil
starvation. Triangles, strain DBY8863 during phosphate starvation; squares,
strain DBY8863 during uracil starvation; circles, isogenic ppm1⌬0 deletion
strain during uracil starvation; open symbols, OD600; closed symbols, glucose
concentration. Values represent averages and SD of two (DBY8863) or four
(ppm1⌬0 deletion strain) independent experiments.
consistent with the repeated recovery of null mutations in the
selection. The situation for tor1 mutants is less clear. The three
independent spontaneous tor1 mutants we recovered had halflives of 3.9 (⫾0.3) days on leucine-limited media. The modest
suppression of the tor1 deletion in the BY background could be
due to strain differences (e.g., because of the relative contribution of Tor2p in TOR complex 1) or, alternatively, mutations
that were not detected in our screen.
Finally, we tested whether the ppm1 mutations had any effect
on the glucose-wasting phenomenon previously associated with
starvation for leucine and uracil, compared with starvation for
phosphate or sulfate. Fig. 5 shows that, during uracil starvation,
our ppm1-deletion strain consumes significantly less excess glucose than its isogenic parent, but more compared with exponential growth of the parent strain during phosphate starvation.
Fig. S3 shows the same for leucine starvation.
Discussion
Survival of Nongrowing Cells Depends on the Nutritional Limitation
and the Carbon Source. Our results show that starvation for
auxotrophic requirements is substantially more lethal than starvation for phosphate or sulfate. Just by changing the limiting
nutrient, we found ⱖ10-fold differences in exponential survival
rate (e.g., between phosphate and uracil starvation in Fig. 1).
Hence, an important aspect of chronological aging has gone
largely unnoticed so far: Yeast viability depends on which
nutrient is limited first. Therefore, it seems clear that rigorous
control of conditions is required while studying chronological
aging specifically because gene effects previously found to affect
chronological aging (10, 12) are often smaller than the conditional effects we have found here. Moreover, all studies on
chronological aging in S. cerevisiae published so far have made
use of auxotrophic strains (usually including leucine, histidine,
and uracil as auxotrophic requirements). Assays that have been
used in determining chronological lifespan almost uniformly
involved growing auxotrophic strains to high density in YPD or
synthetic complete medium and subsequent starvation in the
same medium. Alternatively, cells were transferred to water after
growth to saturation (reviewed in refs. 18 and 19). In both
experimental designs, one cannot tell which nutrient runs out
first, and it is unclear to the absence of what nutrient(s) the cells
responds. As we have shown, the nature of the limiting nutrient
determines lifespan. Thus, the techniques of measurement of
chronological aging require a rigorous reassessment.
The rapid loss of viability during starvation for leucine is, in
part, dependent on the carbon source(s) available during starBoer et al.
vation. Changing the carbon source from glucose to galactose or
ethanol/glycerol, which are less preferred sources of carbon,
drastically increased the lifespan during starvation. These results
strengthen the notion that growth-stimulating pathways that are
activated by glucose (TOR and PKA) have a negative effect on
lifespan (9, 12, 20), whereas glucose represses pathways that
activate entry into a resting state (4, 9). It is significant that the
carbon source matters only during the starvation: The carbon
source available before starvation makes no detectable difference, ruling out prior metabolic activity or growth rate as a factor
influencing viability.
Amino Acid Starvation and the Cell Division Cycle. Survival during
starvation of auxotrophs has been studied previously in a number
of organisms, including Neurospora (21), Escherichia coli (22),
and S. cerevisiae (23, 24), with varying survival rates. In S.
cerevisiae, Henry (24) reported very rapid loss of viability of S.
cerevisiae inositol auxotrophs (hundreds of fold per day) and only
a modest loss of viability upon lysine, adenine and tryptophan
starvation. The rates of loss of viability found by Henry (24) for
amino acid starvation appear to be comparable to our observation for leucine and uracil; for inositol starvation, the rate is
markedly higher. We also tested histidine auxotrophs with very
similar results (data not shown). An exception to the pattern that
yeast auxotrophs die rapidly upon starvation seems to be methionine. When Unger and Hartwell (5) observed that methionine starvation of a methionine auxotroph results in G0/G1
arrest, they reported no loss of viability. They proposed that
methionine or a product of methionine (e.g., methionyl-tRNA)
acts as a more general signal for starvation and also is involved
in signaling sulfate starvation (5).
