Glucose metabolism in Neurospora is altered by heat shock and by disruption of HSP30

Abstract

We compared the metabolism of [1-13C]glucose by wild type cells of Neurospora crassa at normal growth temperature and at heat shock temperatures, using nuclear magnetic resonance analysis of cell extracts. High temperature led to increased incorporation of 13C into trehalose, relative to all other metabolites, and there was undetectable synthesis of glycerol, which was a prominent metabolite of glucose at normal temperature (30 degrees C). Heat shock strongly reduced formation of tricarboxylic acid cycle intermediates, approximately 10-fold, and mannitol synthesis was severely depressed at 46 degrees C, but only moderately reduced at 45 degrees C. A mutant strain of N. crassa that lacks the small alpha-crystallin-related heat shock protein, Hsp30, shows poor survival during heat shock on a nutrient medium with restricted glucose. An analysis of glucose metabolism of this strain showed that, unlike the wild type strain, Hsp30-deficient cells may accumulate unphosphorylated glucose at high temperature. This suggestion that glucose-phosphorylating hexokinase activity might be depressed in mutant cells led us to compare hexokinase activity in the two strains at high temperature. Hexokinase was reduced more than 35% in the mutant cell extracts, relative to wild type extracts. alpha-Crystallin and an Hsp30-enriched preparation protected purified hexokinase from thermal inactivation in vitro, supporting the proposal that Hsp30 may directly stabilize hexokinase in vivo during heat shock.

Use of 1 H Nuclear Magnetic Resonance To Measure Intracellular Metabolite Levels during Growth and Asexual Sporulation in Neurospora crassa

“…Previously published studies have shown that metabolites that can be readily used as carbon sources, such as glutamate and trehalose, are found in dormant spores of N. crassa (61). A role for trehalose as a thermal protectant has been established for N. crassa (56). This previous work also demonstrated that although trehalose is a large component of the metabolite pool in conidia, the relative levels of trehalose are similar in conidia and hyphae of wild-type strains.…”

Stress-Induced Cell Death Is Mediated by Ceramide Synthesis in Neurospora crassa

“…In this way it depletes the cells of ATP, as well as blocking glycolysis and other steps in glucose metabolism (30). Glycolysis and heat shock-induced glycolytic enzymes appear to be especially important for energy generation at high temperature (56,57). Although phosphorylated 2-DG has been reported to act as a high-glucose signal, e.g., in activating Akt kinase (20,61), the chief effect of 2-DG in our assays likely stems from its inhibition of glucose metabolism, since another glucose analog, Lsorbose, that is not phosphorylated (66) also leads to cell death, albeit less dramatically and at higher concentrations.…”

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

“…In response to high temperature, carbohydrate metabolism changes from a focus on oxidative respiration to an increase in glycolysis and fermentation [2–3]. However, addition of an inhibitor of glycolysis, such as 2-DG, would be expected to shift the balance from intense glycolysis to increased oxidative respiration.…”

“…This response is also characterized by increased reliance on glycolysis for energy generation [3] at the expense of mitochondrial oxidative respiration. We have asked whether cell recovery from heat shock could be compromised if glycolysis simultaneously was blocked by glucose starvation, induced by a glucose metabolism inhibitor, 2-deoxyglucose (2-DG) [4].…”