The Natural Osmolyte Trehalose Is a Positive Regulator of the Heat-Induced Activity of Yeast Heat Shock Transcription Factor

Trehalose is a disaccharide of glucose that is found predominantly in bacteria, fungi (including yeasts), plants, and invertebrates. This natural osmolyte was initially characterized as a storage carbohydrate due to the high intracellular concentrations observed during these organisms' resting and anhydrobiotic states (reviewed in reference 55). Since these states involve the ability to survive stressful conditions, trehalose levels were then linked to thermotolerance, the ability of an organism to survive an otherwise lethal heat shock (reviewed in reference 47). Trehalose has been shown to stabilize the structures and enzymatic activities of proteins against thermal denaturation in vitro (15, 27, 63). In addition, trehalose can prevent the aggregation of misfolded proteins, including amyloidogenic proteins (3, 11, 27, 33, 46, 52), and is being considered for clinical trials for Huntington's disease (53). Trehalose is more effective than other sugars in protecting proteins against thermal denaturation and aggregation because of its unusual ability to alter the water environment surrounding a protein, stabilizing the protein in its native conformation (1, 30, 34, 48). In the yeast Saccharomyces cerevisiae, trehalose levels vary depending on the environment of the cell. The levels are almost undetectable during normal exponential growth. After heat shock, trehalose levels increase rapidly and dramatically, along with the accumulation of heat shock proteins (HSPs) (26, 28). This rapid increase in trehalose has been attributed to the increase in both the translation of the genes involved in the synthesis of trehalose (TPS1 and TPS2) (4, 41, 58) and the substrates required for trehalose synthesis (1, 61). In addition, the enzymatic activity of TPS1 increases during the heat shock response (1, 38). High trehalose levels can stabilize enzymatic activity and can prevent the aggregation of exogenous proteins in vivo during heat shock (45, 46). Trehalose is degraded in the cytoplasm by neutral trehalase, encoded by the gene NTH1 (26, 31, 38, 40, 59). The transcription of NTH1 is stress responsive (62); however, the activity of Nth1 is greater during recovery from stress (39), allowing Nth1 to successfully compete with the biosynthetic pathway and reduce intracellular trehalose levels (28). This degradation of trehalose is critical for recovery from heat shock (59) as very high levels of trehalose can interfere with normal protein activity by stabilizing proteins in nonnative conformations and inhibiting the refolding of these denatured proteins by HSPs (12, 43, 44, 46, 56). Taken together, these data have led to a temporal model of trehalose function as a cochaperone during the heat shock response: trehalose functions to protect proteins at the initial stages of the heat shock response before HSPs have been fully induced, but trehalose must be degraded in order for the HSPs to fully assist the cell in recovery from heat shock (47). In S. cerevisiae, the transcriptional response to heat shock is controlled by two sets of transcription factors, the heat shock transcription factor (Hsf1) and the partially redundant transcription factors Msn2 and Msn4 (Msn2/4). The Msn2/4 transcription factors, which are found only in yeast, bind to stress response elements found in the promoters of many heat shock genes (35). The activity of Msn2/4 is regulated by nuclear localization and phosphorylation (20, 29). Hsf1 is part of a family of conserved transcription factors critical to the heat shock response in all eukaryotes (57). All heat shock transcription factors (HSFs) share a conserved central core, consisting of a DNA-binding domain, a flexible linker, and a trimerization domain, which are essential for binding to heat shock elements (42). Stress response elements and heat shock elements are found in overlapping sets of promoters, allowing Msn2/4 and Hsf1 to provide distinct contributions to the heat shock response (2, 7). The mechanism by which Hsf1's transcriptional activity is regulated in response to heat shock is not well understood. In yeast, Hsf1 is localized to the nucleus, with a low level of constitutive transcriptional activity that is necessary for normal cellular processes (50, 60). Following a mild stress, such as heat shock, Hsf1 becomes transcriptionally active and the expression of HSP mRNA dramatically increases. During a prolonged stress, Hsf1 activity gradually decreases to a new plateau, while removal of stress causes Hsf1 activity to return rapidly to near-constitutive levels. The dramatic changes in Hsf1 activity during heat shock involve several components, including hyperphosphorylation (24, 50) and conformational changes (9, 32). Hsf1 is also thought to be negatively regulated through interactions with several heat shock proteins, including Hsp82 and members of the Hsp70 family, under both constitutive and heat shock conditions (6, 10, 13, 23, 51). To date, no specific signal that positively regulates Hsf1 activity during a heat shock has been identified. We have previously shown that high concentrations of trehalose can increase the structure of the S. cerevisiae Hsf1 C-terminal activation domain in vitro, and this structural change is enhanced by temperature (8). We hypothesized that the effects of trehalose and elevated temperature on Hsf1's structure in vitro were linked to its dramatic increase in transcriptional activity after heat shock. In this paper, we show that trehalose is required for the robust increase in transcription of heat shock protein genes by Hsf1 during the initial response to heat shock. In addition, a high trehalose level maintains Hsf1 in a highly active state, preventing the decrease in activity of Hsf1 that occurs during a sustained heat shock response. The enhanced transcriptional response does not require the other heat-responsive transcription factors Msn2/4 but is dependent upon heat and Hsf1. Despite the structural enhancement of the C-terminal activation domain (CAD) observed in vitro, the enhanced transcriptional response does not require the presence of the C-terminal activation domain, suggesting that any structural changes in vivo must be more global. In addition, the increase in transcriptional activity is correlated with an increase in Hsf1 phosphorylation. By showing that trehalose modifies the dynamic range of Hsf1's heat-induced transcriptional activity, we have identified a novel and physiologically relevant function of trehalose as a positive regulator of the transcriptional response to heat shock.