Glucose Starvation Alters Heat Shock Response, Leading to Death of Wild Type Cells and Survival of MAP Kinase Signaling Mutant

<div><p>A moderate heat shock induces <i>Neurospora crassa</i> to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. However, wild type cells do not survive when carbohydrate deprivation is added to heat shock. In contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. In order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins in the mutant and wild type strain during dual stress, and we have identified gene transcripts in both strains whose quantities change in response to heat shock or dual stress. These data and supportive experimental evidence point to reasons for survival of the mutant strain. By using alternative respiratory mechanisms, these cells experience less of the oxidative stress that proves damaging to wild type cells. Of central importance, mutant cells recycle limited resources during dual stress by undergoing autophagy, a process that we find utilized by both wild type and mutant cells during heat shock. Evidence points to inappropriate activation of TORC1, the central metabolic regulator, in wild type cells during dual stress, based upon behavior of an additional signaling mutant and inhibitor studies.</p></div

Categories of transcripts whose levels increase or decrease during stress: Metabolism.

<p>Numbers of up- or down-regulated transcripts discussed in this report, are indicated for wt (filled bars) and os2 (outlined bars) during heat shock (hs) or dual stress (ds). For details of transcript levels for individual proteins see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165980#sec049" target="_blank">Supporting Information</a>.</p

Ratio of reactive oxygen species (ROS) in wt and os2, according to fluorescence measurements.

<p>Germinating conidiospores were incubated 1 hr at control (30°C), HS (45°C), or DS (45°C and 2-DG) conditions. The measurements are expressed as a ratio to those in the wt control. Experiments were performed at least two times with similar results.</p

Categories of transcripts whose levels increase or decrease during stress: Signaling.

<p>Numbers of up- or down-regulated transcripts discussed in this report, are indicated for wt (filled bars) and os2 (outlined bars) during heat shock (hs) or dual stress (ds). For details of transcript levels for individual proteins see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165980#sec049" target="_blank">Supporting Information</a>.</p

Differential activation of Os2 in Western blot of proteins from wt and stk10 cell extracts.

<p>The cells were subjected to DS for 10 min in the presence (+) or absence (-) of rapamycin. Proteins were separated by SDS-gel electrophoresis, and the blot was probed with antibody against mammalian (Thr180/Tyr182) phospho-p38, which detects phosphorylated Os2.</p

Vacuoles of germinating conidiospores of wt and os2 in transmission electron micrographs.

<p>Cells were incubated under 30°C control conditions (a + d) or subjected to heat shock (b + e) or dual stress (c + f) for 2 hr. The vacuoles (lysosomes) are light-colored and indicated by v. The horizontal bar in each micrograph indicates 1 micrometer.</p

Percentage of surviving colonies, representing viable conidiospores, in petri plate assays.

<p>The controls in each case consist of the particular strain incubated at room temperature. At least three plates were counted for each treatment, and experiments were performed at least two times with similar results. Typical examples of reproducible experiments are shown.</p

Phosphorylation of eIF2α in response to DS.

<p>The cells were either incubated only at 30°C or subjected to DS for 2 hr. Proteins in cell extracts were separated by SDS-gel electrophoresis, and the blot was probed with monoclonal antibody against human (Ser51) phospho-eIF2α, which detects the <i>Neurospora</i> homolog.</p

Primers used in this study.

*<p>:5-terminal end phosphorylated.</p

Effect of the deletion of the <i>ilv</i>-<i>leu</i> operon on the essentiality of Gcp for growth.

<p>The growth curves of wild-type control, WCUH29 carrying pYH4-lacI, in nutrient complete CDM (A) and in ILV dropout CDM (B). The growth curves of the <i>ilv</i>-<i>leu</i> operon deletion mutant (JW290211) in nutrient complete CDM (C) and in ILV dropout CDM (D); the growth curves of the <i>ilv</i>-<i>leu</i> operon deletion and defined <i>Pspac</i>-regulated <i>gcp</i> expression strain (JW290311) in nutrient complete CDM (E) and in ILV dropout CDM (F). The growth curves are monitored by kinetically measuring the optical density at OD600 nm in the corresponding culture medium in the presence of different concentrations of inducer, IPTG (0. 10, 25 and 100 µM), in every 15 min at 37°C using a BioTek Synergy plate reader. These figures are one representative of three independent experiments.</p