Caenorhabditis elegans diet significantly affects metabolic profile, mitochondrial DNA levels, lifespan and brood size.

Diet can have profound effects on an organism's health. Metabolic studies offer an effective way to measure and understand the physiological effects of diet or disease. The metabolome is very sensitive to dietary, lifestyle and genetic changes. Caenorhabditis elegans, a soil nematode, is an attractive model organism for metabolic studies because of the ease with which genetic and environmental factors can be controlled. In this work, we report significant effects of diet, mutation and RNA interference on the C.elegans metabolome. Two strains of Escherichia coli, OP50 and HT115 are commonly employed as food sources for maintaining and culturing the nematode. We studied the metabolic and phenotypic effects of culturing wild-type and mutant worms on these two strains of E. coli. We report significant effects of diet on metabolic profile, on mitochondrial DNA copy number and on phenotype. The dietary effects we report are similar in magnitude to the effects of mutations or RNA interference-mediated gene suppression. This is the first critical evaluation of the physiological and metabolic effects on C.elegans of two commonly used culture conditions.

Mitochondrial respiratory chain deficiency in Caenorhabditis elegans results in developmental arrest and increased life span.

The growth and development of Caenorhabditis elegans are energy-dependent and rely on the mitochondrial respiratory chain (MRC) as the major source of ATP. The MRC is composed of approximately 70 nuclear and 12 mitochondrial gene products. Complexes I and V are multisubunit proteins of the MRC. The nuo-1 gene encodes the NADH- and FMN-binding subunit of complex I, the NADH-ubiquinone oxidoreductase. The atp-2 gene encodes the active-site subunit of complex V, the ATP synthase. The nuo-1(ua1) and atp-2(ua2) mutations are both lethal. They result in developmental arrest at the third larval stage (L3), arrest of gonad development at the second larval stage (L2), and impaired mobility, pharyngeal pumping, and defecation. Surprisingly, the nuo-1 and atp-2 mutations significantly lengthen the life spans of the arrested animals. When MRC biogenesis is blocked by chloramphenicol or doxycycline (inhibitors of mitochondrial translation), a quantitative and homogeneous developmental arrest as L3 larvae also results. The common phenotype induced by the mutations and drugs suggests that the L3-to-L4 transition may involve an energy-sensing developmental checkpoint. Since approximately 200 gene products are needed for MRC assembly and mtDNA replication, transcription, and translation, we predict that L3 arrest will be characteristic of mutations in these genes.

The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase.

The Saccharomyces cerevisiae succinate dehydrogenase (SDH) of the mitochondrial electron transport chain oxidizes succinate and reduces ubiquinone. Using a random mutagenesis approach, we identified functionally important amino acid residues in one of the anchor subunits, Sdh4p. We analyzed three point mutations (F69V, S71A, and H99L) and one nonsense mutation (Y89OCH) that truncates the Sdh4p subunit at the third predicted transmembrane segment. The F69V and the S71A mutations result in greatly impaired respiratory growth in vivo and quinone reductase activities in vitro, with negligible effects on enzyme stability. In contrast, the Y89OCH and the H99L mutations elicit large structural perturbations that impair assembly as evidenced by reduced covalent FAD levels, membrane-associated succinate-phenazine methosulfate reductase activities, and thermal stability. We propose that the Phe-69 and the Ser-71 residues are involved in the formation of a quinone-binding site, whereas the His-99 residue is at the interface of the peripheral and the membrane domains. In addition, the properties of the Y89OCH mutation are consistent with the interpretation that the third transmembrane segment is not involved in catalysis but rather plays an important structural role. The mutant enzymes are differentially sensitive to a quinone analog inhibitor, providing further evidence for a two-quinone binding model in the yeast SDH.

The Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase. Identification of Sdh3p amino acid residues involved in ubiquinone binding.

