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 transcriptional response in which thousands of genes are induced or repressed within minutes. Early microarray work demonstrated that approximately a third of the yeast genome is regulated 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
). The general response to nutrient repletion consists of a rapid induction of genes involved in mass accumulation and cell division 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
) and cyclic AMP (cAMP)-dependent protein kinase A (PKA) (40
) signaling pathways 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
Transcriptional profiling and phenotypic evidence both suggest that the TOR pathway is a primary carrier of nutrient signals (4
). 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 G1
phase of the cell cycle (4
). While these experiments demonstrate a role for the TOR pathway in maintaining the expression of genes related to protein synthesis and mass accumulation during 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 responses (40
). A recent study by Broach and colleagues demonstrated that artificial induction of the cAMP pathway by either Ras2 or the Gα homolog Gpa2 mimics the transcriptional response to glucose repletion (49
). However, the researchers 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 cAMP-independent pathways.
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 gluconeogenesis and respiration genes (8
). 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 contribution to this response is unclear.
In this report, we compare the contributions made by TOR, PKA, and glucose transport to the overall transcriptional response to nutrient repletion in yeast. While much of the response 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.