Following the reasoning of Unger and Hartwell (5), the
lethality observed with auxotrophic starvations results, in part,
from a failure to execute a cell-cycle arrest; the prompt arrest of
the cell cycle during starvation for methionine or the natural
nutrients phosphate or sulfate serves to put cells into a ‘‘safe’’
quiescent state. This idea is in accord with our observation that
auxotrophs starved for phosphate or sulfate results in low
lethality. We suppose further that the lethality depends on
metabolic activities (e.g., initiation of DNA replication) inappropriate to the circumstances that happen to a greater degree
when good carbon sources are available.
Loss-of-Function Mutations in a Number of Genes Can Suppress
Lethality During Auxotrophy Starvation. Identifying the genetic
basis of new phenotypes is a serious challenge in biological
studies. With the use of recently developed microarray technology, we were able to identify causal mutations in strains with
acquired longer lifespans. The mutations that we recovered that
suppress the lethality of auxotrophic starvation provide clues to
the identity of regulatory pathways involved. We repeatedly
isolated mutations in three genes (PPM1, TOR1, and SCH9) that
significantly suppress the lethality phenotype. Two independently derived ppm1 deletions suppress lethality, resulting in
survival over time close to that observed for phosphate starvation. The ppm1 deletion also suppresses the elevated glucose
consumption during auxotrophy-limited growth, although not
completely. In the case of TOR1, a deletion suppresses the
lethality phenotype, but significantly less well.
In addition to the use of new microarray technology, we also
made use of recently developed bioinformatic tools. The
bioPIXIE program (25) summarizes evidence for gene interactions by using Bayesian analysis of diverse kinds of experimental
data, including coexpression, synthetic lethality, and more direct
biochemical protein interaction data. Fig. 6 summarizes the
genes in which we found mutations and the closest associated
genes, as inferred by using the bioPIXIE program. We found
that, in many mutants, the suppression of lethality is the result
PNAS 兩 May 13, 2008 兩 vol. 105 兩 no. 19 兩 6933
CELL BIOLOGY
1.2
glucose (g/l)
1.6
25
WT URA
WT PHO
ppm1∆0 URA 20
2
QRI8
0.20 - 0.50
0.51 - 0.75
HMG2
HMG1
0.76 - 0.90
HRD1
0.91 - 1.0
HRD3
TPD3
KOG1
†
SCH9
TOR1
SSA1
PPH22
CDC55
*
RTS1
LST8
HAP1
SSD1
PPM1
Fig. 6. Gene neighborhood of genes affected by mutation in the leucine
survival screen (orange) and the top-ranking associated genes (green). *,
Methylation site; †, added interaction based on evidence published previously
(39). See Materials and Methods for details.
of a loss of function of one of a group of genes that have already
been implicated in signaling pathways that regulate one or more
basic cellular processes: metabolism, the cell cycle, growth rate,
and stress response (26–28).
PP2A Plays an Important Role in Survival upon Auxotrophy Starvation.
The PPM1 gene encodes a carboxyl methyl transferase that
methylates the catalytic subunit of PP2A (in yeast, encoded by
PPH21 or PPH22). This function is the only known type of this
protein, and Ppm1p appears to be the only protein in yeast that
carries out this methylation (29, 30). Ppm1p and its substrate, the
C-terminal domain of the catalytic subunit, are both highly
conserved in eukaryotes from yeast to human. Loss of function
of Ppm1p in yeast greatly reduces the interaction of the catalytic
and regulatory subunits of PP2A (29, 30) and results in a
rapamycin-resistant phenotype (30). The administration of rapamycin mimics nutrient-poor conditions; loss of function of the
TOR complex1 has a similar effect. As is clear from Fig. 6, the
set of genes we recovered are all closely related to each other,
and many of them could, like ppm1 and tor1 mutations, mimic the
perception of a nutrient-poor environment and set off a starvation response, thus leading to an increase in long-term viability
and a decrease in glucose consumption.
