Hsf1 Activation Inhibits Rapamycin Resistance and TOR Signaling in Yeast Revealed by Combined Proteomic and Genetic Analysis

TOR kinases integrate environmental and nutritional signals to regulate cell growth in eukaryotic organisms. Here, we describe results from a study combining quantitative proteomics and comparative expression analysis in the budding yeast, S. cerevisiae, to gain insights into TOR function and regulation. We profiled protein abundance changes under conditions of TOR inhibition by rapamycin treatment, and compared this data to existing expression information for corresponding gene products measured under a variety of conditions in yeast. Among proteins showing abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional (i.e., in similar direction) transcriptomic changes under conditions of heat/oxidative stress. Because the known downstream responses regulated by Tor1/2 did not fully explain the extent of overlap between these two conditions, we tested for novel connections between the major regulators of heat/oxidative stress response and the TOR pathway. Specifically, we hypothesized that activation of regulator(s) of heat/oxidative stress responses phenocopied TOR inhibition and sought to identify these putative TOR inhibitor(s). Among the stress regulators tested, we found that cells (hsf1-R206S, F256S and ssa1-3 ssa2-2) constitutively activated for heat shock transcription factor 1, Hsf1, inhibited rapamycin resistance. Further analysis of the hsf1-R206S, F256S allele revealed that these cells also displayed multiple phenotypes consistent with reduced TOR signaling. Among the multiple Hsf1 targets elevated in hsf1-R206S, F256S cells, deletion of PIR3 and YRO2 suppressed the TOR-regulated phenotypes. In contrast to our observations in cells activated for Hsf1, constitutive activation of other regulators of heat/oxidative stress responses, such as Msn2/4 and Hyr1, did not inhibit TOR signaling. Thus, we propose that activated Hsf1 inhibits rapamycin resistance and TOR signaling via elevated expression of specific target genes in S. cerevisiae. Additionally, these results highlight the value of comparative expression analyses between large-scale proteomic and transcriptomic datasets to reveal new regulatory connections

Activation and repression of glucose-stimulated ChREBP requires the concerted action of multiple domains within the MondoA conserved region

Carbohydrate response element-binding protein (ChREBP) is a glucose-dependent transcription factor that stimulates the expression of glycolytic and lipogenic genes in mammals. Glucose regulation of ChREBP has been mapped to its conserved NH2-terminal region of 300 amino acids, designated the MondoA conserved region (MCR). Within the MCR, five domains (MCR1–5) have a particularly high level of conservation and are likely to be important for glucose regulation. We carried out a large-scale deletion and substitution mutational analysis of the MCR domain of ChREBP. This analysis revealed that MCRs 1–4 function in a concerted fashion to repress ChREBP activity in basal (nonstimulatory) conditions. Deletion of the entire MCR1–4 segment or the combination of four specific point mutations located across this region leads to a highly active, glucose-independent form of ChREBP. However, deletion of any individual MCR domain and the majority of point mutations throughout MCR1–4 rendered ChREBP inactive. These observations suggest that the MCR1–4 region interacts with an additional coregulatory factor required for activation. This possibility is supported by the observation that the MCR1–4 region can compete for activity with wild-type ChREBP in stimulatory conditions. In contrast, mutations in the MCR5 domain result in increased activity, suggesting that this domain may be the target of intramolecular repression in basal conditions. Thus, the MCR domains act in a complex and coordinated manner to regulate ChREBP activity in response to glucose

Glucose Activates ChREBP by Increasing Its Rate of Nuclear Entry and Relieving Repression of Its Transcriptional Activity*S⃞

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that activates genes involved in de novo lipogenesis in mammals. The current model for glucose activation of ChREBP proposes that increased glucose metabolism triggers a cytoplasmic to nuclear translocation of ChREBP that is critical for activation. However, we find that ChREBP actively shuttles between the cytoplasm and nucleus in both low and high glucose in the glucose-sensitive β cell-derived line, 832/13. Glucose stimulates a 3-fold increase in the rate of ChREBP nuclear entry, but trapping ChREBP in the nucleus by mutagenesis or with a nuclear export inhibitor does not lead to constitutive activation. In fact, mutational studies targeting the nuclear export signal of ChREBP also identified a distinct function essential for glucose-dependent transcriptional activation. From this, we conclude that an additional event independent of nuclear translocation is required for activation. The N-terminal segment of ChREBP (amino acids 1-298) has previously been shown to repress activity under basal conditions. This segment has five highly conserved regions, Mondo conserved regions 1-5 (MCR1 to -5). Based on activating mutations in MCR2 and MCR5, we propose that these two regions act coordinately to repress ChREBP in low glucose. In addition, other mutations in MCR2 and mutations in MCR3 were found to prevent glucose activation. Hence, we conclude that both relief of repression and adoption of an activating form are required for ChREBP activation