Glucose, Nitrogen, and Phosphate Repletion in
Saccharomyces cerevisiae: Common Transcriptional
Responses to Different Nutrient Signals
Michael K. Conway, Douglas Grunwald, and Warren Heideman1
Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705
ABSTRACT Saccharomyces cerevisiae are able to control growth in response to changes in nutrient avail-
ability. The limitation for single macronutrients, including nitrogen (N) and phosphate (P), produces stable
arrest in G1/G0. Restoration of the limiting nutrient quickly restores growth. It has been shown that glucose
(G) depletion/repletion very rapidly alters the levels of more than 2000 transcripts by at least 2-fold, a large
portion of which are involved with either protein production in growth or stress responses in starvation.
Although the signals generated by G, N, and P are thought to be quite distinct, we tested the hypothesis
that depletion and repletion of any of these three nutrients would affect a common core set of genes as part
of a generalized response to conditions that promote growth and quiescence. We found that the response
to depletion of G, N, or P produced similar quiescent states with largely similar transcriptomes. As we
predicted, repletion of each of the nutrients G, N, or P induced a large (501) common core set of genes and
repressed a large (616) common gene set. Each nutrient also produced nutrient-specific transcript changes.
The transcriptional responses to each of the three nutrients depended on cAMP and, to a lesser extent, the
TOR pathway. All three nutrients stimulated cAMP production within minutes of repletion, and artificially
increasing cAMP levels was sufficient to replicate much of the core transcriptional response. The recently
identified transceptors Gap1, Mep1, Mep2, and Mep3, as well as Pho84, all played some role in the core
transcriptional responses to N or P. As expected, we found some evidence of cross talk between nutrient
signals, yet each nutrient sends distinct signals.
protein kinase A
Yeast starved for macronutrients, such as glucose (G), nitrogen (N), or
phosphorous (P), arrest growth and cell division and become quies-
cent, with cell wall thickening, reduced transcription and translation,
and increased stress tolerance (Gray et al. 2004; Rowley et al. 1993).
Upon nutrient repletion, yeast immediately return to growth and di-
vision (Unger and Hartwell 1976).
Glucose addition to starved, quiescent yeast rapidly alters the
expression of more than a third of the yeast genome by at least 2-fold
(Martinez et al. 2004; Radonjic et al. 2005; Slattery and Heideman
2007; Wang et al. 2004). Genes needed for ribosome biogenesis (RiBi),
ribosomal proteins (RP) translation, mass accumulation, and cell di-
vision are induced (Jorgensen et al. 2004). In contrast, environmental
stress response (ESR) (Gasch et al. 2000), gluconeogenic, respirative,
and alternative metabolism genes are repressed.
This large-scale change depends on the Gpa2 G-protein asso-
ciated with the Gpr1 glucose receptor (Santangelo 2006; Wang
et al. 2004). The response is also largely dependent on cAMP pro-
duction, as well as a functional TOR pathway (Slattery et al. 2008).
Glucose produces this massive rearrangement of the transcriptome,
even in cells lacking the ability to take up and metabolize glucose
(Slattery et al. 2008). These results point to a cell surface receptor-
Most study of nutrient sensing in S. cerevisiae has focused on
specific metabolic challenges, such as glucose repression or regulation
of amino acid synthesis. Thus, we have limited knowledge of how
these different nutrients cause cells to return to growth (Forsberg
and Ljungdahl 2001; Magasanik and Kaiser 2002; Wykoff and O’Shea
2001; Zaman et al. 2008).
Copyright © 2012 Conway et al.
Manuscript received April 18, 2012; accepted for publication June 20, 2012
This is an open-access article distributed under the terms of the Creative
Commons Attribution Unported License (http://creativecommons.org/licenses/
by/3.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Supporting information is available online at http://www.g3journal.org/lookup/
1Corresponding author: School of Pharmacy, University of Wisconsin, 777 Highland
Avenue, Madison, WI 53705. E-mail: email@example.com
Volume 2| September 2012|
Work from Thevelein et al. (2005) has shown a role in trehalase
activation for cell surface N and P sensors that is largely dependent
on PKA. Because these proteins needed for trehalase stimulation
also serve as nutrient transporters, they have been termed transceptors
(Donaton et al. 2003; Popova et al. 2010; Van Zeebroeck et al. 2009).
One such transceptor is the Gap1 amino acid transporter (Jauniaux
and Grenson 1990; Magasanik and Kaiser 2002). Trehalase activation
by amino acids is Gap1-dependent. A similar finding was made in
studying the role of the Pho84 phosphate transporter. Phosphate
activation of trehalase activation is Pho84-dependent (Popova et al.
2010). Finally, the Mep family of ammonium transporters are impor-
tant for ammonium stimulation of trehalase activity (Van Nuland
et al. 2006).
Because the states of quiescence and growth appear have require-
ments that are the same regardless of the missing nutrient, we hy-
pothesized that N, P, and G depletion and repletion would control
large common gene sets needed for growth and quiescence. In this
article, we confirm this hypothesis and show that all three nutrients
produce responses that are similar in appearance and share common
mechanisms. While the initial signals appear to come from different
receptors, including Gpr1, Gap1, Pho84, and Mep proteins, the nu-
trient signals produce a response that is largely cAMP-dependent.
Furthermore, all three nutrients elevate cAMP levels when repleted.
These results indicate that different nutrient signals converge to con-
trol common states of quiescence and growth.
MATERIALS AND METHODS
Yeast strains and growth media
S288C (MATa SUC2 gal2 mal mel flo1 flo8-1 hap1 ho bio1 bio6) was
used for glucose, nitrogen, and phosphate experiments. TC41-1
(MATa leu2-3 leu2-112 trp1-1 his3-532 his4 cyr1::URA3 cam) and
the isogenic CYR1+ wild-type HR125 were used for PKA and TOR
nutrient repletion experiments (Heideman et al. 1990). BY4742
(MATa his3D1 leu2D0 lys2D0 ura3D0) was used for transceptor work
as wild-type and gap1D and pho84D mutants made in BY4742 were
from the yeast knockout collection obtained from Open Biosystems,
with either gene deleted by KanMX. Deletions were confirmed by
Cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose,
Sunrise Chemicals) or synthetic medium (SD) with 2% glucose
containing 6.7 g/l yeast nitrogen base (US Biological) supplemented
with adenine, uracil, and amino acids. Cells were cultured at 30? with
shaking. Nitrogen deprivation medium (SD-N) consisted of SD made
up with nitrogen-free Yeast Nitrogen Base (Difco) and without amino
acids or uracil. Phosphate deprivation medium (SD-P) consisted of SD
made with yeast nitrogen base in which potassium chloride was
substituted for potassium phosphate. Rapamycin (10 mg/ml stock in
ethanol, LC Laboratories) and cAMP (1M stock in water, pH 7,
Sigma) treatments were as described previously (Newcomb et al.
2003; Russell et al. 1993; Slattery et al. 2008). When added, cAMP
was used at 1 mM and rapamycin at 200 nM.
Glucose depletion was as previously described (Slattery and Heideman
2007). Cells were grown in SD medium for 48–72 hr until they had
arrested as a quiescent G1 phase population. Nitrogen starvation was
achieved by inoculating cells from an overnight SD culture into SD-N
at a density of 0.4 OD660. When growth stopped (24 hr, approximately
1.5 OD660), cells were transferred to a fresh volume of SD-N to a den-
sity of 0.5 OD660. These cells were incubated an additional 24 hr and
generally reached a density of OD6600.75–1. Finally, this culture was
resuspended in fresh SD-N at OD660of 1.0 and incubated for 12–
24 hr. Depletion was confirmed by determining that cells would not
proliferate in fresh SD-N but would grow in SD. Phosphate depletion
was carried out in the same manner, except that phosphate depletion
medium was used. In both cases, nutrient repletion was accomplished
by pelleting in a Beckman J6 centrifuge at 30? at 2500 rpm for 5 min
and resuspension in an equivalent volume of fresh SD medium.
