Coenzyme Q (CoQ) is an essential player in the respiratory electron transport chain and is the only lipid-soluble antioxidant synthesized endogenously in mammalian and yeast cells. In humans, genetic ...mutations, pathologies, certain medical treatments, and aging, result in CoQ deficiencies, which are linked to mitochondrial, cardiovascular, and neurodegenerative diseases. The only strategy available for these patients is CoQ supplementation. CoQ supplements benefit a small subset of patients, but the poor solubility of CoQ greatly limits treatment efficacy. Consequently, the efficient delivery of CoQ to the mitochondria and restoration of respiratory function remains a major challenge. A better understanding of CoQ uptake and mitochondrial delivery is crucial to make this molecule a more efficient and effective therapeutic tool. In this study, we investigated the mechanism of CoQ uptake and distribution using the yeast Saccharomyces cerevisiae as a model organism. The addition of exogenous CoQ was tested for the ability to restore growth on non-fermentable medium in several strains that lack CoQ synthesis (coq mutants). Surprisingly, we discovered that the presence of CoQ biosynthetic intermediates impairs assimilation of CoQ into a functional respiratory chain in yeast cells. Moreover, a screen of 40 gene deletions considered to be candidates to prevent exogenous CoQ from rescuing growth of the CoQ-less coq2Δ mutant, identified six novel genes (CDC10, RTS1, RVS161, RVS167, VPS1, and NAT3) as necessary for efficient trafficking of CoQ to mitochondria. The proteins encoded by these genes represent essential steps in the pathways responsible for transport of exogenously supplied CoQ to its functional sites in the cell, and definitively associate CoQ distribution with endocytosis and intracellular vesicular trafficking pathways conserved from yeast to human cells.
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•CoQ biosynthetic intermediates impair assimilation of exogenously supplied CoQ.•CDC10, RTS1, RVS161, RVS167, NAT3, and VPS1 genes are necessary for CoQ trafficking.•These proteins mediate transport of exogenous CoQ to functional sites in the cell.•CoQ transport is associated with endocytosis and intracellular vesicular trafficking.
Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated ...with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure–activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D1-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D2-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20–50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function.
The objective of this study was to characterize the lipidome and electron transport chain activities in purified non-synaptic (NS) and synaptic (Syn) mitochondria from C57BL/6J mouse cerebral cortex. ...Contamination from subcellular membranes, especially myelin, has hindered past attempts to accurately characterize the lipid composition of brain mitochondria. An improved Ficoll and sucrose discontinuous gradient method was employed that yielded highly enriched mitochondrial populations free of myelin contamination. The activities of Complexes I, II, III, and II/III were lower in Syn than in NS mitochondria, while Complexes I/III and IV activities were similar in both populations. Shotgun lipidomics showed that levels of cardiolipin (Ptd₂Gro) were lower, whereas levels of ceramide and phosphatidylserine were higher in Syn than in NS mitochondria. Coenzyme Q₉ and Q₁₀ was also lower in Syn than in NS mitochondria. Gangliosides, phosphatidic acid, sulfatides, and cerebrosides were undetectable in brain mitochondria. The distribution of Ptd₂Gro molecular species was similar in both populations and formed a unique pattern, consisting of seven major molecular species groups, when arranged according to mass to charge ratios. Remodeling involving choline and ethanolamine phosphoglycerides could explain Ptd₂Gro heterogeneity. NS and Syn mitochondrial lipidomic heterogeneity could influence energy metabolism, which may contribute to metabolic compartmentation of the brain.
Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae. Here, we report a role for the chromatin remodeler SWI/SNF in ...respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism.
The antiferromagnetic structures of the layered oxychalcogenides (Sr1−x Ba x )2CoO2Cu2S2 (0 ≤ x ≤ 1) have been determined by powder neutron diffraction. In these compounds Co2+ is coordinated by four ...oxide ions in a square plane and two sulfide ions at the apexes of an extremely tetragonally elongated octahedron; the polyhedra share oxide vertexes. The magnetic reflections present in the diffraction patterns can in all cases be indexed using a √2a × √2a × c expansion of the nuclear cell, and nearest-neighbor Co2+ moments couple antiferromagnetically within the CoO2 planes. The ordered magnetic moment of Co2+ in Sr2CoO2Cu2S2 (x = 0) is 3.8(1) μB at 5 K, consistent with high-spin Co2+ ions carrying three unpaired electrons and with an additional significant unquenched orbital component. Exposure of this compound to moist air is shown to result in copper deficiency and a decrease in the size of the ordered moment to about 2.5 μB; there is a strong correlation between the size of the long-range ordered moment and the occupancy of the Cu site. Both the tetragonal elongation of the CoO4S2 polyhedron and the ordered moment in (Sr1−x Ba x )2CoO2Cu2S2 increase with increasing Ba content, and in Ba2CoO2Cu2S2, which has Co2+ in an environment that is close to purely square planar, the ordered moment of 4.5(1) μB at 5 K is over 0.7 μB larger than that in Sr2CoO2Cu2S2, so the unquenched orbital component in this case is even larger than that observed in octahedral Co2+ systems such as CoO. The experimental observations of antiferromagnetic ground states and the changes in properties resulting from replacement of Sr by Ba are supported by ab initio calculations on Sr2CoO2Cu2S2 and Ba2CoO2Cu2S2. The large orbital moments in these systems apparently result from spin−orbit mixing of the unequally populated d xz , d yz , and d z 2 orbitals, which are reckoned to be almost degenerate when the CoO4S2 polyhedron reaches its maximum elongation. The magnitudes of the ordered moments in high-spin Co2+ oxide, oxychalcogenide, and oxyhalide systems are shown to correlate well with the tetragonal elongation of the coordination environment. The large orbital moments lead to an apparently magnetostrictive distortion of the crystal structures below the Neél temperature, with the symmetry lowered from tetragonal I4/mmm to orthorhombic Immm and the size of the distortion correlating well with the size of the long-range ordered moment for all compositions and for temperature-dependent data gathered on Ba2CoO2Cu2S2.
