EUKARYOTIC CELL, Feb. 2008, p. 358–367
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 7, No. 2
Protein Kinase A, TOR, and Glucose Transport Control the Response
to Nutrient Repletion in Saccharomyces cerevisiae?†
Matthew G. Slattery,1Dritan Liko,2and Warren Heideman2*
Pharmaceutical Sciences, School of Pharmacy,1and Department of Biomolecular Chemistry,2University of Wisconsin,
Received 6 September 2007/Accepted 8 December 2007
Nutrient repletion leads to substantial restructuring of the transcriptome in Saccharomyces cerevisiae. The
expression levels of approximately one-third of all S. cerevisiae genes are altered at least twofold when a
nutrient-depleted culture is transferred to fresh medium. Several nutrient-sensing pathways are known to play
a role in this process, but the relative contribution that each pathway makes to the total response has not been
determined. To better understand this, we used a chemical-genetic approach to block the protein kinase A
(PKA), TOR (target of rapamycin), and glucose transport pathways, alone and in combination. Of the three
pathways, we found that loss of PKA produced the largest effect on the transcriptional response; however, many
genes required both PKA and TOR for proper nutrient regulation. Those genes that did not require PKA or
TOR for nutrient regulation were dependent on glucose transport for either nutrient induction or repression.
Therefore, loss of these three pathways is sufficient to prevent virtually the entire transcriptional response to
fresh medium. In the absence of fresh medium, activation of the cyclic AMP/PKA pathway does not induce
cellular growth; nevertheless, PKA activation induced a substantial fraction of the PKA-dependent genes. In
contrast, the absence of fresh medium strongly limited gene repression by PKA. These results account for the
signals needed to generate the transcriptional responses to glucose, including induction of growth genes
required for protein synthesis and repression of stress genes, as well as the classical glucose repression and
hexose transporter responses.
The transition from quiescence to proliferative growth is an
important biological process for organisms of every variety, a
process that, when misregulated, can have serious implications.
The laboratory yeast Saccharomyces cerevisiae regularly makes
this transition when nutrient-starved, quiescent cultures are
resuspended in fresh glucose medium. Clonal expansion in
response to favorable nutrient conditions requires both an
increase in cellular mass (cell growth) and an increase in cell
number (cell proliferation). Accordingly, the cell that initiates
growth and division soonest is at a selective advantage, as it will
generate the most progeny as resources are depleted.
Refeeding of quiescent yeast leads to a robust transcrip-
tional response in which thousands of genes are induced or
repressed within minutes. Early microarray work demon-
strated that approximately a third of the yeast genome is reg-
ulated as cells growing in rich medium deplete glucose and
shift to slow growth on ethanol (7). More recent work has
focused on the transition from slow growth or stationary phase
to resumption of growth in glucose media (27, 34, 49). The
general response to nutrient repletion consists of a rapid in-
duction of genes involved in mass accumulation and cell divi-
sion along with repression of genes necessary for respiration,
gluconeogenesis, and stress resistance (42).
Yeast cells have multiple pathways for sensing the presence
of nutrients. The TOR (17, 26, 51) and cyclic AMP (cAMP)-
dependent protein kinase A (PKA) (40, 41, 49) signaling path-
ways have both been implicated in regulating genes that are
induced during nutrient repletion, and there is evidence for
signals generated by the transport of glucose into the cell and
subsequent aerobic fermentation (16, 30).
Transcriptional profiling and phenotypic evidence both sug-
gest that the TOR pathway is a primary carrier of nutrient
signals (4, 9). Treatment of logarithmically growing yeast with
the small molecule rapamycin, a potent inhibitor of the Tor
proteins, leads to downregulation of many nutrient-sensitive
protein synthesis genes and arrests cells in the G1phase of the
cell cycle (4, 9, 12). While these experiments demonstrate a
role for the TOR pathway in maintaining the expression of
genes related to protein synthesis and mass accumulation dur-
ing exponential growth, the effect of TOR blockade on the
massive changes in transcript levels caused by refeeding has
not been tested.
