Recent work in our lab and others has revealed that a certain threshold of mitochondrial respiration (40% of wild-type capacity) is necessary to promote chronological lifespan (CLS) extension via its dual roles in metabolic reprogramming to accumulate storage carbohydrates (trehalose and glycogen) and in the activation of the stress response to maintain homeostasis. The activation of the stress response includes the expression of molecular heat shock proteins (HSPs) and the antioxidant defence system mediated by the transcription factors Msn2/4, Gis1 and Hsf1. Here, we reveal that an intermediate threshold of heme biosynthesis is sufficient to activate the stress response and maintain mitochondrial redox homeostasis but not mitochondrial respiration.

In the first result Chapter (Chapter 3), using the hem1∆ mutants supplemented with different concentrations of 5-ALA, multiple thresholds of heme biosynthesis were shown to be necessary for fermentative cell growth, starvation-induced HSP gene expression and redox homeostasis, and mitochondrial biogenesis and respiratory growth. The essentiality of heme synthesis was probably due to its roles in synthesis of unsaturated lipids. An intermediate threshold (~55% of the WT level) was required for the full activation of the HSP gene expression, restoration of mitochondrial superoxide and fermentative cell growth to WT levels. Higher heme levels than the intermediate threshold were required for inducing respiratory growth and mitochondrial biogenesis.

In the second result Chapter (Chapter 4), transcriptome studies were conducted to reveal why the intermediate heme threshold was sufficient to activate the stress response and mitochondrial redox homeostasis. It has been found that the intermediate heme threshold is necessary to promote the transcription of ribosome biogenesis to support fermentative growth, while higher heme levels than the threshold are required to coordinate ribosome biogenesis and metabolic reprogramming in response to glucose starvation to promote respiratory growth. The intermediate heme threshold was sufficient to activate the expression of the HSP genes mediated by Msn2/4 and Gis1, and the Hap4-dependnet genes involved in oxidative phosphorylation. However, biochemical assays indicated that the intermediate heme threshold was not sufficient for the full activation of the antioxidant defence system. These data suggest that mitochondrial redox homeostasis is maintained at the intermediate threshold possibly due to limited respiration to generate ROS and the moderate activation of the anti-oxidant defence system to remove ROS, both of which are dependent on heme levels.

In the final result Chapter (Chapter 5), genetic and biochemical assays were conducted to find why heme deficiency leads to defective stress response and redox imbalance. Firstly, heme deficiency below the intermediate threshold leads to hyperpolarization of mitochondria and the accumulation of active Ras on mitochondria during glucose starvation. Removal of Ras2 from heme-deficient cells enhanced catalase and mitochondrial superoxide dismutase activities, restored hydrogen peroxide to WT levels, but had little impact on the levels of labile heme or mitochondrial superoxide. These data suggest that heme deficiency below the intermediate threshold leads to redox imbalance due to excessive generation of mitochondrial superoxide and compromised antioxidant defence system. Secondly, unlike the cox mutants, the ER-resident NADPH oxidase Yno1 was not responsible for ROS accumulation in the heme-deficient mutants. Finally, exogenous hemin was shown to rescue the redox imbalance in mitochondrial respiratory mutants independently of the SOD and catalase activities or the heme oxygenase Hmx1.

Put together, the above findings suggest that the intermediate heme threshold is necessary to maintain redox homeostasis through at least two distinct mechanisms: preventing aberrant Ras2 signalling to activate the Msn2/4- and Gis1-depedent stress response, and activating Hap4-dependent gene expression involved in oxidative phosphorylation to prevent excessive generation of mitochondrial superoxide. Labile heme may also participate in ROS removal independently of the antioxidant defence system. Given the role of heme in mitochondrial function and redox homeostasis, the intermediate heme threshold may provide a new perspective for understanding mitochondrial dysfunction and high levels of ROS in age-related diseases and exploring their potential therapies.