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. 2008 Feb;7(2):358-67.
doi: 10.1128/EC.00334-07. Epub 2007 Dec 21.

Protein kinase A, TOR, and glucose transport control the response to nutrient repletion in Saccharomyces cerevisiae

Matthew G Slattery  1 , Dritan Liko, Warren Heideman
Affiliations

Protein kinase A, TOR, and glucose transport control the response to nutrient repletion in Saccharomyces cerevisiae

Matthew G Slattery et al. Eukaryot Cell. 2008 Feb.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Experimental design. cyr1Δ (TC-41) cells were grown for 48 h in YPD supplemented with 1 mM cAMP, centrifuged, and resuspended 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 hxt1-7Δ gal2Δ (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.
FIG. 2.
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 hxt1-7Δ gal2Δ, as indicated.
FIG. 3.
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 hxt1-7Δ gal2Δ (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).
FIG. 4.
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.
FIG. 5.
FIG. 5.
Effect of cAMP on nutrient-starved cells. (A) cyr1Δ cultures 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 examination using a 60× objective with differential interference contrast microscopy. (B and C) Samples were collected after 1 h for microarray 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 represented in panel B, and the nutrient repressed genes from Fig. 4 are represented in panel C.
FIG. 6.
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).
FIG. 7.
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.
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 represent P values of <1 × 10−11, and the pound sign represents P values of <1 × 10−3.
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