Activation of the cAMP/PKA pathway by either the Ras
GTPases or the G-protein-coupled receptor Gpr1 promotes
protein synthesis and cell division, while repressing stress re-
sponses (40, 41). A recent study by Broach and colleagues
demonstrated that artificial induction of the cAMP pathway by
either Ras2 or the G? homolog Gpa2 mimics the transcrip-
tional response to glucose repletion (49). However, the re-
searchers also found that this large-scale response to glucose
occurs in cells containing a cAMP-insensitive PKA mutant,
indicating a significant role for one or more unidentified
Glucose entry into the yeast cell also generates signals that
regulate transcription. This mechanism is most evident in the
case of carbon catabolite repression, in which glucose transport
ultimately leads to Mig1-mediated repression of gluconeogen-
* Corresponding author. Mailing address: School of Pharmacy, Univer-
sity of Wisconsin, 777 Highland Avenue, Madison, WI 53705. Phone:
† Supplemental material for this article may be found at http://ec
?Published ahead of print on 21 December 2007.
esis and respiration genes (8, 40, 41). Glucose transport and
glycolysis also appear to be necessary for the induction of
certain cell cycle transcripts (30). Nevertheless, the role of
glucose import in the response to nutrient repletion has not
been studied at the whole-genome level, and its overall con-
tribution to this response is unclear.
In this report, we compare the contributions made by TOR,
PKA, and glucose transport to the overall transcriptional re-
sponse to nutrient repletion in yeast. While much of the re-
sponse is dependent on PKA, signals generated by TOR and
glucose internalization also play important roles in the total
response. We find that simultaneous inhibition of PKA and
TOR is sufficient to prevent a majority of the transcriptional
responses. Those responses that are not blocked by loss of
PKA and TOR are abolished when glucose transport is
blocked. Taken together, our results demonstrate that PKA,
TOR, and glucose uptake are sufficient to account for the
entire transcriptional response to glucose repletion.
MATERIALS AND METHODS
Yeast strains and growth conditions. The media used were YPD (1% yeast
extract, 2% peptone, 2% glucose) and YP-glycerol-lactate (1% yeast extract, 2%
peptone, 2% glycerol, 2% lactate). Nutrient-depleted YPD was created by fil-
tering media from a 96-h wild-type culture. The strains used in this study were
TC41 (MATa leu2-3,112 ura3-52 trp1-1 his3-532 his4 cyr1::URA3 cam) (11),
MC996A (MATa his3-11,15 leu2-3,112 ura3-52) (36), and KY73 (MATa his3-
11,15 leu2-3,112ura3-52 hxt1?::HIS3::hxt4?
hxt3?::LEU2::hxt6? hxt7?::HIS3 gal2?) (20). Rapamycin (10-?g/ml stock in eth-
anol; LC Laboratories) and cAMP (1 M stock in water, pH 7; Sigma) treatments
were as described previously (30, 38).
Experimental setup. TC41 (cyr1?) cells were grown to glucose exhaustion (48
h; optical density at 600 nm [OD600] ? 4) in YPD containing 1 mM cAMP. The
cyr1? cells were then transferred to spent YPD medium, harvested from a
4-day-old wild-type culture that had been grown in YPD lacking cAMP (final
OD600? 5); the cells were incubated in this nutrient-depleted YPD for 24 h. This
produced a quiescent culture of viable cyr1? cells in nutrient-depleted medium
lacking cAMP. We challenged quiescent cyr1? cells with fresh YPD medium,
either with cAMP to allow the normal activation of PKA or without cAMP to
prevent PKA activation. The TOR pathway was manipulated by adding the TOR
inhibitor rapamycin (Fig. 1).
Specifically, the quiescent culture was then split in two; TOR signaling was
inhibited with rapamycin in one half while the other half was untreated. After 15
min, aliquots from each culture were collected for microarray analysis (the
baseline samples). Cells from the untreated quiescent culture were then diluted
(final OD600? 0.5) in fresh YPD supplemented with 5 mM cAMP (PKA?/
TOR?) or YPD lacking cAMP (pka?/TOR?). Concurrently, cells from the
rapamycin-treated quiescent culture were diluted in fresh YPD containing 5 mM
cAMP and 200 nM rapamycin (PKA?/tor?) or YPD lacking cAMP but contain-
ing 200 nM rapamycin (pka?/tor?). Cells were collected after 1 h for microarray
To assess the transcriptional response to fresh medium in a strain that is
unable to take up and metabolize glucose, we used a strain derived from
MC996A (KY73) that lacks the HXT1-7 and GAL2 hexose transporter genes (20,
30). Because KY73 (henceforth referred to as hxt?) cannot import and metab-
olize glucose, it was necessary to grow these cells to starvation (3 days) in
glycerol-lactate rather than in glucose prior to the shift to fresh YPD. We also
performed the glycerol–lactate-to-YPD shift experiment in a wild-type strain
(MC996A) that is isogenic to the hxt?strain.