A series of layered oxychalcogenide and oxypnictide solids is described that contain oxide layers separated by distinct layers, which contain the softer chalcogenide (S, Se, Te) or pnictide (P, As, ...Sb, Bi) anions. The relationships between the crystal structures adopted by these compounds are described, and the physical and chemical properties of these materials are related to the structures and the properties of the elements. The properties exhibited by the oxychalcogenide materials include semiconductor properties, for example, in LaOCuCh (Ch = chalcogenide) and derivatives, unusual magnetic properties exhibited by the class Sr2MO2Cu2−δS2 (M = Mn, Co, Ni), and redox properties exhibited by the materials Sr2MnO2Cu2m−0.5S m+1 (m = 1−3) and Sr4Mn3O7.5Cu2Ch2 (Ch = S, Se). Recent results in the oxychalcogenide area are reviewed, and some new results on the intriguing series of compounds Sr2MO2Cu2−δS2 (M = Mn, Co, Ni) are reported. Oxypnictides have received less recent attention, but this is changing: a new frenzy of research is underway following the discovery of high-temperature superconductivity (>40 K) in derivatives of the layered oxyarsenide LaOFeAs. The early results in this exciting new area will be reviewed.
Terpenoid quinones are liposoluble redox-active compounds that serve as essential electron carriers and antioxidants. One such quinone, rhodoquinone (RQ), couples the respiratory electron transfer ...chain to the reduction of fumarate to facilitate anaerobic respiration. This mechanism allows RQ-synthesizing organisms to operate their respiratory chain using fumarate as a final electron acceptor. RQ biosynthesis is restricted to a handful of prokaryotic and eukaryotic organisms, and details of this biosynthetic pathway remain enigmatic. One gene, rquA, was discovered to be required for RQ biosynthesis in Rhodospirillum rubrum. However, the function of the gene product, RquA, has remained unclear. Here, using reverse genetics approaches, we demonstrate that RquA converts ubiquinone to RQ directly. We also demonstrate the first in vivo synthetic production of RQ in Escherichia coli and Saccharomyces cerevisiae, two organisms that do not natively produce RQ. These findings help clarify the complete RQ biosynthetic pathway in species which contain RquA homologs.
•A complete pathway for rhodoquinone biosynthesis in rquA-producing species has been elucidated.•Recombinant RquA is active in two non rhodoquinone-producing species, Escherichia coli and Saccharomyces cerevisiae.•Expression of RquA in E. coli and S. cerevisiae facilitates the in vivo synthesis of rhodoquinone.•Ubiquinone is a required substrate of RquA, and the product of the RquA reaction is rhodoquinone.
Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae ...mutants designated coq1-coq8. Here we provide genetic evidence that several of the Coq polypeptides interact with one another. Deletions in any of the COQ genes affect the steady-state expression of Coq3p, Coq4p, and Coq6p. Antibodies that recognize Coq1p, a hexaprenyl diphosphate synthase, were generated and used to determine that Coq1p is peripherally associated with the inner membrane on the matrix side. Yeast Δcoq1 mutants harboring diverse Coq1 orthologs from prokaryotic species produce distinct sizes of polyprenyl diphosphate and hence distinct isoforms of Q including Q7, Q8, Q9, or Q10 (Okada, K., Kainou, T., Matsuda, H., and Kawamukai, M. (1998) FEBS Lett. 431, 241–244). We find that steady-state levels of Coq3p, Coq4p, and Coq6p are rescued in some cases to near wild-type levels by the presence of these diverse Coq1 orthologs in the Δcoq1 mutant. These data suggest that the lipid product of Coq1p or a Q-intermediate derived from polyprenyl diphosphate is involved in stabilizing the Coq3, Coq4, and Coq6 polypeptides.
Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the ...hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5′ and 3′, respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q10. Here we show that the Q9 content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q9 content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.
► Two C. elegans coq-3 null mutants lack coenzyme Q but have distinct phenotypes. ► One null coq-3 mutant shows limited fertility, while the other is sterile. ► Unlike clk-1 mutants, neither coq-3 mutant is rescued with dietary Q supplements. ► coq-3 mutants rescued for de novo Q synthesis are as fertile as wild-type worms. ► Augmenting de novo synthesis of Q is more effective than dietary therapies with Q.
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