Succinate dehydrogenase (SDH) participates in the mitochondrial electron transport chain by oxidizing succinate to fumarate and transferring the electrons to ubiquinone. In yeast, it is composed of a catalytic dimer, comprising the Sdh1p and Sdh2p subunits, and a membrane domain, comprising two smaller hydrophobic subunits, Sdh3p and Sdh4p, which anchor the enzyme to the mitochondrial inner membrane. To investigate the role of the Sdh3p anchor polypeptide in enzyme assembly and catalysis, we isolated and characterized seven mutations in the SDH3 gene. Two mutations are premature truncations of Sdh3p with losses of one or three transmembrane segments. The remaining five are missense mutations that are clustered between amino acids 103 and 117, which are proposed to be located in transmembrane segment II or the matrix-localized loop connecting segments II and III. Three mutations, F103V, H113Q, and W116R, strongly but specifically impair quinone reductase activities but have only minor effects on enzyme assembly. The clustering of the mutations strongly suggests that a ubiquinone-binding site is associated with this region of Sdh3p. In addition, the biphasic inhibition of quinone reductase activity by a dinitrophenol inhibitor supports the hypothesis that two distinct quinone-binding sites are present in the yeast SDH.

The Saccharomyces cerevisiae succinate dehydrogenase anchor subunit, Sdh4p: mutations at the C-terminal lys-132 perturb the hydrophobic domain.

The yeast succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits, Sdh1p-Sdh4p, that couples the oxidation of succinate to the transfer of electrons to ubiquinone. One of the membrane anchor subunits, Sdh4p, has an unusual 30 amino acid extension at the C-terminus that is not present in SDH anchor subunits of other organisms. We identify Lys-132 in the Sdh4p C-terminal region as necessary for enzyme stability, ubiquinone reduction, and cytochrome b562 assembly in SDH. Five Lys-132 substituted SDH4 genes were constructed by site-directed mutagenesis and introduced into an SDH4 knockout strain. The mutants, K132E, K132G, K132Q, K132R, and K132V were characterized in vivo for respiratory growth and in vitro for ubiquinone reduction, enzyme stability, and cytochrome b562 assembly. Only the K132R substitution, which conserves the positive charge of Lys-132, produces a wild-type enzyme. The remaining four mutants do not affect the ability of SDH to oxidize succinate in the presence of the artificial electron acceptor, phenazine methosulfate, but impair quinone reductase activity, enzyme stability, and heme insertion. Our results suggest that the presence of a positive charge on residue 132 in the C-terminus of Sdh4p is critical for establishing a stable conformation in the SDH hydrophobic domain that is compatible with ubiquinone reduction and cytochrome b562 assembly. In addition, our data suggest that heme does not play an essential role in quinone reduction.

The Saccharomyces cerevisiae succinate-ubiquinone reductase contains a stoichiometric amount of cytochrome b562.

The Saccharomyces cerevisiae succinate-ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b-type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b562 in the yeast SDH. The cytochrome is detected as a peak present in fumarate-oxidized, dithionite-reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92+/-0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b562 is a component of the yeast SDH.

The Saccharomyces cerevisiae TCM62 gene encodes a chaperone necessary for the assembly of the mitochondrial succinate dehydrogenase (complex II).

The assembly of the mitochondrial respiratory chain is mediated by a large number of helper proteins. To better understand the biogenesis of the yeast succinate dehydrogenase (SDH), we searched for assembly-defective mutants. SDH is encoded by the SDH1, SDH2, SDH3, and SDH4 genes. The holoenzyme is composed of two domains. The membrane extrinsic domain, consisting of Sdh1p and Sdh2p, contains a covalent FAD cofactor and three iron-sulfur clusters. The membrane intrinsic domain, consisting of Sdh3p and Sdh4p, is proposed to bind two molecules of ubiquinone and one heme. We isolated one mutant that is respiration-deficient with a specific loss of SDH oxidase activity. SDH is not assembled in this mutant. The complementing gene, TCM62 (also known as SCYBR044C), does not encode an SDH subunit and is not essential for cell viability. It encodes a mitochondrial membrane protein of 64,211 Da. The Tcm62p sequence is 17.3% identical to yeast hsp60, a molecular chaperone. The Tcm62p amino terminus is in the mitochondrial matrix, whereas the carboxyl terminus is accessible from the intermembrane space. Tcm62p forms a complex containing at least three SDH subunits. We propose that Tcm62p functions as a chaperone in the assembly of yeast SDH.