For dual nutrient depletion experiments, cultures were grown in
N- or P-free medium until they ceased dividing and were then
transferred to medium also lacking G and incubated for an additional
48 hr to deplete any glucose remaining in the medium. This
produced GN- and GP-depleted cells; we confirmed that repletion
of only a single nutrient did not produce growth (data not shown).
The pho84D cells were starved for P as described above and chal-
lenged with KH2PO4or Gly3P (both 10 mM). The Gap1 cells were
nitrogen depleted as described above and repleted with SD-N with 10
mM L-citrulline added. The MEP-deletion strains were N-depleted as
described above and repleted by addition of SD-N plus 10 mM am-
The cyr1D strain TC41 was nutrient depleted using the techniques
previously described (Slattery and Heideman 2007), in which the
nutrient depletion followed the schedule described above except that
during nutrient the first 24 hr of depletion the cells were cultured with
1 mM cAMP and 0.5· auxotrophic supplements to be certain that the
cells could remain growing enough to deplete the missing macronu-
trient. This was followed by 24 hr in depletion medium with 1 mM
cAMP and by an additional 24 hr with no cAMP. This procedure was
used to avoid halting growth and metabolism prematurely by cAMP
withdrawal before true nutrient depletion had occurred.
Twenty-five optical density (OD) units of cells were harvested into ice-
cold TCA to a final concentration of 5% and vortexed briefly to mix.
The lysates were neutralized with NaHCO3to pH ?6.5–7 and snap-
frozen on liquid nitrogen. Immediately prior to assay, samples were
thawed on ice and centrifuged at 4? for 1 min, and then 20 mL of
supernatant was used in the R&D Systems cAMP Parameter Assay Kit
(KGE002B) as indicated by the manufacturer.
RNA isolation and microarray hybridization
Samples of cells were collected in independent experiments to produce
true biological replicates. For these experiments, cultures from in-
flasks to nutrient limitation as described above. In some cases, duplicate
cultures were grown on separate days, and in others, the cultures were
grown in the same shaker started on the same day. Samples were kept
separate, and the results from the duplicates are shown.
For each hybridization, 10 ODU cells were collected and pelleted at
5000 RPM in a Beckman J6 centrifuge for 2 min at 30?. Then the
supernatant was removed and the pellets were frozen with liquid
RNA was isolated using MasterPure Yeast RNA Purification Kits
(Epicentre Technologies), and the quality was assayed by gel
electrophoresis. cRNA synthesis was carried out using the GeneChip
Expression 39 Amplification One-Cycle Target Labeling and Control
Reagents kit from Affymetrix following the manufacturer’s instruc-
tions. cRNA samples were hybridized to GeneChip Yeast Genome 2.0
Arrays for 16 hr. Arrays were washed, stained, and scanned according
|M. K. Conway, D. Grunwald, and W. Heideman
to the manufacture’s recommendation. Affymetrix .CEL files were
RMA normalized with R and the Bioconductor Suite (Gentleman
et al. 2004). Data analysis was performed within TIGR Multiexperi-
ment Viewer, v4.5.1 (Saeed et al. 2003; 2006), in-house Perl scripting,
R, and Bioconductor.
Genes that were differentially expressed between the fed and
starved states from each nutrient condition were selected using the
following criteria: a P value less than 0.05; a false discovery rate less
than 0.01 (q-value); and a 2-fold change in expression. The individual
nutrient lists were compared to identify the genes in common, as well
as those unique to each combination, resulting in the Venn diagrams
shown in Figures 3 and 4 and supporting information, Table S1 and
GO enrichment analysis was performed through FUNSPEC tool
(Robinson et al. 2002) at http://funspec.med.utoronto.ca/. Under- and
overrepresented DNA motifs were identified using the RSAT online
motif discovery tool at http://rsat.ulb.ac.be/ (van Helden 2003), where
oligomer length was scanned between 4 and 8 bases and the best-
consensus sequence scores were collected. Transcription factor target
and motif enrichment significance were calculated using a hypergeo-
metric distribution test.
Different nutrient limitations produce
a similar transcriptome
Starvation for glucose (G), nitrogen (N), or phosphate (P) each
2000; Giots et al. 2003; Gray et al. 2004; Johnston et al. 1977; Mazon
1978). This leads to the idea that while the signals from these nutrients
are distinct, they converge in some way to control quiescence and
We first compared transcripts in yeast starved for G, N, or P.
Prototrophic S288C cells were transferred from complete medium to
synthetic medium lacking G, N, or P and incubated until they had
arrested growth as described in Materials and Methods. Total RNA
was isolated and used to probe Affymetrix microarrays. The experi-
ments were conducted using duplicate yeast cultures started from
separate colonies. Paired biological replicate signal intensities for each
mRNA for each starvation condition are shown as a heat map (Figure
1A). In this arrangement, genes at the top of the map with the bright-
est color have the highest hybridization signals, whereas those at the
bottom had the lowest. Replicate experiments are shown side by side,
but in most cases, the replicate values were so similar that they cannot
be visually distinguished.
Qualitatively, the heat map in Figure 1A indicates that all three
nutrient limitations produce regions of strong similarity in transcript
abundance pattern; with the G-depleted samples somewhat distinct
from the N- and P-depleted samples. We calculated Pearson correla-
tion coefficients and produced linear regression plots to compare the
patterns of transcript abundances produced by each type of depletion
(Figure 1B). Each transcript is plotted as a dot positioned with its
abundance in one nutrient condition plotted on the Y-axis, and abun-
dance in the other nutrient condition on the X-axis. Overall, there was
high correlation between the three starvation states (Chua et al. 2006;
Grigull et al. 2004), but as noted, the responses to N and P limitation
were more similar to each other than either was to the response pro-
duced by G limitation. Comparison of the N and P starvation
responses produced a correlation of 0.93, whereas comparison of G
limitation to N and P starvation yielded Pearson correlations of 0.79
and 0.77 respectively.
In general, these results indicate that the quiescent states produced
by the separate nutrient limitations are quite similar in terms of global
gene expression, a conclusion recently reported by Klosinska et al.
(2011). Although we used an S288C strain and Broach and co-workers
in that study used a W303 derivative, both experiments yielded similar
results. Comparing our single time point values with the published
results, we found the highest correlations to their 5760 (96 hr) time
Figure 1 Comparing transcript levels in G-, N-, or P-depleted cells.
Wild-type (S288C) cultures were transferred into S medium lacking G,
N, or P and were cultured until growth was arrested, and then samples
were collected for Affymetrix microarray analysis as described in
Materials and Methods. (A) Heat map plotting normalized log2-
transformed hybridization intensity data for G-, N-, and P-limited sam-
ples arranged by k-means clustering. Independent biological replicate
samples are shown as side-by-side columns; the values are so similar
between replicates that these columns cannot be readily distinguished
by eye in most cases. (B) For each transcript, average intensity data
from (A) is plotted such that the expression level in one nutrient con-
dition is on the X-axis, and intensity value in another condition is on
the Y-axis. G vs. N starvation yielded a Pearson correlation of 0.79;
G vs. P starvation a correlation of 0.77; and N compared with P, a
correlation of 0.92.
Volume 2 September 2012|Growth Response to Nutrients in Yeast|
point samples, with Pearson correlations of 0.78, 0.75, and 0.64 be-
tween the G-, N-, and P-depletion results, respectively.