Labeled cRNA preparation and microarray hybridization. Total yeast RNA
was isolated using an Epicentre MasterPure yeast RNA purification kit. cDNA
and labeled cRNA were generated from total yeast RNA by using a GeneChip
one-cycle target labeling kit (Affymetrix) according to the manufacturer’s pro-
tocol. Briefly, first-strand cDNA was generated using a T7-oligo(dT) primer and
SuperScript II reverse transcriptase. Second-strand cDNA synthesis was per-
formed using Escherichia coli DNA ligase, E. coli DNA polymerase I, and RNase
H, followed by incubation with T4 DNA polymerase. After cleanup of cDNA,
biotin-labeled antisense cRNA was generated using an IVT labeling kit. Cleanup
and fragmentation of labeled cRNA was performed using a GeneChip sample
cleanup module. Labeled cRNA was then mixed with hybridization controls and
hybridized to a yeast genome S98 array (Affymetrix) at 45°C with rotation (60
rpm) for 16 h. Microarrays were then washed and stained with streptavidin-
phycoerythrin in a model 400 GeneChip fluidics station.
Microarray data analysis. Affymetrix yeast genome S98 arrays were scanned
using an Agilent GeneArray scanner and Microarray Suite 5.0. The MAS-gen-
erated .CEL files were analyzed using DCHIP 1.3 (22). Intensity values were
normalized across all 24 microarrays by using DCHIP’s invariant set normaliza-
tion method (21). Model-based analysis, including log2transformation of expres-
sion indexes by use of the perfect match-mismatch difference model, was per-
formed using values from duplicate microarrays for each time point (21).
Clustering was performed using the k-means algorithm in TIGR Multiexperi-
ment Viewer, version 3.1 (39). Array data for each transcript, as well as lists of
the transcripts contained in each cluster, are included in the supplemental ma-
Verification of experimental design. First, we compared the transcriptional
response of quiescent cyr1? cells shifted to YPD-5 mM cAMP with the response
to YPD reported for wild-type cells (42, 49). We found that the addition of
YPD-5 mM cAMP produced an expression profile in the cyr1? strain that was
consistent with the normal response to YPD previously described (not shown). In
addition, since we needed to grow the hxt?cells in the absence of glucose, we
were also concerned that cells grown to quiescence in YP-glycerol-lactate me-
dium might not respond to fresh medium in the same way as cells grown to
post-log phase in YPD. Again, we found that the starting medium did not
produce an appreciable difference in the response (not shown). Of the YPD-
responsive transcripts described in the legends to Fig. 3 and 4, more than 85%
(P ? 1 ? 10?140) responded in the same direction (at least 1.5-fold as reported
for the 60? point) in a previously published nutrient repletion experiment (42).
Change in response to nutrients is dependent upon expression levels at star-
vation and expression levels after nutrient addition. To ensure that the effect of
blocking a given pathway is due to expression differences after nutrient addi-
tion—not differences in expression during starvation—we had to confirm that
our starvation expression profiles were similar. In other words, starved cyr1? cells
FIG. 1. Experimental design. cyr1? (TC-41) cells were grown for
48 h in YPD supplemented with 1 mM cAMP, centrifuged, and resus-
pended in nutrient-depleted YPD without cAMP to (final OD600? 5;
see Materials and Methods). After 24 h, the culture was split in two
and TOR signaling was inhibited with rapamycin (200 ng/ml, final
concentration) in one half, while the other half was untreated. After 15
min, aliquots from each starved culture were collected for microarray
analysis. Immediately afterward, the two cultures were diluted (final
OD600? 0.5) into fresh YPD maintaining the original concentration of
rapamycin, and each culture was split to yield a total of four cultures,
two with rapamycin and two without. cAMP was added to one culture
of each pair to yield the following conditions: 5 mM cAMP and no
rapamycin (PKA?/TOR?), no cAMP or rapamycin (pka?/TOR?), 5
mM cAMP and 200 nM rapamycin (PKA?/tor?), and no cAMP and
200 nM rapamycin (pka?/tor?). Cells were then incubated for 1 h, and
aliquots were collected for microarray analysis. Wild-type (WT;
MC996A) and isogenic hxt1-7? gal2? (KY73) cells were grown for 3
days in YP-glycerol-lactate and transferred to fresh YPD at an OD600
of 0.5. Samples were collected before and after the shift to YPD for
microarray analysis. Each experiment was done in duplicate.
VOL. 7, 2008TRANSCRIPTIONAL RESPONSE TO FRESH MEDIUM IN YEAST 359
had to have an expression profile similar to that of starved cyr1? that had been
treated with rapamycin, and the starved hxt?cells had to be comparable to the
corresponding wild-type control. We found that our starting quiescent strains
had very similar expression profiles. Though the addition of rapamycin mimics
nutrient starvation in growing cells (4, 9), rapamycin did not alter the expression
profile of starved cyr1? cells (R2? 0.97). Similarly, the nutrient-starved wild-type
and hxt?profiles were also consistent with each other (R2? 0.93).