Interactions of a high mobility group-like protein with human mitochondrial DNA.

A number of nuclear-encoded proteins are known to bind to the mitochondrial genome and may be involved in mitochondrial replication and in mitochondrial diseases. Mitochondrial diseases such as Kearns-Sayre syndrome (KSS) are flanked by common direct repeats. To study protein binding to these mitochondria DNA regions we used gel mobility shift binding assays. Proteins present in nuclear or in mitochondrial extracts from normal or KSS-derived fibroblasts bound to a 280-bp mitochondrial DNA fragment encompassing a deletion breakpoint in the mitochondrial genome. In addition, nuclear and mitochondrial protein bound to a nearby surrounding fragment and to a synthetic oligonucleotide with a related consensus DNA sequence. Southwestern blot analysis showed that the DNA binding ability resided in a 35-kDa protein. The amino terminal sequence of the 35-kDa protein was very similar to human high mobility group proteins. These results suggest that this protein may be involved in mitochondrial DNA deletions.

The COQ5 gene encodes a yeast mitochondrial protein necessary for ubiquinone biosynthesis and the assembly of the respiratory chain.

Saccharomyces cerevisiae is a facultative anaerobe capable of meeting its energy requirements by fermentation and is thus an ideal system for studying the biogenesis of respiring mitochondria. We have isolated a respiration-deficient mutant exhibiting a pleiotropic loss of the mitochondrial electron transport chain. The corresponding wild-type gene, COQ5, was cloned, sequenced, and able to restore respiratory growth. Deletion of the chromosomal COQ5 gene results in a respiration deficiency and reduced levels of respiratory protein components. Exogenously added decylubiquinone can partially restore electron transport chain function to mitochondrial membranes from the deletion mutant. The COQ5 nucleotide sequence predicts a polypeptide of 307 amino acids containing a mitochondrial targeting signal. COQ5p is 43% identical to the polypeptide predicted by the Escherichia coli open reading frame, o251 (1). The COQ5 gene, when introduced into E. coli, complements the respiratory deficiency of an ubiE mutant that maps near o251, suggesting that it is the yeast homolog of the ubiE gene product. We conclude that the COQ5 gene encodes the mitochondria-localized 2-hexaprenyl-6-methoxy-1,4-benzoquinone methyltransferase of the yeast ubiquinone biosynthetic pathway.

The carboxyl terminus of the Saccharomyces cerevisiae succinate dehydrogenase membrane subunit, SDH4p, is necessary for ubiquinone reduction and enzyme stability.

The succinate dehydrogenase (SDH) of Saccharomyces cerevisiae is composed of four nonidentical subunits encoded by the nuclear genes SDH1, SDH2, SDH3, and SDH4. The hydrophilic subunits, SDH1p and SDH2p, comprise the catalytic domain involved in succinate oxidation. They are anchored to the inner mitochondrial membrane by two small, hydrophobic subunits, SDH3p and SDH4p, which are required for electron transfer and ubiquinone reduction. Comparison of the deduced primary sequence of the yeast SDH4p subunit to SDH4p subunits from other species reveals the presence of an unusual 25-30 amino acid carboxyl-terminal extension following the last predicted transmembrane domain. The extension is predicted to be on the cytoplasmic side of the inner mitochondrial membrane. To investigate the extension's function, three truncations were created and characterized. The results reveal that the carboxyl-terminal extension is necessary for respiration and growth on nonfermentable carbon sources, for ubiquinone reduction, and for enzyme stability. Combined with inhibitor studies using a ubiquinone analog, our results suggest that the extension and more specifically, residues 128-135 are involved in the formation of a ubiquinone binding site. Our findings support a two-ubiquinone binding site model for the S. cerevisiae SDH.