Nutrient repletion triggers massive changes
in transcript abundance
The cellular pathways for sensing G, N, or P are thought to be quite
distinct (Santangelo 2006; Schneper et al. 2004; Zaman et al. 2008).
Yet, repletion of each of these nutrients causes growth, protein
synthesis, and reversal of quiescence. We hypothesized that repletion
of each nutrient would produce a set of transcriptional changes
overlapping those produced by G (Radonjic et al. 2005; Slattery and
Heideman 2007; Wang et al. 2004).
To test this, we added back the limiting nutrient to quiescent G-,
N- or P-depleted S288C cells and collected samples for microarray
analysis at 60 min. By 90 min, cells had begun to produce buds,
indicating a return to growth (not shown). Figure 2A shows the
changes in gene expression produced by each nutrient, expressed as
fold change (log2) relative to quiescent samples. Red indicates in-
creased expression, and green indicates repression caused by nutrient
Large groups of genes were upregulated by all three nutrient
repletions, as revealed by clusters toward the top of the figure, whereas
other groups, shown toward the bottom, were downregulated by all
three repletions. Other gene sets were nutrient-specific.
As with the data in the first figure, we used dot plots to compare
the fold change induced by one nutrient with the changes induced by
another (Figure 2B). As before, the similarities between the responses
were reflected in the correlation coefficients, and the N- and P-
repletion responses were more similar to each other than they were
to the G response. N and P repletion produced responses with a cor-
relation of 0.82, whereas the response to N repletion produced a cor-
relation coefficient of 0.69 compared with the response to G.
Comparison of the responses to G and P repletion produced a corre-
lation of 0.67.
Growth genes are upregulated by all three nutrients
To identify a core set of genes induced in response to all three
nutrients, we selected sets of genes that were induced by each of the
nutrient repletions. These sets were made up of transcripts that met
the following criteria: P , 0.05, a false discovery rate (q-value) less
than 0.01, and at least a 2-fold induction by the nutrient repletion
(log2 ratio greater than or equal to 1).
This arbitrary cutoff yielded the following gene sets: 1601 increased
by glucose; 877 increased by nitrogen; and 1003 increased by
phosphate. Of the genes upregulated by both nitrogen and phosphate,
83% (P , 2.139 · 102257) were also upregulated by glucose, pro-
ducing an overlapping set of 501 genes induced by each of the three
nutrients (Figure 3).
This common set of 501 genes was greatly enriched for protein
synthesis genes. GO functional classification showed significant enrich-
ment for rRNA processing (,1e214), tRNA processing (,1e214),
ribosome biogenesis (,1e214), rRNA synthesis (,1.84e212), and re-
lated growth functions (Table 1). This set was also significantly enriched
for genes mapping to the nucleolus (,1e214), a key site for ribosome
development. Of the 236 recognized RiBi transcripts we measured, 160
(67%, P , 1.145e2117) were found in this common cluster (Jorgensen
et al. 2004). G, N, and P repletions all induced ribosomal protein (RP)
transcripts; however, the fold induction by G averaged well above the
2-fold cutoff, while for most RP genes, induction by N or P fell slightly
below this cutoff (Table S1).
We also looked for short over-enriched motifs within promoter
regions of each gene in each cluster using RSAT, an online motif
discovery tool for de novo promoter enrichment analysis (Thomas-
Chollier et al. 2008; van Helden 2003). In a related search, we exam-
ined transcription factor target enrichments and binding sequence
Figure 2 Large-scale transcriptional responses to G, N, and P re-
pletion. Wild-type (S288C) cells were starved for G, N, or P until
growth was arrested as in Figure 1, and then the missing nutrient was
repleted as described in Materials and Methods. Samples for micro-
array analysis were taken at the nutrient-limited state and 60 min after
repletion of each missing nutrient. (A) Heat map showing the log2 fold
change for each gene, comparing the nutrient repleted with the initial
quiescent samples. Independent biological replicate samples are
shown as side-by-side columns, and the transcripts are arranged by
k-means clustering. Red indicates increased expression; green indi-
cates reduced. (B) For each transcript, fold change data from (A) is
averaged and plotted such that the fold change in response to one
nutrient condition is on the X-axis, and the fold change in response to
another nutrient is plotted on the Y-axis. The red diagonal line indi-
cates the linear regression: G compared with N repletion yielded
a Pearson correlation of 0.69, whereas compared with P, yielded
0.67. N vs. P repletion had a correlation of 0.83.
| M. K. Conway, D. Grunwald, and W. Heideman
enrichments based on previous genome-wide studies (Macisaac et al.
2006; Robert et al. 2004; Zhu et al. 2009). These enrichments are
summarized in Table 1. We find the set of 501 genes induced in
common by G, N, and P to be significantly enriched for RRPE
(AAAWTTTT) and PAC (GATGAG) elements (Dequard-Chablat
et al. 1991; Hughes et al. 2000; Slattery and Heideman 2007). Of
the 501 G-, N-, and P-induced genes, 64% (P , 2.3e285) contain at
least one RRPE, 53% (P , 6.17e244) at least one PAC, and approx-
imately 42% (P , 3.67e290) contain both an RRPE and a PAC. This is
not surprising in that a connection between genes induced by G re-
pletion and the presence of PAC and RRPE elements has previously
been made (Fingerman et al. 2003; Hughes et al. 2000; Slattery and
Heideman 2007; Wade et al. 2001).
Nutrient-specific differences: genes not upregulated
In addition to genes displaying a common response, we anticipated
nutrient-specific expression patterns. A set of 780 genes met our
criteria for G induction, but not for N or P. These transcripts induced
2-fold specifically by G repletion showed a connection to growth
metabolism, perhaps distinct from the N and P sets because of the
dual energy and carbon source potential provided by G. This set was
significantly enriched for RP genes (P , 1.42e214), containing 97 of
the 137 RP genes (P , 3.83e252). As mentioned above, the RP genes
were also significantly induced by N or P repletion, but they failed to
pass the 2-fold cut off. On closer examination, we found that the RP
transcripts had the greater fold induction in G largely because of
a greater repression during G starvation (See Discussion and Figure
S1). In this G-induced set, we found enrichment for gene targets for
Rap1, Fhl1, and Sfp1, regulators of RP transcription, and we also found
enrichment for their corresponding binding motifs (Fingerman et al.
2003; Lieb et al. 2001; Marion et al. 2004; Pina et al. 2003; Schawalder
et al. 2004). GO enrichments were also observed for translation-related,
protein-trafficking, and ER-localization functions (Table 1).
As might be expected, the subset of 115 N-induced genes was
enriched for genes involved with amino acid biosynthesis and
nitrogen metabolism. This set was enriched for targets bound by
Gcn4, Bas1, Met32, Met4, and Cbf1, and it was also enriched for their
binding sequence motifs (Table 1).
The 183 genes specifically induced by P were less obviously
connected with P metabolism by GO enrichment analysis, appearing
enriched for transcriptional control and DNA binding (Table 1). Ino4
and Ino2 targets are enriched, suggesting a need for phospholipid syn-
thesis after P deprivation, an observation noted previously (Greenberg
and Lopes 1996).
Stress genes are downregulated by all three nutrients
We used the cutoff screen described above to identify a set of 616 genes
downregulated in common by G, N, or P (Figure 4). Each nutrient
decreased the expression of a large group of stress-related genes. Func-
tional classifications showed significant enrichment in the following
groups: oxidative stress response (P , 6.32e29), heat shock response
(P , 3.16e27), autoproteolytic processing (P , 1.07e206), metabolism
of energy reserves (P , 3.1e25), and autophagy (P , 1e214) (Table 2).