Analysis of gene sets. Overrepresented DNA motifs were extracted using the
oligonucleotide analysis pattern discovery program in the Regulatory Sequence
Analysis Tools resource (46). Each gene set was tested for overrepresented 6-
through 8-mers, and when multiple motifs were deemed significant (Regulatory
Sequence Analysis Tools significance index greater than 1), only the top three
dissimilar, nonoverlapping motifs were chosen. Overrepresented functional cat-
egories were determined using the MIPS functional catalogue database (37). The
three most significant nonredundant functional categories (P ? 0.005; each
included at least 10 genes) are given for each gene set. The significance of the
overlap between two gene sets was calculated using the exact hypergeometric
distribution calculator at http://www.alewand.de/stattab/tabdiske.htm.
RESULTS AND DISCUSSION
Experimental approach. It has long been known that several
signal transduction pathways allow yeast to sense the presence
of glucose in the medium and that these signals affect tran-
scription. However, it is not known how many signal transduc-
tion pathways are needed to produce the total response to
nutrient repletion, nor is it known how much each individual
pathway contributes to the overall response. To test this, we
blocked these pathways in nutrient-depleted, quiescent cells.
We then followed global changes in transcript levels after ad-
dition of fresh medium. A schematic of this approach is shown
in Fig. 1.
A detailed description of the experimental setup is described
in Materials and Methods. Briefly, we used a strain (TC41)
lacking CYR1, encoding adenylyl cyclase, to manipulate the
cAMP/PKA pathway. This strain is completely dependent on
exogenous cAMP for growth (38). The small molecule rapa-
mycin was used to block the TOR pathway, and a strain
(KY73) lacking the hexose transporters HXT1-7 and GAL2
was used to block glucose entry into the cell. Ultimately, this
experimental approach allowed us to test the response of qui-
escent, nutrient-depleted cells to fresh YPD under conditions
in which PKA and TOR were blocked (PKA?/TOR?, pka?/
TOR?, PKA?/tor?, and pka?/tor?) and when glucose trans-
port was blocked (hxt?).
Overall effect of pathway blockade. The dot plots in Fig. 2A
show how the loss of specific pathways affects the response to
fresh YPD for each of ?6,000 transcripts. In these graphs, each
transcript is represented as a dot. The position on the x axis
represents the change (n-fold) in response to fresh YPD in
normal cells. The position on the y axis represents the change
(n-fold) in cells in which specific pathways are blocked. The
changes are expressed as log2values. When the response is
unchanged by pathway blockade, then each point has the same
value on the x and y axes and falls on a diagonal line with a
slope of 1 (indicated by the dotted lines). While blockade of
each pathway had an effect, it can be seen that rapamycin
blockade of the TOR pathway had a relatively small effect,
while loss of PKA and TOR together produced the greatest
The R-squared values for each of the four dot plots are
shown in Fig. 2B. Individual loss of the PKA, TOR, or glucose
transport pathways affected the genome-wide nutrient reple-
tion response to various degrees. PKA signaling played the
largest role of the three individual pathways; blockade of hex-
ose transport or TOR produced smaller changes. Simultaneous
inhibition of PKA and TOR eliminated a substantial portion of
the overall response and produced the lowest correlation with
the normal response.
Nutrient-induced genes. While the dot plots show involve-
ment of the different pathways in the response to nutrients, the
plots do not tell us which genes are regulated by the different
pathways, nor do they show a clear picture of the pattern of
overlap between the gene sets regulated by the different path-
ways. To get a better picture of this, we generated heat maps by
k-means clustering of the transcripts that are normally altered
by YPD (changed 1.5-fold or more in PKA?/TOR?or wild-
type cells). This identified distinct groups of genes regulated by
different pathway combinations. These groups tended to have
distinct functional and regulatory characteristics. Furthermore,
FIG. 2. Global effects of signaling pathway inhibition. (A) Dot
plots represent the log2-transformed change (n-fold) for each gene
after nutrient repletion under the indicated conditions. Solid lines
represent the trend line for each data set, and dashed lines represent
a hypothetical perfect correlation (R2? 1). (B) R-squared values
represent the overall correlation of PKA?/TOR?and the individual
PKA/TOR blockades or the correlation between the wild type and
hxt1-7? gal2?, as indicated.
360SLATTERY ET AL.EUKARYOT. CELL
the patterns seen with induced genes were distinct from those
seen with the repressed genes.
