Acute depression of mitochondrial protein synthesis during anoxia: Contributions of oxygen sensing, matrix acidification, and redox state

Mitochondrial protein synthesis is acutely depressed during anoxia-induced quiescence in embryos of Artemia franciscana. Oxygen deprivation is accompanied in vivo by a dramatic drop in extramitochondrial pH, and both of these alterations strongly inhibit protein synthesis in isolated mitochondria. Here we show that the oxygen dependence is not explained simply by blockage of the electron transport chain or by the increased redox state. Whereas oxygen deprivation substantially depressed protein synthesis within 5 min and resulted in a 77% reduction after 1 h, aerobic incubations with saturating concentrations of cyanide or antimycin A had little effect during the first 20 min and only a modest effect after 1 h (36 and 20% reductions, respectively). Yet the mitochondrial NAD(P)H pools were fully reduced after 2-3 min with all three treatments. This cyanide- and antimycin-insensitive but hypoxia-sensitive pattern of protein synthesis depression suggests the presence of a molecular oxygen sensor within the mitochondrion. Second, we show for the first time that acidification of extramitochondrial pH exerts inhibition on protein synthesis specifically through changes in matrix pH. Matrix pH was 8.2 during protein synthesis assays performed at the extramitochondrial pH optimum of 7.5. When this proton gradient was abolished with nigericin, the extramitochondrial pH optimum for protein synthesis displayed an alkaline shift of ∼0.7 pH unit. These data suggest the presence of proton-sensitive translational components within the mitochondrion

Comparison of the transcriptomic "stress response" evoked by antimycin A and oxygen deprivation in saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Acute changes in environmental parameters (e.g., O₂, pH, UV, osmolarity, nutrients, etc.) evoke a common transcriptomic response in yeast referred to as the "environmental stress response" (ESR) or "common environmental response" (CER). Why such a diverse array of insults should elicit a common transcriptional response remains enigmatic. Previous functional analyses of the networks involved have found that, in addition to up-regulating those for mitigating the specific stressor, the majority appear to be involved in balancing energetic supply and demand and modulating progression through the cell cycle. Here we compared functional and regulatory aspects of the stress responses elicited by the acute inhibition of respiration with antimycin A and oxygen deprivation under catabolite non-repressed (galactose) conditions.</p> <p>Results</p> <p>Gene network analyses of the transcriptomic responses revealed both treatments result in the transient (10 – 60 min) down-regulation of MBF- and SBF-regulated networks involved in the G1/S transition of the cell cycle as well as Fhl1 and PAC/RRPE-associated networks involved in energetically costly programs of ribosomal biogenesis and protein synthesis. Simultaneously, Msn2/4 networks involved in hexose import/dissimilation, reserve energy regulation, and autophagy were transiently up-regulated. Interestingly, when cells were treated with antimycin A well before experiencing anaerobiosis these networks subsequently failed to respond to oxygen deprivation. These results suggest the transient stress response is elicited by the acute inhibition of respiration and, we postulate, changes in cellular energetics and/or the instantaneous growth rate, not oxygen deprivation <it>per se</it>. After a considerable delay (≥ 1 generation) under anoxia, predictable changes in heme-regulated gene networks (e.g., Hap1, Hap2/3/4/5, Mot3, Rox1 and Upc2) were observed both in the presence and absence of antimycin A.</p> <p>Conclusion</p> <p>This study not only differentiates between the gene networks that respond to respiratory inhibition and those that respond to oxygen deprivation but suggests the function of the ESR or CER is to balance energetic supply/demand and coordinate growth with the cell cycle, whether in response to perturbations that disrupt catabolic pathways or those that require rapidly up-regulating energetically costly programs for combating specific stressors.</p

Oxidative phosphorylation and the realkalinization of intracellular pH during recovery from anoxia in Artemia franciscana embryos

The contribution of mitochondrial oxidative phosphorylation to the realkalinization of intracellular pH (pHi) and resynthesis of purine nucleotides during recovery from anoxia was investigated in embryos of Artemia franciscana by assessing the sensitivity of mitochondrial respiration to pH, calculating proton consumption by oxidative phosphorylation, and measuring changes in pHi using 31P nuclear magnetic resonance. Following short-term anoxia, pHi increased from 6.7 to 7.7 during 20 min of aerobic recovery and was temporally correlated with a large increase in ATP. State 3 respiration rates of isolated mitochondria were not substantially compromised at the acidic pH corresponding to the pHi during anoxia (pH 6.3-6.8) compared to values obtained at pH 7.7. Both state 3 respiration rates and respiratory control ratios exhibited broad, substrate-specific pH optima, whereas state 4 respiration rates increased gradually with increasing pH. P:O flux ratios were near their mechanistic limits and did not vary appreciably with pH below 7.5. Estimates of intracellular buffering capacity indicate that between 18 and 37 mmol H+ (1 cytosol)-1 must be consumed to elevate pHi from 6.7 to 7.7. Phosphorylation of mono- and diphosphate purine-nucleotides during the first 20 min of recovery may account for the consumption of up to 4.79 mmol H+ (1 cytosol)-1. An additional 4.77 to 8.18 mmol H+ (1 cytosol)-1 may be consumed through the oxidation of mono- or dicarboxylic acids, respectively, in the Krebs cycle. Taken together, these data are consistent with a role for oxidative phosphorylation in the realkalinization of pHi and resynthesis of purine nucleotides in A. franciscana embryos during recovery from anoxia. © 1995

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery