Mitochondrial activity in illuminated leaves of chlorophyll-deficient mutant rice (<Emphasis Type="Italic">OsCHLH</Emphasis>) seedlings

The rice CHLH gene encodes the Mg²⁺-chelatase H subunit, which is involved in chlorophyll biosynthesis. Growth of the chlorophyll-deficient oschlh mutant is supported by mitochondrial activity. In this study, we investigated the activity of mitochondrial respiration in the illuminated leaves during oschlh seedling development. Growth of mutant plants was enhanced in the presence of 3% sucrose, which may be used by mitochondria to meet cellular energy requirements. ATP content in these mutants was, however, significantly lowered in light conditions. Low cytosolic levels of NADH in illuminated oschlh mutant leaves further indicated the inhibition of mitochondrial metabolism. This down-regulation was particularly evident for oxidative stress-responsive genes in the mutant under light conditions. Hydrogen peroxide levels were higher in oschlh mutant leaves than in wild-type leaves; this increase was largely caused by the impairment of the expression of the antioxidant genes, such as OsAPX1, OsRAC1, and OsAOXc in knockout plants. Moreover, treatment of mesophyll protoplasts with ascorbic acid or catalase recovered ATP content in the mutants. Taken together, these results suggest that the light-mediated inhibition of mitochondrial activity leads to stunted growth of CHLH rice seedlings.     

The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana

The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis. However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis. Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

Heat Shock Protein 22 (Hsp22) Regulates Oxidative Phosphorylation upon Its Mitochondrial Translocation with the Inducible Nitric Oxide Synthase in Mammalian Heart

Objectives

Stress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 overexpression in vivo stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion in vivo significantly reduces respiration. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS.

Methods and Results

Adenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a N-terminal 20 amino acid putative mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, WT-Hsp22 accumulated in mitochondria by 2.5 fold (P<0.05) and increased oxygen consumption rates by 2-fold (P<0.01). This latter effect was abolished upon addition of the selective iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2.5-fold (P<0.01), and also increased iNOS mitochondrial localization by 4.5 fold vs β-gal (P<0.05). Upon comparable overexpression, the N20-Hsp22 mutant did not show significant mitochondrial translocation or stimulation of mitochondrial respiration. Moreover, although N20-Hsp22 did increase global iNOS expression by 4.6-fold, it did not promote iNOS mitochondrial translocation.

Conclusion

Translocation of both Hsp22 and iNOS to the mitochondria is necessary for Hsp22-mediated stimulation of oxidative phosphorylation.     

Mitochondrial UCP4 attenuates MPP+- and dopamine-induced oxidative stress,mitochondrial depolarization,and ATP deficiency in neurons and is interlinked with UCP2 expression

Mitochondrial uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis. We explored the neuroprotective role of UCP4 with its stable overexpression in SH-SY5Y cells, after exposure to either MPP⁺ or dopamine to induce ATP deficiency and oxidative stress. Cells overexpressing UCP4 proliferated faster in normal cultures and after exposure to MPP⁺ and dopamine. Differentiated UCP4-overexpressing cells survived better when exposed to MPP⁺ with decreased LDH release. Contrary to the mild uncoupling hypothesis, UCP4 overexpression resulted in increased absolute ATP levels (with ADP/ATP ratios similar to those of controls under normal conditions and ADP supplementation) associated with increased respiration rate. Under MPP⁺ toxicity, UCP4 overexpression preserved ATP levels and mitochondrial membrane potential (MMP) and reduced oxidative stress; the preserved ATP level was not due to increased glycolysis. Under MPP⁺ toxicity, the induction of UCP2 expression in vector controls was absent in UCP4-overexpressing cells, suggesting that UCP4 may compensate for UCP2 expression. UCP4 function does not seem to adhere to the mild uncoupling hypothesis in its neuroprotective mechanisms under oxidative stress and ATP deficiency. UCP4 overexpression increases cell survival by inducing oxidative phosphorylation, preserving ATP synthesis and MMP, and reducing oxidative stress.     