Figure 3 shows the heat map generated by clustering of the
1,011 transcripts normally induced by YPD. Each transcript is
shown as a horizontal line, with YPD induction indicated by
the degree of red color saturation. The normal YPD induction
responses are shown in the PKA?/TOR?column for the PKA/
TOR experiments and in the wild-type column, indicating the
isogenic wild-type control strain for the hxt?mutant. Some
clusters with obvious similarities were grouped together, with
the 11 clusters organized into groups A through E.
The 436 transcripts in group A were dependent primarily on
PKA signaling. A significant fraction of these genes (32%; P ?
1.5 ? 10?18) are involved in posttranscriptional RNA-related
processes such as the rRNA and tRNA processing/modifica-
tion needed for increased protein translation (classified as
“transcription” by MIPS). The promoters in these two clusters
are enriched in the nutrient-responsive RRPE (rRNA process-
FIG. 3. Effect of signaling pathway inhibition on nutrient-induced genes. k-means clustering of the 1011 genes induced by YPD (1.5-fold or
greater induction in both PKA?/TOR?and wild-type genes) generated seven clusters, which were then divided into the following gene sets:
PKA-dependent genes (A), PKA- and TOR-dependent genes (B), PKA-, TOR-, and glucose transport-dependent genes (C), TOR- and glucose
transport-dependent genes (D), and glucose transport-dependent genes (E). In all cases, the line graphs on the left represent the average
expression profile for each cluster. The line on the left side of each graph connects, from left to right, the PKA?/TOR?, PKA?/tor?, pka?/TOR?,
and pka?/tor?data points; the line on the right connects, from left to right, the wild-type (MC996A) and hxt1-7? gal2? (KY73) data points. The
heat maps for each cluster represent, from left to right, the log2-transformed changes (n-fold) after nutrient repletion for PKA?/TOR?,
PKA?/tor?, pka?/TOR?, pka?/tor?, the wild type, and hxt?, respectively. Columns to the right of the heat maps describe enriched functional
categories and promoter DNA motifs associated with each gene set (see Materials and Methods).
VOL. 7, 2008TRANSCRIPTIONAL RESPONSE TO FRESH MEDIUM IN YEAST 361
ing element; AAAWTTTT) (15, 42), PAC (polymerase A and
C; GATGAG) (6), and GRE (glucose response element;
A4GA3) motifs (31, 33, 43). This is consistent with the idea that
transcriptional regulation via these cis-acting elements must
involve factors that are downstream of PKA (49).
Group B consists of 299 genes that are dependent upon both
PKA and TOR for full YPD induction. For these genes, the
TOR input was more apparent in the absence of PKA signal-
ing. Protein synthesis genes make up a significant fraction of
this set (29%; P ? 2.4 ? 10?34). The RRPE, PAC, and GRE
motifs were also overrepresented in these promoters.
For the 276 genes in the remaining clusters (groups C to E),
YPD induction was dependent at least in part on glucose
transport. Interestingly, groups C and E contain a significant
percentage of cell cycle genes (30% and 26%, respectively).
Consistent with this, the cell cycle-regulated and glucose-re-
sponsive MCB (ACGCG) and/or SCB (CRCGAAA) regula-
tory motifs (2, 32, 49) are overrepresented in the promoters of
these three groups.
The fact that PKA and TOR are required for the induction
of genes required for protein synthesis is not surprising. How-
ever, these results show that the PKA-dependent genes can be
divided into TOR-dependent and TOR-independent sub-
groups. Furthermore, it is clear that PKA is not required for a
significant group of YPD-induced genes. These PKA-indepen-
dent genes, however, are almost all dependent on glucose
Nutrient-repressed genes. Nutrient-repressed transcripts
displayed regulatory patterns similar to those seen with nutri-
ent-induced transcripts (Fig. 4). As with induction, blocking
signals generated by PKA, TOR, and glucose transport was
sufficient to prevent virtually the entire response. However, in
this case almost all genes displayed some degree of depen-
dence on all three pathways, although for some groups a single
pathway often predominated. Glucose transport played a
larger role, but PKA and TOR were still the major regulators.
Concurrent inhibition of both PKA and TOR prevented the
repression of almost three-quarters of the nutrient-responsive
genes (groups A to C). For the remaining genes that showed
nutrient repression despite the double PKA and TOR block,
this repression was dependent on glucose transport.
Over half of the nutrient-repressed genes fell into group A
and were primarily PKA dependent. This group includes many
genes that are necessary for generating and using reserve car-
bohydrates, recycling of cellular machinery, and responding to
stressful environmental conditions. The Msn2/4-regulated
STRE (AGGGG) motif is highly represented in the promoters
of these genes, consistent with the finding that Msn2/4 activity
is negatively regulated by PKA activity (23, 28).