We also found this set enriched for peroxisome (P , 6.65e29) and
vacuolar lumen concentration (P , 9.5e24). Perhaps most noteworthy,
this set also contains 197 of the approximately 272 (P , 1.07e2134)
environmental stress response (ESR) genes originally identified by
Gasch et al. (2000). Consistent with this, the group is enriched for
STRE elements (Martinez-Pastor et al. 1996) in the 59 intragenic
regions (Table 2). This group is also enriched for Hsf1, Msn2, and
Msn4 targets important for stress gene regulation (Gasch et al. 2000).
We found 537 genes downregulated by G repletion but not N or P.
This group was enriched for sugar transport (P , 3.15e28), aerobic
respiration (P , 3.5e24), transcriptional control (P , 1.92e28), and
homeostasis of metal ions (P , 2.6e24). This cluster was enriched for
gene products localizing to the mitochondrial inner membrane, con-
sistent with GO enrichment for aerobic respiration. Gal4, Hap1, Sok2,
and Sut1 targets were also enriched in this set, and motifs for Mig1
and Rtg3 were enriched in the 59 intragenic regions (Table 2).
The 136 genes specifically downregulated by N alone were
enriched for catabolism of nitrogenous compounds (P , 8.29e25)
and amino acid/amino acid derivative transport. While this set was
enriched for nitrogen catabolite repression (NCR) genes (P , 1.9e29),
the majority of NCR genes were found in the set repressed in common
by G, N, and P, an observation in concordance with the fact that many
NCR genes are elevated in starvation (Wu et al. 2004). Finally, the 59
intragenic regions of this cluster were enriched for Stp1, Gln3, Bas1,
Gat1 binding targets, and their corresponding sequence motifs.
Figure 3 Genes induced by G, N, and P repletion. Microarray data
from Figure 2 was used to identify genes induced at least 2-fold by G,
N, or P repletion using cutoffs described in Materials and Methods.
The Venn diagram shows the intersections between the sets of genes
induced at least 2-fold by each nutrient. This intersection of three sets
produced seven different groups, and a heat map of fold-change
responses is shown for each of these seven sets. Independent biolog-
ical replicate samples are shown as side-by-side columns, and the
transcripts are arranged by k-means clustering. The number of individ-
ual transcripts in each set is shown in the figure, and the transcripts are
listed for each set in Table S1.
Volume 2September 2012|Growth Response to Nutrients in Yeast|
n Table 1 Transcription factor target and binding motif enrichment, gene ontology enrichment, and overrepresented promoter motifs found in the induced Venn diagram genes
Enriched TF Motifs
Enriched GO Terms
G, N, P
ABF1, YOX1, REB1, LEU3?,
CHA4?, RPH1?, RIM101?,
PT23??, DAL80??, ARR1??
CHA4, STB2, REB1, ABF1,
AZF1, PHO2, XBP1, MOT3?,
rRNA processing (11.04.01),
tRNA modification (11.06.02),
Ribosome biogenesis (12.01),
RNA binding (16.03.03),
rRNA synthesis (11.02.01),
Translation initiation (12.04.01)
SFP1, FHL1, RAP1, SWI6,
MBP1, STB1, SWI4, FKH2,
Ribosomal proteins (12.01.01)
ER to Golgi transport (20.09.07.03)
Triterpene metabolism (01.06.06.11)
Chromosome segregation/division 10.03.04.05)
Glycosylation, deglycosylation 14.07.02.02)
Translation elongation (12.04.02)
GCN4, BAS1, MET32,
RTG3, MET4, CBF1, STP4,
GAT3, STB5?, PUT3?
BAS1, ARG81, ARG80, GCN4,
MET32, MET4, RTG3,
CBF1, SFL1, AFT2
Amino acid metabolism (GO:0006519),
Nitrogen metabolic process (GO:0006807),
Purine nucleotide/nucleoside/ nucleobase
Sulfate assimilation (01.02.03.01),
NAD/NADP binding (16.21.07),
Aminoadipic acid pathway (01.01.06.06.01.03)
INO4, INO2, STP1,
ARG81, RPN4?, HAP3?,
ARG80?, ABF1?, STB5?,
ABF1, SPT23, CBF1, SKN7,
SWI5, AZF1, NRG1,
RPN4, YAP5, MSN4?
Transcriptional control (11.02.03.04),
DNA binding (16.03.01),DNA conformation modification (e.g. chromatin) (10.01.09.05),
Transcription repression (11.02.03.04.03),
General transcription activities (11.02.03.01)
GCN4, STB5, STB1,
TYE7?, RGT1?, GLN3?,
INO4?, SWI6?, STB2?, OPI1?
ARR1, HAP2, RAP1, SPT2,
YAP3, CHA4, HAP4,
GCN4, FKH2, YHP1?
Biosynthesis of tryptophan (01.01.09.06.01),
Pentose-phosphate pathway (02.07),
Biosynthesis of histidine (01.01.09.07.01),
N-directed glycosylation, deglycosylation (14.07.02.02),
Purine nucleotide/nucleoside/nucleobase anabolism (01.03.01.03),
Aminoadipic acid pathway (01.01.06.06.01.03)
STE12, DIG1, FKH2,
SWI4, FKH1, SWI6, STB1,
HAP5?, MATA1?, CHA4?
RLM1, YER051W, SFL1, SWI4,
STB1, SWI5, GLN3?,
MBP1?, THI2?, STB4?
Sexual reproduction (GO:0019953),
Pheromone response, mating-type determination (34.11.03.07),
Ori recognition and priming complex formation (10.01.03.03),
Chitin anabolism (01.05.03.03.04),
G2/M transition of mitotic cell cycle (10.03.01.01.09)
ARG81, YAP7, AZF1,
NRG1, ARG80, SPT23?,
MET32?, GZF3?, MET31?,
CRZ1, XBP1, RTG3, SKO1,
RGT1, CHA4, GAL80, RCS1?, STP1?, RDS1?
Ribosome biogenesis (GO:0042254),
rRNA processing (GO:0006364)(GO:0000447)(GO:0000472)
Ribosomal small subunit biogenesis (GO:0042274),Endonucleolytic cleavage in 59-ETS of pre-rRNA
Each of the seven gene clusters identified in Figure 3A was searched for transcription factor target gene enrichment, transcription factor motifs in gene promoter regions, gene ontology (GO) enrichment, and
overrepresented promoter motifs. The column TF Bound indicates transcription factors whose targets were enriched in that cluster based on data from Harbison et al. (2004) and Macisaac et al. (2006). The column
Enriched TF Motifs indicates transcription factor consensus motifs found to be enriched in gene promoters of each cluster based on the work of Zhu et al. (2009). In both columns, unmarked transcription factors had
enrichment with a P value less than 0.05, whereas?indicates enrichment with a P value less than 0.2, and??a P value greater than 0.2 but less than 0.5. Sliding the P value cut off allowed us to include targets that could
have been masked because of how they were identified originally; for example, by cut off or condition. The column Enriched GO Terms shows enriched gene ontology terms via MIPS functional or GO biological process
categories (see Materials and Methods). Those shown were limited to the top six categories (or less if fewer were identified) all with a P value at least less than 0.0025. Overrepresented Motifs shows DNA sequences
identified in each cluster that were overrepresented in the promoters of those genes. (see Materials and Methods for analysis method).
|M. K. Conway, D. Grunwald, and W. Heideman
Interestingly, the set specifically repressed by P was enriched for
genes associated with glycolysis and gluconeogenesis (P , 7.084e211),
as well as sugar, glucoside, polyol, and carboxylate catabolism (P ,
9.988e27). The 59 intragenic regions of this set were enriched with
Gcr1, Gcr2, and Hap4 targets. As expected, the set was also enriched
for Pho4 targets; however, Pho4 target genes were spread between
several repressed clusters, especially the common G, N, P cluster.