Zidovudine and dideoxynucleosides deplete wild-type mitochondrial DNA levels and increase deleted mitochondrial DNA levels in cultured Kearns-Sayre syndrome fibroblasts.

Kearns-Sayre syndrome is the most commonly diagnosed mitochondrial cytopathy and produces severe neuromuscular symptoms. The most frequent cause is a mitochondrial DNA deletion that removes a 4977-base pair segment of DNA that includes several genes encoding for respiratory chain subunits. Treatment of AIDS patients with nucleoside analogs has been reported to cause mtDNA depletion and myopathies. Here, we report that azidothymidine, dideoxyguanosine, and dideoxycytidine cause a depletion of wild-type mtDNA while increasing the levels of deleted mitochondria DNA in Kearns-Sayre syndrome fibroblasts. The result of these effects is a large increase in the relative amounts of delta mtDNA in comparison to wild type mtDNA. We found that Kearns-Sayre syndrome fibroblasts are a mixed population of cells with deleted mtDNA comprising from 0 to over 20% of the total mtDNA in individual cells. Treatment of cloned cell lines with dideoxycytidine did not result in increased levels of delta mtDNA. The results suggest that nucleoside analogs may act to increase the average delta mtDNA levels in a mixed population of cells by preferentially inhibiting the proliferation of cells with little or no delta mtDNA. This raises the possibility that modulation of deleted mtDNA levels may occur by similar mechanisms in vivo, in response to the influence of exogenous agents.

Covalent attachment of FAD to the yeast succinate dehydrogenase flavoprotein requires import into mitochondria, presequence removal, and folding.

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial respiratory chain enzyme that utilizes the cofactor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron-sulfur protein, and two hydrophobic subunits. The FAD is covalently attached to a histidine residue near the amino terminus of the flavoprotein. In this study, we have investigated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between apo- and holoflavoproteins. Cofactor attachment, both in vivo and in vitro, occurs within the mitochondrial matrix once the presequence has been cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate. Furthermore, this modification does not occur with C-terminally truncated flavoprotein subunits that are fully competent for import. Taken together, these data suggest that cofactor addition occurs to an imported protein that has folded sufficiently to recognize both FAD and its substrate.

A requirement for matrix processing peptidase but not for mitochondrial chaperonin in the covalent attachment of FAD to the yeast succinate dehydrogenase flavoprotein.

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial heterotetramer containing a flavoprotein subunit with an 8alpha-N(3)-histidyl-linked FAD cofactor. The covalent linkage of the FAD is necessary for activity. We have developed an in vitro assay that measures the flavinylation of the flavoprotein precursor in mitochondrial matrix fractions. Flavoprotein modification does not depend on translocation across a membrane, but it does require proteolytic processing by the mitochondrial processing peptidase prior to flavin attachment. Since ATP depletion, N-ethylmaleimide, or proteinase treatments of matrix fractions inhibit flavoprotein modification, at least one additional matrix protein component appears to be required. Having previously suggested that the flavoprotein begins folding before FAD attachment occurs, we tested whether the mitochondrial chaperonin, heat shock protein 60, might be necessary. Co-immunoprecipitation of the flavoprotein and the chaperonin demonstrate that the proteins do indeed interact. However, immunodepletion of the chaperonin from matrix fractions does not inhibit FAD attachment. Nonprotein components are also required for flavoprotein modification. In addition to ATP, effector molecules such as succinate, fumarate, or malate also stimulate modification. Together, these results suggest that FAD addition is an early event in succinate dehydrogenase assembly.

Quantification of mitochondrial DNA in heteroplasmic fibroblasts with competitive PCR.