Nutrient repression of the 148 genes in groups B and C was
dependent on the TOR pathway. Group C includes a number
of genes that are involved in amino acid metabolism and are
part of the nitrogen catabolite repression response. Nitrogen
catabolite repression genes are normally repressed in the pres-
ence of a preferred nitrogen source, a condition that would be
provided by fresh YPD. The sequence GATAA is found up-
stream of genes in this set at a higher than expected frequency;
this DNA motif is known to bind the GATA transcription
factor Gln3, a well-characterized downstream effector of the
TOR pathway (5).
The two clusters in group D were largely dependent on
glucose transport for nutrient repression. This group contains
a number of transcripts encoding proteins that are necessary
for oxidative metabolism, such as enzymes of the TCA cycle
and electron transport chain. The promoters of this gene set
are enriched in the sequences GGGGTA and CCAAT, motifs
that are bound by the transcriptional repressor Mig1 and the
transactivating HAP complex, respectively (29, 47). It is worth
noting that the presence of these two promoter motifs in this
highly glucose transport-dependent gene set is consistent with
current models of glucose repression. The repressive activity of
the Mig1 repressor is positively regulated by intracellular glu-
cose via the Hxk2 and Snf1 kinases (1). This group of tran-
scripts that are primarily dependent on glucose entry into the
cell accounts for the classical glucose repression genes that are
among the longest-studied set of glucose-sensitive genes. Sim-
ilarly, the expression of HAP4, the transactivator subunit of the
HAP complex, is negatively regulated by glucose (35).
While findings such as the connection between PKA and
downregulation of stress response genes are not surprising,
again our results map out the groups of genes for which specific
pathways are needed for repression by YPD. This shows the
degree of redundancy in regulation by the different pathways,
and again, we find that virtually all of the gene repression
produced by transfer to YPD can be abolished by blockade of
some combination of the three pathways.
Effects of PKA activation in the absence of nutrients. Acti-
vation of PKA via cAMP can account for a large fraction of the
transcriptional response to fresh medium. However, in our
experiments, as in nature, glucose and other nutrients were
present when cAMP was added. It seems possible that the
transcriptional response to cAMP might depend on ongoing
metabolism and that the response to cAMP might be depen-
dent on other signals generated by the actions and metabolism
of glucose. One reason for thinking that this could be true is
the simple fact that cAMP alone is not sufficient to promote
either growth or cell division when nutrients are limiting (14,
25). This is shown in Fig. 5A, in which quiescent cyr1? cells
were transferred to either YPD-cAMP or cAMP alone. As can
be seen, the YPD-cAMP allowed the cells to reenter the cell
division cycle, with thinner cell walls and reduced vacuole size.
In contrast, we could see little if any difference in appearance
between the cells receiving cAMP alone and the starting qui-
escent cells, leading one to wonder whether the massive
changes in transcript abundance were also occurring in these
To examine the transcriptional response to cAMP alone, we
repeated the experiment from Fig. 2 of adding 5 mM cAMP to
quiescent cyr1? cells, but without the inclusion of fresh YPD.
We found that gene induction produced by cAMP alone cor-
related well with the induction produced by cAMP-YPD (Fig.
5B). This shows that the metabolism afforded by nutrient re-
pletion was not needed for the induction of these genes. As
would be expected, the set of PKA-dependent genes identified
in Fig. 3 (groups A and B) tended to be the ones induced by
cAMP alone, while the groups from Fig. 3 that were dependent
on glucose transport rather than on PKA (groups C to E) did
not tend to respond to cAMP alone (Fig. 6A and B).
On the other hand, genes that were repressed by cAMP-
YPD were far less likely to be repressed by cAMP alone,
362SLATTERY ET AL.EUKARYOT. CELL
producing a correlation value of only 0.13 (Fig. 5C). This
indicates that in contrast to induction, PKA-dependent gene
repression requires some additional signal generated by YPD
that is not provided by cAMP alone. Many of the genes down-
regulated by PKA are involved in stress responses; perhaps as
cells reach quiescence additional PKA-independent mecha-
nisms maintain the expression of these genes, despite PKA
Wang et al. reported a related experiment in which cells
growing slowly in glycerol medium were challenged by activat-
ing expression of upstream components of the PKA pathway,
either constitutively activated Ras2 or Gpa2 (49). Glycerol is a
much poorer nutrient source for S. cerevisiae than glucose;
however, the cells in glycerol were steadily growing, while the
cells in our experiment had reached a plateau. Nonetheless,
our results with gene induction by cAMP in the cells in ex-
hausted medium are largely in agreement with those of Wang
et al., with a significant portion of the induction being inde-
pendent of nutrient conditions. This indicates that for most
genes, induction by PKA activation is independent of the rate
FIG. 4. Effect of signaling pathway inhibition on nutrient-repressed genes. The 1,474 genes repressed by YPD (1.5-fold or greater repression
in both PKA?/TOR?and the wild type) were clustered as in Fig. 3 to generate the following four gene sets: PKA-dependent genes (A), PKA- and
TOR-dependent genes (B), TOR-dependent genes (C), and glucose transport-dependent genes (D). The line graphs, heat maps, and descriptive
columns are as described in the legend to Fig. 3.