Requirement for cAMP and TOR
The large transcriptional response to G is dependent on sensory
pathways requiring Gpa2, cAMP/PKA, and to a lesser extent, TOR
signaling (Slattery et al. 2008; Wang et al. 2004). Addition of G to
post-log cells has long been linked to activation of PKA via cAMP
(Matsumoto et al. 1985). However, the receptors that link G signals to
cAMP production are quite distinct from those that sense N and P
(Zaman et al. 2008). Nonetheless, the similarities between the tran-
scriptional responses to G, N, and P suggested that PKA and TOR
might be involved in the responses to all three nutrients.
To test this, we selectively blocked PKA, TOR, or both, and we
measured the effect on the transcriptional response to repletion of G,
N, or P. We blocked cAMP production using a cyr1D strain (TC41)
and blocked TOR with rapamycin (Mitts et al. 1990; Russell et al.
1993; Slattery et al. 2008). For each of the three nutrients, both gene
induction and repression were highly dependent on cAMP/PKA and
TOR. The heat map in Figure 5A focuses on the core 501 upregulated
and 616 downregulated genes responding to G, N, or P. Loss of both
PKA and TOR signaling essentially blocked the large-scale transcrip-
tional response to nutrient repletion. We found that most transcript
changes showed a greater dependence on cAMP/PKA than on Tor.
The plots in Figure 5B compare the responses to nutrient repletion
between cells with normal cAMP/PKA and TOR with the responses in
cells when different pathways are blocked. Each transcript on the array
is plotted as a dot, with the X-axis position indicating the fold change
caused by nutrient repletion with both TOR and cAMP/PKA intact;
the position on the Y-axis indicates the response when the indicated
pathways are blocked. The different pathway manipulations are rep-
resented by different colors, and each nutrient repletion, G, N, or P, is
presented as a separate graph.
As a control, we plotted the response to each nutrient in a wild-
type CYR1+strain against our cyr1D strain supplied with cAMP. This
produced an almost perfect diagonal line, shown as dark blue; the line
obscures the dots. This indicates that our cyr1D mutant can respond
normally when cAMP is provided.
TOR blockade (red dots) caused some loss of response, while loss
of cAMP (dark green) had a striking impact on the response to each
nutrient. With both TOR and PKA knocked out (turquoise), the
responses to nutrient addition were substantially reduced: the G and
N responses were almost completely blocked, and a small amount of
the response to P repletion remained.
Nutrient signaling through cAMP
Although the connection between glucose and cAMP production is well
known (Boy-Marcotte et al. 1987; Camonis et al. 1986; Matsumoto
et al. 1982; 1985; Thevelein and Beullens 1985), we were surprised
to find that the response to N and P repletion was cAMP-dependent.
If N and P produce the transcriptional response through cAMP/PKA,
then directly stimulating this pathway by adding cAMP should by-
pass the nutrients and replicate the response. On the other hand, cAMP
might be necessary without playing a direct role in transmitting the
signal. In this permissive role, cAMP alone should be inactive
without N or P.
To test this, cyr1D cells were N or P depleted with cAMP present to
ensure growth into quiescence, and then moved to starvation minus
cAMP for 24 hr. The cultures were then divided, and the missing
nutrient plus cAMP was added to one aliquot, while cAMP alone
was added to the other, and then the samples were prepared for micro-
array analysis. Changes in gene expression produced by cAMP alone are
plotted on the Y-axes, and the responses to the nutrient with cAMP on
the X-axes (Figure 6). The response to cAMP produced transcriptional
changes that were largely similar to those produced by the nutrient itself
with cAMP present. The correlation between cAMP alone and cAMP
and N was 0.79, while the correlation with cAMP and P was 0.87.
Of note is the fact that when cAMP alone was added, these cells
did not grow and under the microscope maintained a quiescent
appearance. The transcriptional response is not sufficient to produce
growth in the absence of the missing nutrient.
Thus, the responses to N and P were largely cAMP-dependent.
Furthermore, cAMP was able to produce similar transcriptional
responses to those produced by N or P repletion. We have previously
observed very similar results with G repletion (Slattery et al. 2008).
The obvious next step was to examine the effect of G, N, and P
repletion on cAMP levels. While glucose tended to produce a more
robust response, in repeated experiments, we consistently found that
all three nutrients increased cAMP levels when added back to
Figure 4 Genes repressed by G, N, and P repletion. Microarray data
from Figure 2 was used to identify transcripts reduced by at least
2-fold by G, N or P repletion compared with quiescence levels as
described in Materials and Methods. The Venn diagram shows the
intersections between the sets of genes repressed at least 2-fold by
each nutrient. This intersection of three sets produced seven different
groups, and a heat map of fold-change responses is shown for each of
these seven sets. Independent biological replicate samples are shown
as side-by-side columns, and the transcripts are arranged by k-means
clustering. The number of individual transcripts in each set is shown in
the figure, and the transcripts are listed for each set in Table S2.
Volume 2 September 2012|Growth Response to Nutrients in Yeast|
n Table 2 Transcription factor target and binding motif enrichment, gene ontology 25 enrichment, and overrepresented promoter motifs found in the repressed Venn diagram genes
Enriched TF Motifs
Enriched GO Terms
HSF1, SN4, MSN2, ROX1,
MIG1, STB5, NRG1,
SNT2, PHO2?, GLN3?
MSN2, MSN4, ADR1, SUT1,
STP1, GZF3, SKN7,
PUT3, MET31, MATA1
Oxidative stress response (32.01.01),
Heat shock response (32.01.05),
Autoproteolytic processing (14.07.11.01),
Regulation of glycolysis and gluconeogenesis (02.01.03),
Protein/peptide degradation (14.13),
Metabolism of energy reserves (e.g. Glycogen,
GAL4, RCS1, HAP1, YAP1,
BAS1, SOK2, SUT1,
SKO1, HAP4, HAP3
MIG1, SUT1, ARG80, AFT2,
CBF1, RCS1, RTG3,CAD1, PHO4, ADR1
Transcriptional control (11.02.03.04),
Sugar transport (20.01.03.01),
Homeostasis of metal ions (34.01.01.01)
Aerobic respiration (02.13.03),
Modification by acetylation, deacetylation (14.07.04),
Transcription initiation (11.02.03.01.01),
STP1, GLN3, DAL82, STB4,
GAT1, UME6, DIG1?,
PUT3?, PDR1?, YDR520C?
UME6, XBP1, GZF3, SOK2,
CST6, RTG3, STB5?,
CRZ1?, GAT1?, GTS1?
Catabolism of nitrogenous compounds 01.02.02.09),
Meiosis I (10.03.02.01),
Amine/polyamine transport (20.01.11),
Meiotic recombination (10.01.05.03.01),
Amino acid/amino acid derivatives transport (20.01.07),
Cytoskeleton-dependent transport (20.09.14)
GCR2, GCR1, TYE7, HAP4,
SUM1, RLR1, RTG3,STB2, CBF1?, MSN2?
GCR1, RCS1, MET4, GCR2,
PHO4, ARG81, RAP1,
MATA1, BAS1, DIG1
Glycolysis and gluconeogenesis (02.01),
Sugar, glucoside, polyol and carboxylate
Cell growth / morphogenesis (40.01),
C-compound and carbohydrate metabolism (01.05),
Purine nucleotide/nucleoside/nucleobase anabolism
THI2, CIN5, STB1?, UME6?,
YAP7?, GAT1??, ARR1??,
CST6??, ARG80??, PHD1??