We found that, contrary to the wild type, survival upon
auxotrophy starvation is independent of rapamycin in the
ppm1⌬0 strain (Fig. S4). The rapamycin-resistant phenotype of
a ppm1⌬0 mutant has been observed previously, and it is likely
mediated through Tap42p, which binds both TOR complex1 and
the unbound PP2A catalytic subunits Pph21p and Pph22p (30,
31). Inactivation of TOR complex1 leads to the dissociation of
the PP2A complex from Tap42p (31–33), whereas inactivation of
Ppm1p leads to the dissociation of the PP2A catalytic subunits
from the PP2A regulatory and scaffolding subunits (29, 30, 34).
The mutation we found in TPD3 also might lead to reduced
stability of the PP2A trimeric complex. Hence, we can speculate
that ultimately it is elevated levels of unbound PP2A catalytic
subunit that result in an auxotrophy starvation-resistant phenotype. However, how TOR complex1, Sch9p and PP2A, or
unbound Pph21p or Pph22p act in the sensitivity to auxotrophy
starvation is not yet clear.
Starving Yeast Auxotrophs and Human Tumor Cells May Waste Glucose
(Warburg Effect) Through the Failure of Conserved Growth Control
Pathways. An interesting comparison can be made between the
results we have found in yeast and the properties of human tumor
cells. An inference arises from the coincidence of three factors:
6934 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0802601105
cell-cycle dysregulation [found in both yeast auxotrophs (2, 13)
and in tumor cells], the genes involved (conserved growth
regulators in both yeast and animal cells and implicated in tumor
etiology), and the relatively new observation of excess glucose
consumption (which we call ‘‘wasting’’) known as the Warburg
effect (35). This elevated glucose consumption is routinely used
to identify tumors in vivo with the use of the glucose analogue
2-[18F]fluoro-2-deoxy-D-glucose. It seems that both tumor and
yeast cells growing under auxotrophy starvation grow in an
uncontrolled state of increased metabolism without the proper
checks and balances. In both yeast and humans, TOR1 could be
an important effector of this phenotype because mammalian
TOR1 (mTOR1) is a protooncogene, and constitutively active
mTOR complex1 leads to tumor growth. We found that loss of
function of yeast TOR1 leads to increased survival upon auxotrophy starvation, implicating active Tor1 in the sensitivity toward auxotrophy starvation. Inhibiting TOR complex1 activity
with rapamycin increases survival upon auxotrophy starvation
(Fig. S4). The same analogy can be drawn for mammalian
kinases Akt1p and S6K1, also protooncogenes (36, 37), and their
yeast homologue Sch9p, which we recovered twice in our screen.
In light of this and given the highly evolutionary conserved
C-terminal domain of protein phosphatase 2A, it would be of
great interest to see how the protein phosphatase methyltransferase affects cell signaling, in both yeast and human.
To conclude, our results show that the survival of yeast cells
that have stopped growing for nutritional reasons depends on
why they ceased growing. If natural nutrients, like phosphate or
sulfate, are depleted, a prompt and orderly arrest of the cell
division cycle, accompanied by a cessation of glucose fermentation, ensures that cells are able to survive for weeks. Depletion
of nutrients made necessary because of an auxotrophic mutation,
like leucine or uracil (but probably not methionine), does not
lead to an orderly arrest. Glucose consumption remains high
even after cessation of growth, and the cells die exponentially in
a matter of days. This rapid lethality can be suppressed by the loss
of a number of proteins already implicated in sensation of
nutrient sufficiency and/or growth control, such as the products
of the PPM1, SCH9, and TOR1 genes.
Materials and Methods
Yeast Strains and Growth Conditions. FY derivative strain DBY8863 (MATa
leu2-⌬1 ura3-52) and SK1 derivative strain NKY1060 (MATalpha ho::LYS2 lys2
ura3 leu2::hisG) were used for the selection of mutants, linkage analysis, and
growth experiments. DBY8863 and BY4742 (MATalpha his3⌬1 leu2⌬0 lys2⌬0
ura3⌬0) and their single-deletion derivatives for ppm1⌬0 and tor1⌬0 (BY4742
only) were used for the confirmation of survival phenotypes.
All minimal media were slightly adapted from Saldanha et al. (13). Deviations were as follows: for phosphate limitation, 1 g/liter KCl and 10 mg/liter
KH2PO4; for sulfate limitation, 2.6 mg/liter (NH4)2SO4; for uracil limitation, 4
mg/liter uracil; and for leucine limitation, 40 mg/liter leucine. Where needed,
excess additions of 240 mg/liter leucine, 40 mg/liter uracil, 40 mg/liter histidine, and 60 mg/liter lysine were used. All media contained 20 g/liter glucose
or, when indicated, 20 g/liter galactose or 8.1 g/liter ethanol and 12.6 g/liter
glycerol as a carbon source. YPD contained 10 g/liter bacto yeast extract, 20
g/liter bacto peptone, and 20 g/liter glucose.