VOL. 7, 2008TRANSCRIPTIONAL RESPONSE TO FRESH MEDIUM IN YEAST 363
of metabolism. In contrast, our results showing a nutrient re-
quirement for gene repression in response to PKA activation
do not so well match those of Wang et al. While PKA activa-
tion produced normal gene repression in cells growing on
glycerol, we found that this response was significantly reduced
when starting with quiescent cells and adding no new nutrients.
One obvious possible reason for this discrepancy, as mentioned
above, is the fact that the cells in glycerol were growing, while
the cells in our experiments were not able to.
Regulatory proteins downstream of TOR, PKA, and glucose
transport. A complete picture of the transcriptional response
to nutrient repletion requires identification and placement of
downstream pathways. With regard to the nutrient-induced
genes, it is clear that the proteins Sfp1 and Sch9 play a signif-
icant regulatory role (Fig. 7A). Sfp1 nuclear localization, and
presumably Sfp1 activity, is positively regulated by the TOR
and PKA pathways (17, 24), while evidence indicates that Sch9
is directly activated by TOR (17, 45). Tyers and colleagues
have found that Sfp1 and Sch9 regulate essentially the same
gene set, which they have termed the RP (ribosomal protein)
and Ribi (ribosome biogenesis) regulons (17). We compared
these genes to our YPD-induced clusters, and consistent with
FIG. 5. Effect of cAMP on nutrient-starved cells. (A) cyr1? cul-
tures were cultured for 48 h and transferred to spent YPD without
cAMP as described in the legend to Fig. 1. After 24 h of incubation,
the cells were either shifted to YPD-5 mM cAMP or treated with 5
mM cAMP alone. (A) Samples were collected after 2 h for exam-
ination using a 60? objective with differential interference contrast
microscopy. (B and C) Samples were collected after 1 h for mi-
croarray analysis comparing transcripts in the quiescent, starved
cells with transcripts receiving either cAMP alone or YPD-cAMP.
Both dot plots compare the log2-transformed transcript changes
(n-fold) observed in response to YPD-cAMP with the response to
cAMP alone. The nutrient-induced genes from Fig. 3 are repre-
sented in panel B, and the nutrient repressed genes from Fig. 4 are
represented in panel C.
FIG. 6. Nutrient-induced genes and nutrient-independent effects
of cAMP. (A) The dot plot is the same as that described for Fig. 5B
except restricted to the PKA- and/or TOR-dependent induced genes
(groups A and B in Fig. 3). (B) Same as panel A except restricted to the
glucose transport-dependent induced genes (groups C to E in Fig. 3).
364SLATTERY ET AL.EUKARYOT. CELL
the idea that PKA and TOR are upstream of these regulators,
we find a highly significant fraction of Sfp1/Sch9 target genes in
our PKA- and PKA/TOR-dependent clusters (Fig. 7C).
The Snf3/Rgt2 glucose sensors are known to activate glucose
transporter transcription through the Rgt1 transcription factor
(16) and are therefore expected to regulate a subset of the
nutrient-induced genes (Fig. 7B). Kaniak et al. have identified
a core set of Snf3/Rgt2/Rgt1 targets (18); approximately 10%
of these genes fall into our PKA-dependent gene set, and 10%
are in the PKA/TOR/glucose-dependent gene set (Fig. 7C).
Though the Snf3/Rgt2 pathway is traditionally thought of as a
separate glucose-sensing pathway, a PKA input is supported by
the recent finding that Rgt1 is also directly targeted by PKA
The Rim15 kinase regulates a set of genes that are repressed
during growth on YPD. Rim15 activates these starvation-in-
duced genes through the Msn2/4 and Gis1 transcription fac-
tors, and Rim15 activity is negatively regulated by both TOR
and PKA (Fig. 8A) (44). Accordingly, we found that half of the
previously identified Rim15 targets (3) are downregulated by
YPD in a PKA-dependent manner (Fig. 8C). Rim15 targets
were not significantly dependent on TOR for their repression.
Interestingly, a small but statistically significant fraction of
Rim15-dependent genes were highly dependent on glucose
import (Fig. 8C). Perhaps glucose metabolism itself contrib-
utes to inhibition of Rim15 activity.