SUT1, GTS1, DIG1, SOK2,
STE12, GAT1, GZF3,
IME1, MSN2, MET31
Development of asco- basidio- or zygospore (43.01.03.09),
Amino acid/amino acid derivatives transport (20.01.07),Allantoin and allantoate transport (20.01.23),Autophagy (GO:0006914)
MET31,MSN4 MET4, MSN2
GCN4, ACE2, BAS1
MET32, CBF1, UGA3
MSN2, MSN4, MIG1, BAS1,
ARG81, XBP1, CRZ1,
GZF3, STB4, UGA3
Sugar, glucoside, polyol and carboxylate
Tricarboxylic-acid pathway (02.10),
Electron transport and membrane-associated
energy conservation (02.11),
Stress response (32.01),
Aerobic respiration (02.13.03)
RPN4, FKH2, FKH1,NDD1,
CIN5?, REB1?, HSF1?
RPN4, YHP1, MSN2, SUT1,
GAT3, HSF1, NDD1,
IME1?, MSN4?, ADR1?
Modification by ubiquitination, deubiquitination (14.07.05),
Vacuolar/lysosomal transport (20.09.13),
Actin cytoskeleton (42.04.03), ATP binding (16.19.03),
Tetracyclic and pentacyclic triterpenes (cholesterin,
steroids and hopanoids) metabolism (01.06.06.11),
Each of seven gene clusters identified in Figure 3B was searched for transcription factor target gene enrichment, transcription factor motifs in gene promoter regions, gene ontology (GO) enrichment, and over-
represented promoter motifs. Otherwise, columns are as described in Table 1.
| M. K. Conway, D. Grunwald, and W. Heideman
quiescent cells (Figure 7). The sustained cAMP levels following G
repletion are consistent with our previous results (Russell et al. 1993).
Cross talk between nutrient signals
The fact that cAMP is required for the transcriptional responses to G,
N, and P, that the nutrients produce increases in cAMP, and that
cAMP alone can simulate the repletion response indicates that nutrients
regulate these large changes in part through cAMP/PKA. However, this
model poses problems: How can a cell halt growth when just one
nutrient is depleted? How does the cell know which nutrient is sending
the signal? Indeed, we wondered whether there were cases where
adding one nutrient could substitute for the lack of another.
To examine this, we simultaneously depleted cells for two nutrients,
and then repleted only one of the pair. We measured changes in
transcripts when we added each nutrient alone or with its missing
partner. For comparison, we also repeated the depletion experiments in
which a single nutrient was depleted and repleted (Figure 8A).
As expected, The GP- and GN-depleted cells showed robust
responses to repletion of both limiting nutrients together (Figure 8, B
and C). These responses were largely similar to those studied earlier
when a single nutrient was depleted and repleted (Figure 8A). However,
addition of N alone to a GN-depleted culture produced almost no re-
sponse; nor did addition of P to a GP-depleted culture. In contrast,
Figure 5 The effect of PKA and TOR signaling in the transcriptional
response to G, N, and P. Cells carrying a cyr1D mutation (TC41) were
starved for G, N, or P until growth arrested, and cAMP was removed as
described in Materials and Methods. Samples for microarray analysis
were taken at the starved state and 60 min after repletion of each
missing nutrient. In some samples, rapamycin (200 nM) was added
during repletion to block the TOR pathway, and in some samples,
cAMP (1 mM) was omitted from the repletion medium to block the
cAMP/PKA pathway. (A) Heat map showing the average log2 fold
change for each of the 501 (top panel) or 616 genes (bottom panel)
identified in Figures 3 and 4 as induced or repressed in all three
nutrient-repletion conditions. Heat maps were k-means clustered.
Red indicates induction relative to the starved state; green indicates
repression; and black, no change. The + symbols indicate active and
the 2 symbols indicate blocked pathways, produced by adding and
subtracting rapamycin and cAMP. (B) For each transcript, the average
fold change in expression induced by nutrient repletion in the cyr1D
mutant +cAMP and 2rapamycin is plotted on the X-axis (+PKA,
+TOR). Responses under different conditions are shown on the Y-axis
as follows: red is fold change produced in cyr1D cells treated with
rapamycin (2TOR); green is fold change produced in cyr1D cells with-
out cAMP (2PKA); light blue is fold change produced by nutrients in
cyr1D cells without cAMP and with rapamycin (2PKA-TOR); and dark
blue is fold change produced by nutrients in the isogenic wild-type
cells (HR125). The slope of each line indicates the severity of signaling
inhibition across the genome relative to the +PKA, +TOR samples.
Figure 6 Response of N- or P-starved cells to cAMP addition. Cells
carrying a CYR1 deletion (TC41) were grown until limited for N or P as
described in Materials and Methods, and then challenged with either
cAMP alone or cAMP with the limiting nutrient. Samples were col-
lected at the starved state and 60 min after repletion for microarray
analysis. For each transcript, the average fold change in expression
induced by cAMP plus nutrient is plotted on the X-axis, and the re-
sponse to cAMP alone is plotted on the Y-axis. Comparison of the
response to cAMP alone with cAMP plus N produced a Pearson cor-
relation of 0.79, and comparison of cAMP alone with cAMP plus P
yielded a correlation of 0.87.
Volume 2 September 2012| Growth Response to Nutrients in Yeast|
addition of G alone, while not producing growth, produced a large
transcriptional response in the doubly depleted cultures, despite the
fact that the cells were still growth limited by N or P.
Origin of nutrient signals
Gap1 activates trehalase in response to amino acid stimulation
(Donaton et al. 2003). To determine whether Gap1 carries signals
regulating the large-scale response to nitrogen repletion, we chal-
lenged wild-type and gap1D cells with citrulline and then collected
RNA for microarray analysis. We used citrulline because it is a rela-
tively specific Gap1 agonist (Donaton et al. 2003; Grenson et al. 1970).
We found that addition of citrulline to N-depleted wild-type cells
produced a marked change in transcript levels relative to the starved
state as shown in the heat map in Figure 9. However, this response
was almost entirely silenced by loss of GAP1, indicating that the signal
induced by citrulline repletion requires Gap1.
The dot plot in Figure 9B shows for each transcript the fold change
produced by L-citrulline plotted on the X-axis for wild-type cells and
on the Y-axis for the gap1D mutant samples. For most transcripts that
responded to citrulline, loss of Gap1 had a profound effect.
The box and whisker plots in Figure 9C show the effect of citrul-
line on the sets of 501 and 616 core genes induced or repressed at least
2-fold by G, N, or P in Figures 3 and 4. It can be seen that the
responses produced by L-citrulline average less than 2-fold, so L-
citrulline produces a less robust response than the original N reple-
tion, which included both ammonium and amino acids. However,
more than 75% of the 501 genes in this set were induced by citrulline,
and this was almost completely dependent on Gap1. Repression was
The ammonium permeases Mep1, Mep2, and Mep3 are all in-
volved in ammonium activation of trehalase, although Mep2 produces
the most prominent signal (Van Nuland et al. 2006). Mep2 has also
been implicated in regulating the ammonium control of pseudohyphal
growth (Lorenz and Heitman 1998).
As in the previous experiment, we used N-starved wild-type or
mep1D, mep2D, or mep3D, mutant cells and challenged them with 10
mM ammonium. The heat map shows that each mutation has some
impact on the overall response to ammonium, with the mep3D mu-
tant producing the most blunted response (Figure 10). The relative
impacts that the mutations had are shown in the dot plot in Figure
10B, and the effects on the 501 induced and 616 repressed genes
described above are shown in the box and whisker plots of Figure
10C. Overall, it appears that, as with trehalase, all three MEP genes
contribute to the transcriptional response to ammonium repletion.
phorous sensor (Popova et al. 2010). We challenged wild-type and
pho84D cells with two P sources: KH2PO4, the a Pho84-transportedP
source used in the previous repletions, and glycerol-3-phosphate
Figure 7 G, N, and P repletions increase cAMP. Prototrophic yeast
(S288C) were starved for G, N, or P and then repleted with the missing
nutrient as described in Materials and Methods. Levels of cAMP were
measured at the indicated time after repletion using an ELISA assay,
with each well receiving extract from the same number of cells (5 OD
units). Error bars represent the standard error the mean (n = 3). The
experiment was repeated multiple times with similar results.