Kearns-Sayre syndrome (KSS) is a disease with severe clinical symptoms that often arises from a mitochondrial DNA deletion of 4977 bp. Quantification of defective mitochondrial DNA is important since the severity of symptoms in KSS is thought to be related to increased content of abnormal mitochondrial DNA. We developed a rapid, quantitative and competitive PCR assay to measure both wild-type and mutant forms of mitochondrial DNA in cells from KSS patients. The assay can accurately measure absolute numbers of mitochondrial DNA per cell by normalizing to a single copy nuclear gene.

The covalent attachment of FAD to the flavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria.

Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron-transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron-sulfur protein subunit with three different iron-sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8 alpha-[N(3)-histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinate-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.

Structure and regulation of SDH3, the yeast gene encoding the cytochrome b560 subunit of respiratory complex II.

Using an expression library, we have isolated yeast genes activated in the presence of the yeast CCAAT box-binding protein HAP2. One of these genes, SDH3, encodes the cytochrome b560 subunit of respiratory complex II. The SDH3 protein contains three potential transmembrane domains and is more than 30% identical to bovine cytochrome b560 and to a mitochondrially encoded protein from Marchantia polymorpha. Disruption of SDH3 shows that this gene is required for growth on non-fermentable carbon sources. Expression of SDH1, SDH3, and SDH4 is activated in the presence of the HAP2 transcriptional activator.

Isolation and characterization of the Saccharomyces cerevisiae SDH4 gene encoding a membrane anchor subunit of succinate dehydrogenase.

Succinate dehydrogenase (EC 1.3.99.1) is an intrinsic bacterial or inner mitochondrial membrane protein that catalyses the oxidation of succinate and donates electrons to the respiratory chain via quinone acceptors. It is a heterotetramer composed of a flavoprotein, an iron-sulfur, and two hydrophobic subunits. We purified succinate dehydrogenase by blue native gel electrophoresis, determined the amino-terminal sequence of the Sdh4p subunit and used this information to clone the SDH4 gene. It encodes a precursor protein of 181 amino acids that is converted to the 150-amino acid mature Sdh4p protein with a mass of 16,638 Da. Hydrophobicity analysis predicts that Sdh4p forms three transmembrane alpha-helices. We have constructed an SDH4 mutant by targeted gene disruption; it retains the ability to grow on rich glycerol medium. Western blot analysis of SDH4 disruption mutant membrane fractions indicates that membrane attachment of the flavoprotein and iron-sulfur subunits is impaired but not abolished. This membrane-bound enzyme is able to reduce ubiquinone, although less efficiently than the wild-type enzyme. These findings indicate that Sdh4p contributes both to the membrane attachment of the catalytic flavoprotein and iron-sulfur subunits and to electron transfer to ubiquinone.

Isolation and nucleotide sequence of the Saccharomyces cerevisiae gene for the succinate dehydrogenase flavoprotein subunit.

Succinate dehydrogenase (EC 1.3.99.1) of the mitochondrial inner membrane is a four-subunit membrane-bound enzyme that catalyzes the oxidation of succinate to fumarate and the transfer of electrons into the electron transport chain to oxygen. The catalytic domain of the enzyme is composed of a flavoprotein subunit which contains a covalently attached FAD cofactor and an iron-sulfur subunit with three nonidentical iron-sulfur clusters. We have isolated a complete genomic clone for the flavoprotein subunit of the succinate dehydrogenase from Saccharomyces cerevisiae and determined its nucleotide sequence. The sequence predicts a protein of 70,185 Da (640 amino acids) that shows more similarity to the Escherichia coli succinate dehydrogenase flavoprotein subunit than it does to the only other mitochondrial homologue, the human flavoprotein subunit. The yeast flavoprotein subunit precursor was synthesized in a cell-free translation system and shown to possess a mitochondrial targeting sequence that directs its import into isolated, energized mitochondria where it is processed by the matrix-localized protease. The genes for the flavoprotein and the iron-sulfur subunits reside on different chromosomes and hence form different transcriptional units.

Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.