Snf1 kinase activity also regulates a number of YPD-re-
pressed genes. Snf1 acts through the Cat8, Sip4, and Adr1
transcriptional activators and the Mig1 repressor (40, 48, 50).
It has been proposed that Snf1 activity is intimately linked to
glucose metabolism via readouts such as Hxk2 enzymatic ac-
tivity or the AMP/ATP ratio (Fig. 8B) (8, 40). However, it now
appears that Snf1 also responds to stresses unrelated to carbon
source, and there is increasing evidence for glycolysis-indepen-
dent input into Snf1 activity (13). When we compared a pub-
lished list of Snf1-dependent genes (50) to our YPD-repressed
clusters, we found significant overlap between Snf1 target
genes and both PKA-dependent and glucose transport-depen-
dent clusters (Fig. 8C). This indicates that intracellular glucose
might regulate Snf1, which is consistent with traditional models
(Fig. 8B), but also suggests a significant role for PKA in reg-
ulating Snf1. Hedbacker et al. demonstrated that PKA regu-
lates Snf1 localization (10), and it is possible that this plays a
larger-than-expected role in modulating Snf1 activity.
FIG. 7. Nutrient-induced genes and additional growth regulators.
(A) Model of signaling through Sfp1 and Sch9. (B) Model of signaling
through Rgt1. (C) Dark gray bars, percentage of Sfp1/Sch9-dependent
genes (345 genes total, from reference 17) in each of the five gene
groups from Fig. 3; light gray bars, percentage of Snf3/Rgt2/Rgt1-
dependent genes (29 genes total, from reference 18) present in each of
the five gene groups from Fig. 3; white bars, percentages of the genome
as a whole present in the gene groups described in the legend to Fig.
3. Asterisks represent P values of ?1 ? 10?25, and the pound sign
represents P values of ?2 ? 10?2.
FIG. 8. Nutrient-repressed genes and additional growth regulators.
(A) Model of signaling through Rim15. (B) Model of signaling through
Snf1. (C) Dark gray bars, percentage of Snf2-dependent genes (425
genes total, from reference 50) in each of the four gene groups from
Fig. 4; light gray bars, percentage of Rim15-dependent genes (54 genes
total, from reference 3) present in each of the four gene groups from
Fig. 4; white bars, percentages of the genome as a whole present in
the gene groups described in the legend to Fig. 4. Asterisks repre-
sent P values of ?1 ? 10?11, and the pound sign represents P values
of ?1 ? 10?3.
VOL. 7, 2008TRANSCRIPTIONAL RESPONSE TO FRESH MEDIUM IN YEAST 365
Overall, these findings support a model in which PKA and
TOR promote growth gene regulation through the proteins
Sfp1 and Sch9 and lend support to the idea that PKA contrib-
utes to Rgt1-mediated glucose induction. Neither Sfp1/Sch9
nor Rgt1 appears to be significantly dependent on glucose
transport alone, so the regulators downstream of glucose me-
tabolism remain to be discovered. In addition, our results sug-
gest that PKA also promotes nutrient repression through the
Snf1 and Rim15 kinases, but a signal generated by glucose
transport or metabolism also feeds into these pathways.
Summary. The transfer of starved yeast to fresh medium has
a dramatic, reproducible effect on gene expression (27, 34, 42,
49). Our study provides a detailed map showing which genes
are regulated by the different members of a set of important
nutrient-sensing pathways. As expected, the cAMP-PKA path-
way appears to be the primary regulator of these gene expres-
sion changes. A subset of the PKA-dependent genes was also
affected by loss of TOR. Approximately 25% of the nutrient-
repressed genes and 10% of the nutrient-induced genes are
regulated by YPD even in the absence of both PKA and TOR
activity, and this regulation is dependent on glucose import.
Thus, virtually the entire response to YPD can be prevented by
blockade of just these three pathways.
Our results show the role that PKA, TOR, and Hxk2/Snf1
play as hubs lying upstream of local regulators, such as Sch9,
Sfp1, and Msn2/4. The mechanisms by which these compo-
nents interact to carry signals generated at these hubs out to
the genes to be regulated remain to be discovered.
This work was supported by National Science Foundation Grant
MCB-0542779 (W.H.) and a predoctoral fellowship from the Ameri-
can Foundation for Pharmaceutical Education (M.G.S.).
We acknowledge Audrey Gasch, Michael Conway, Dan Kvitek, J.
Joseph Brown, and James Rabbitte, Jr., for assistance and helpful
discussions. We also thank Ted Young for providing Snf1 microarray
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