Mitochondrial Aminopeptidase Deletion Increases Chronological Lifespan and Oxidative Stress Resistance while Decreasing Respiratory Metabolism in S. cerevisiae

Recessive mutations in XPNPEP3, encoding a mitochondrial x-prolyl aminopeptidase, have been identified in families with a rare hereditary tubulointerstitial kidney disease. The yeast ortholog of XPNPEP3, Icp55p, participates in the proteolytic processing and stabilization of mitochondrial proteins and its deletion accelerates the degradation of its protein targets. We used icp55 deletion strains of S. cerevisiae to model loss of XPNPEP3 enzymatic function and study its phenotypic consequences on mitochondrial function. We found that Icp55p is not required for respiratory competence; however, compared to controls deletion strains had reduced mitochondrial oxygen consumption when grown in glucose containing media. The reduced mitochondrial respiration of icp55 deletion strains in glucose media requires the mitochondrial peptide transporter, Mdl1p, and was corrected by Tor1p inhibition with rapamycin. Under similar growth conditions the abundance of the mitochondrial ATP synthase complex was decreased in the icp55 deletion strain and was corrected by concurrent deletion of tor1. The icp55 deletion strain demonstrated an increased chronological lifespan and decreased reactive oxygen species. These changes were additive to similar changes known to occur in tor1 deletion strains suggesting independent mechanisms. Together, these results demonstrate that loss of Icp55p function reduces mitochondrial oxygen consumption and ATP synthase complex assembly in glucose media, while also promoting stress resistance, decreasing reactive oxygen species and increasing chronological lifespan through mechanisms that are distinct from decreased Tor1p activity.     

Arabidopsis Voltage-Dependent Anion Channel 1 (AtVDAC1) Is Required for Female Development and Maintenance of Mitochondrial Functions Related to Energy-Transaction

The voltage-dependent anion channels (VDACs), prominently localized in the outer mitochondrial membrane, play important roles in the metabolite exchange, energy metabolism and mitochondria-mediated apoptosis process in mammalian cells. However, relatively little is known about the functions of VDACs in plants. To further investigate the function of AtVDAC1 in Arabidopsis, we analyzed a T-DNA insertion line for the AtVDAC1 gene. The knock-out mutant atvdac1 showed reduced seed set due to a large number of undeveloped ovules in siliques. Genetic analyses indicated that the mutation of AtVDAC1 affected female fertility and belonged to a sporophytic mutation. Abnormal ovules in the process of female gametogenesis were observed using a confocal laser scanning microscope. Interestingly, both mitochondrial transmembrane potential (ΔΨ) and ATP synthesis rate were obviously reduced in the mitochondria isolated from atvdac1 plants.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

Mitochondrial complex III in larval stage of Echinococcus multilocularis as a potential chemotherapeutic target and in vivo efficacy of atovaquone against primary hydatid cysts

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc₁ complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC₅₀). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug.     

Mitochondrial respiration of complex II is not lower than that of complex I in mouse skeletal muscle

Skeletal muscle (SKM) requires a large amount of energy, which is produced mainly by mitochondria, for their daily functioning. Of the several mitochondrial complexes, it has been reported that the dysfunction of complex II is associated with several diseases, including myopathy. However, the degree to which complex II contributes to ATP production by mitochondria remains unknown. As complex II is not included in supercomplexes, which are formed to produce ATP efficiently, we hypothesized that complex II-linked respiration was lower than that of complex I. In addition, differences in the characteristics of complex I and II activity suggest that different factors might regulate their function. The isolated mitochondria from gastrocnemius muscle was used for mitochondrial respiration measurement and immunoblotting in male C57BL/6J mice. Student paired t-tests were performed to compare means between two groups. A univariate linear regression model was used to determine the correlation between mitochondrial respiration and proteins. Contrary to our hypothesis, complex II-linked respiration was not significantly less than complex I-linked respiration in SKM mitochondria (complex I vs complex II, 3402 vs 2840 pmol/[s × mg]). Complex I-linked respiration correlated with the amount of complex I incorporated in supercomplexes (r = 0.727, p < 0.05), but not with the total amount of complex I subunits. In contrast, complex II-linked respiration correlated with the total amount of complex II (r = 0.883, p < 0.05), but not with the amount of each complex II subunit. We conclude that both complex I and II play important roles in mitochondrial respiration and that the assembly of both supercomplexes and complex II is essential for the normal functioning of complex I and II in mouse SKM mitochondria.