Figure 8 Cross talk between nutrient signals. S288C cells were starved
for the indicated nutrients and repleted with either a single nutrient or
both of the limiting nutrients as described in Materials and Methods.
(A) As a point of reference, the results for depletion and repletion of
single nutrients (G, N, and P) are shown, and the k-means clustering
pattern of this set of single nutrient repletions was used to order the
other two panels. (B) Cells limited for both G and N were repleted with
G, N, or both, and then cells were collected for microarray analysis
60 min after nutrient addition. Duplicate experiments were conducted,
and the heat map shows the average log2 fold-change ratio induced
by the nutrients for each transcript. Red indicates induction relative to
the starved state; green indicates repression; and black, no change. (C)
Samples were depleted for both G and P and repleted as indicated.
| M. K. Conway, D. Grunwald, and W. Heideman
(Gly3P) as a specific agonist for Pho84 (Popova et al. 2010). We found
that loss of PHO84 produced different effects depending on the P
source. As shown by the heat maps and dot plots in Figure 11, the
response to Gly3P was considerably attenuated by loss of PHO84, while
the KH2PO4response was not.
Figure 11C shows a box and whisker plot focused on the sets
of 501 and 616 transcripts upregulated and downregulated, respec-
tively, by all three nutrients. The effect of PHO84 deletion on the
KH2PO4response was quite subtle: the response to Gly3P repletion
was more distinct. Overall, these data suggest that PHO84 plays a role
in, but is not solely responsible for, signaling the transcriptional re-
sponse to P.
Growth and quiescence
The ability of yeast cells to cease division and attain a stress-resistant
state in response to different nutrient limitations was recognized some
time ago (Lillie and Pringle 1980; Paris and Pringle 1983). This some-
what commonplace occurrence has several implications. First, it
appears that quiescence and growth, while influenced by different
conditions, are two fundamental states for yeast cells. This conclusion
was recently strengthened by Klosinska et al. (2011). Another impli-
cation is that cells must use different nutrient signals to control entry
into growth and quiescence.
A simple idea would be that as any nutrient becomes limiting,
metabolic activity subsides and that drop is sensed. While this plan
relieves the cell of having to sense many different important nutrients,
two arguments can be made against it. First, allowing critical resources
to reach such low levels so as to limit metabolism would be expected
to substantially reduce fitness; in fact, nutrients are stored prior to
quiescence. Perhaps more compelling are the numerous nutrient-
sensing mechanisms that are now being discovered. Indeed, many of
the responses described in this article can be observed under con-
ditions in which the cell cannot, or does not, metabolize the nutrient
producing the signal.
One important difference that we noted between nutrient-limited
states was that cells deprived of glucose respond more conservatively
than those limited for N or P. This is best observed in the group of 137
RP genes, which are considerably more repressed when cells are
limited for G than by N or P. In fact, it was because of this failure to
repress the RP genes during N or P starvation that the N induction of
this set by N or P failed to make the $ 2· cut off (Figure 3 and Figure
S1). A similar effect was observed with the 230 RiBi transcripts, being
on average about 2-fold more repressed in G-depleted cells than in N-
or P-depleted cells (Figure S1). This effect contributes to the observa-
tion that the fold changes produced by N and P resembled each other
more closely than they resembled the response to G.
While N and P are needed primarily as elemental building blocks,
limitation for G affects both the availability of carbon and cellular
energy. We speculate that because cells are adept at recycling nutrients
as long as energy is plentiful, they can afford to maintain higher levels
of RP and RiBi gene expression if G is present than they do if starved
for G and the energy G provides. The RP and RiBi transcripts rep-
resent a large fraction of the total mRNA.
A common response triggered by different
Glucose has been studied in relation to growth, metabolic regulation,
respiration, and glucose repression (Carlson 1999; Hedbacker and
Carlson 2008). Many studies of N signalng have focused on the role of
Many studies have focused on nitrogen metabolism and the TOR path-
way (Cardenas et al. 1999; Cooper 2002; Crespo et al. 2002; Hardwick
et al. 1999; Hinnebusch 2005). A similar approach has been used in
studying P signals, focusing on phosphate in metabolism (Wykoff and
O’Shea 2001). This has produced models in which G, N, and P initiate
signals in yeast through very distinct pathways (Forsberg and Ljungdahl
2001; Magasanik and Kaiser 2002; Schneper et al. 2004). With this
in mind, it was possible that the transcriptional responses might
converge in a minor way but be largely distinct and non-overlapping.
A set of experiments by Thevelein et al. (2005) indicated that all
three nutrients were tied together in a PKA-dependent process (Giots
et al. 2003; Popova et al. 2010; Van Zeebroeck et al. 2009). However,
Figure 9 Effect of GAP1 deletion on the transcriptional response to
N. Wild-type (BY4742) or the isogenic gap1D cells were starved for N
as in Figure 1 and then challenged with 10 mM L-citrulline. Samples
for microarray analysis were collected at the starved state and 60 min
after L-citrulline repletion. (A) The heat map shows the log2 fold-
change results for each transcript of duplicate independent experi-
ments in parallel columns: red indicates induction relative to the
starved state, and green indicates repression. The k-means method
was used to arrange gene clusters. (B) Dot plot in which the average
log2 response to L-citrulline is plotted for each transcript. The fold-
change response in wild-type cells is plotted on the X-axis, and the
response in the gap1D mutant is shown on the Y-axis. (C) Box plots
summarizing the expression of the 501 induced or 616 repressed
genes from Figures 3 and 4 in either the wild-type or the gap1D
strains. The black band in the box indicates the median; the upper
box limit, the 75thpercentile; the lower box limit, the 25thpercentile;
and each whisker, the minimum and maximum value within 1.5· of
the interquartile range, respectively. The asterisk indicates a signifi-
cant difference (P , 0.05).
Volume 2 September 2012| Growth Response to Nutrients in Yeast|
this work left two points in question. First, trehalase activation, while
providing an indirect measurement of PKA activity, does not shed
light on the scope of the response. Second, only G, and not N or P,
was thought to increase cAMP.
We found that the response to G, N, and P involves a core set of at
least 1117 genes: approximately 18% of the yeast genome. We
conclude that the need to induce genes for growth and to repress
unneeded stress genes is compelling and generalized. The gene set
induced was enriched for ribosome biogenesis and translation genes;
these would be required for growth regardless of the initiating nutrient
signal. By the same token, the set of 616 repressed genes was enriched
with stress response genes (Gasch et al. 2000). Cells initiating growth
in newly repleted medium under favorable conditions are best off
putting their energy into other processes and reducing stress-response
transcripts, again regardless of the repleted nutrient.
Role of cAMP and PKA
Glucose induces cAMP production (Boy-Marcotte et al. 1987;
François et al. 1987; Russell et al. 1993). However, while N and P
have been shown to produce responses indicative of PKA activation,
there has been little if any positive link between the repletion of these
nutrients and cAMP production (Donaton et al. 2003; Popova et al.
2010; Van Nuland et al. 2006; Van Zeebroeck et al. 2009). In our
hands, all three repletions consistently produced increases in cAMP
levels that were sustained over an hour. This is consistent with the
trehalase activation as well as the cAMP requirement for the large-
scale transcriptional response to all three nutrients and the ability of
cAMP itself to mimic nutrient repletion in the absence of the missing
Clearly, induction and repression of mRNAs is not enough to
make cells grow: metabolism, protein synthesis, and many other
processes are required in parallel. Thus, simply creating a large
transcriptional response by itself without also providing the missing
nutrient did not produce growth.
While the TOR pathway is clearly important in nutrient signaling
(Cardenas et al. 1999; Cooper 2002; Wullschleger et al. 2006), we
found TOR inhibition produced a smaller overall effect than loss of
cAMP production. In fact the largest effect was observed when both
pathways were blocked, suggesting overlapping and partially redun-
dant roles for the two kinase pathways (Martin et al. 2004; Roosen
et al. 2005; Santangelo 2006; Zurita-Martinez and Cardenas 2005).
The response to G repletion can be explained by stimulation of the
glucose receptor Gpr1, coupled to the Gpa2 G-protein to stimulate
cAMP production by adenylyl cyclase, encoded by CYR1. While not
fully understood, this mechanism of cAMP generation has long been
studied (Santangelo 2006; Zaman et al. 2008, 2009). The connections
between N, P, and cAMP/PKA are less well understood.
Gap1 has recently been discovered to produce signals in response
to amino acid substrates to control Gap1 processing as well as treha-
lase activation (Cain and Kaiser 2011; Kriel et al. 2011; Merhi et al.
Figure 10 Role of Mep transceptors in the response to
N. Wild-type (BY4742) and the isogenic mep1D,
mep2D, and mep3D cells were starved for N, and then
challenged with 10 mM ammonium sulfate. Samples for
microarray analysis were taken at the starved state and
60 min after repletion. (A) The heat map shows the
results of duplicate experiments presented in parallel
columns for the wild-type and the MEP mutants. Results
are shown as log2 fold change, with red indicating in-
duction by ammonium, and green repression relative to
the initial quiescent samples. The patterns were
obtained using k-means clustering. (B) Dot plot with
log2 fold change produced by ammonium in wild-type
plotted on the X-axis, and the responses in each of the
MEP mutants plotted on the Y-axis. Each dot represents
a gene, and the black line indicates the pattern
expected for a perfect correlation. The mep1D response
is shown in green; the mep2D , in red; and the mep3D,
in light blue. (C) Box plots summarizing the results for
the core sets of 501 induced and 616 repressed genes
as in Figure 9C. The black band in the box indicates the
median; the upper box limit, the 75thpercentile; the
lower box limit, the 25thpercentile; and each whisker,
the minimum and maximum value within 1.5· of the
interquartile range, respectively. The asterisk indicates
a significant difference (P , 0.05).
| M. K. Conway, D. Grunwald, and W. Heideman
2011; Van Zeebroeck et al. 2009). Citrulline has been identified as
a specific Gap1 agonist that can generate a signal inducing trehalase
without being metabolized as a nitrogen source (Donaton et al. 2003).
We found the response to L-citrulline was Gap1-dependent. There-
fore, in addition to regulating the PKA-dependent posttranslational
increase in trehalase, we found that Gap1 plays a role in our cAMP-
dependent transcriptome response to N repletion.
Mep2 is involved in controlling pseudohyphal growth and treha-
lase activation (Rutherford et al. 2008; Van Nuland et al. 2006).
Therefore, Mep protein involvement in the transcriptional response
to ammonium is not surprising. We found that each of the Mep1–3
proteins played a role in the response to ammonium.
There has also been growing evidence that Pho84 can serve as
a transceptor regulating both aspects of phosphate metabolism and
PKA-dependent trehalase activation (Giots et al. 2003; Lagerstedt et al.
2004; Lundh et al. 2009; Thevelein et al. 2005). Gly3P has been found
to be a transceptor agonist that can activate Pho84 without transport
by Pho84 (Popova et al. 2010). Loss of Pho84 had little impact on the
response to orthophosphate repletion, but it did inhibit the response
to Gly3P. Because Gly3P signaling is largely Pho84-dependent, this
suggests that the receptors for orthophosphate are redundant: other
receptors maintain the response in the absence of Pho84. We specu-
late that Gly3P is not a good agonist for the redundant receptors.
Prior to the advent of microarrays, a prevalent idea was that yeast
growth fueled transcription in general. We show that large changes in
mRNA abundance precede growth rather than result from growth and
that they are readily decoupled from growth. For example, both
citrulline and Gly3P are known agonists for their respective trans-
ceptors, yet they are poor nutrients. However, both of these agents
produced a large-scale response. A strain unable to take in and grow
on glucose still produces a normal large-scale transcriptional response
to G repletion (Slattery et al. 2008). In dually starved cells, addition of
G alone produced a transcriptional response that was not sufficient to
induce growth. Finally, the addition of cAMP mimicked the nutrient
repletion responses in cells that remained nutrient depleted. These
results are evidence for a sensing system that can directly determine
when a missing nutrient is restored.
Because G, N, and P each appear to signal through PKA in a cAMP-
dependent manner, it is not clear how the cell can differentiate
between the nutrients. In fact, one would predict that cross talk would
occur. We found some evidence for cross talk in which G alone
induced transcriptional responses in dually GN- or GP-limited cells. N
or P alone did not produce a response when G was absent. We have
found that G upshifts can produce some transcriptional responses in
N- or P-limited cultures (data not shown). Griffioen et al. (1996)
observed that G upshift produced RP gene induction that was initially
independent of growth. With the information available at present, we
conclude that the three nutrients examined produce a common re-
sponse through mechanisms that have common elements, yet in at
least some circumstances, they can maintain specificity. We speculate
that the response to G, which provides energy as well as material, has
features that are qualitatively different from the other responses. How
the cell actually distinguishes between nutrients remains a mystery.
We thank B. Jansch for help in developing the manuscript. Funding
for this research was provided by the DOE Great Lakes Bioenergy
Research Center supported by the US Department of Energy.
Figure 11 Role of PHO84 in the transcriptional response to P. Wild-type
(BY4742) or isogenic pho84D cells were starved for P, and then challenged
with KH2PO4or Gly3P as described in Materials and Methods. Samples for
microarray analysis were taken from the initial quiescent culture and at
60 min after repletion. (A) The heat map shows the results of duplicate
experiments presented in parallel columns for the wild-type and the
pho84D mutants. Results are shown as log2 fold change, with red indicat-
ing induction by the P source, and green repression relative to the initial
quiescent samples. The patterns were obtained using k-means clustering.
(B) Dot plots showing responses to KH2PO4and Gly3P in wild-type and
pho84D mutant cells. The log2 foldchange for the wild-type cells is plotted
on the X-axis, and the response in the pho84D mutant is plotted on the
Y-axis. Each dot represents a gene, and the black line indicates the pattern
expected for a perfect correlation. (C) Box plots summarizing the results for
the coresets of501 inducedand616repressedgenesas in Figures 9Cand
10C. The presence of the wild-type PHO84 gene is indicated by a + sign.
The black band in the box indicates the median; the upper box limit, the
75thpercentile; the lower box limit, the 25thpercentile; and each whisker,
the minimum and maximum value within 1.5· of the interquartile range,
respectively. The asterisk indicates a significant difference (P , 0.05).
Volume 2September 2012|Growth Response to Nutrients in Yeast|
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Glucose signaling in Saccharomyces cerevisiae.
Sense and sensibility: nutritional
Coordinated regulation of growth
Protein kinase A, TOR,
Cyclic AMP and the stimulation of
Control of cell division in Saccharomyces
TOR signaling in growth
Phosphate transport and sensing in
Tor and cyclic AMP-
Communicating editor: C. S. Hoffman
Volume 2September 2012|Growth Response to Nutrients in Yeast|