For growth experiments, cultures were grown at 30°C, agitated at 200 rpm,
in 250-ml erlenmeyer flasks with a working volume of 50 ml. Cultures were
pregrown overnight on the same media and diluted at t ⫽ 0 to an OD600 of
0.04. For glucose determinations, 1 ml of culture was centrifuged at 13,000 ⫻
g, and supernatants were stored at ⫺20°C. Glucose was determined enzymatically (in triplicate) by using a D-Glucose commercial kit (R-Biopharm).
Survival Assays. A single colony was inoculated and grown in 3 ml of YPD. After
24 h, this culture was diluted 50-fold in nutrient-limited medium. After 24 h,
this culture was diluted 150-fold in media lacking entirely the appropriate
nutrient (time 0), and samples were plated on YPD agar plates to determine
colony-forming units. In sulfate-limited conditions, there was some growth
after the last transfer due to the presence of trace amounts of copper,
Boer et al.
Enrichment for Mutants with Enhanced Survival upon Leucine Starvation. Strains
DBY8863 and NKY1060 were grown in leucine-limited medium, and viability
was assayed over time. When the total fraction of viable cells was ⬍5 ⫻ 10⫺4,
⬇ 100 –1,000 viable cells were transferred to phosphate-limited medium to
limit selection for strains that simply grow faster or recover from arrest more
quickly in the selection media. After several days, this culture was used as an
inocculum for a new round of leucine starvation. Between five and seven
rounds of enrichment were performed, after which single colonies were
isolated and assayed for viability.
resistant and sensitive pools (segregants with intermediate resistance were
omitted) and hybridized to Affymetrix tiling arrays (15). For estimation of the
linkage peak, only the probes were used that have known differences between FY and SK1 strains (38).
Mutation Detection. DNA of one clone of each enrichment was hybridized to
Affymetrix yeast tiling arrays and analyzed for mutations as described previously (15). SNP predictions in ORFs were confirmed by PCR and Sanger sequencing (in duplicate).
Assembly of the Gene Neighborhood (Fig. 6). To place the mutations found in
the genetic screen into a biological context, we used a program called bioPIXIE
(25). All genes affected by mutations (Table 2, excluding CPR3, GCD1, ACF4,
and KEX1 because in this strain it was established that PPM1 is causal) were
used as input in bioPIXIE to assemble the gene neighborhood and to generate
the accompanying gene rank list (Table S1). The top nine ranking genes of the
gene neighborhood remained identical when only using a subset of all genes
(Table 2, column 1) as input. Hence, this subset contributes most strongly to
the gene neighborhood. The graph obtained from bioPIXIE was redrawn
retaining the gene interaction information between the top-ranking genes in
the neighborhood and the genes in Table 2. The connection between TOR1
and SCH9 was added based on evidence published previously (39).
Linkage Analysis. Linkage analysis was performed by using a strategy described previously (14). In short, strain DBY11202 was backcrossed twice to get
a 2:2 segregation (resistant; high viability upon leucine starvation, sensitive;
low viability). One of the resistant segregants was crossed to the sensitive,
divergent SK1 derivative strain NKY1060. After sporulation, segregants were
scored for viability upon leucine starvation. DNA was isolated from the
ACKNOWLEDGMENTS. We thank D. Gresham, D. Ruderfer, J. Schacherer, and
J. Shapiro for help and advice on tiling array analysis; R. Koszul (Harvard
University, Cambridge, MA) for providing strain NKY1060; and various colleagues for critical reading of the manuscript. This work was supported by the
National Institute of General Medical Sciences Center for Quantitative Biology/National Institutes of Health Grant P50 GM-071508 and National Institutes
of Health Grant R01 GM-046406 (to D.B.).
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PNAS 兩 May 13, 2008 兩 vol. 105 兩 no. 19 兩 6935
CELL BIOLOGY
manganese, and zinc sulfate, and, hence, time 0 was set 2 days after the last
transfer.