Insertion and deletion mutations (indels) are important mechanisms of generating protein diversity. Indels in coding sequences are under considerable selective pressure to maintain reading frames and to preserve protein function, but once generated, indels provide raw material for the acquisition of new protein properties and functions. We reported recently that coding sequence insertions in the Candida albicans NDU1 protein, a mitochondrial protein involved in the assembly of the NADH:ubiquinone oxidoreductase are imperative for respiration, biofilm formation and pathogenesis. NDU1 inserts are specific to CTG-clade fungi, absent in human ortholog and successfully harnessed as drug targets. Here, we present the first comprehensive report investigating indels and clade-defining insertions (CDIs) in fungal proteomes. We investigated 80 ascomycete proteomes encompassing CTG clade species, the Saccharomycetaceae family, the Aspergillaceae family and the Herpotrichiellaceae (black yeasts) family. We identified over 30,000 insertions, 4,000 CDIs and 2,500 clade-defining deletions (CDDs). Insert sizes range from 1 to over 1,000 residues in length, while maximum deletion length is 19 residues. Inserts are strikingly over-represented in protein kinases, and excluded from structural domains and transmembrane segments. Inserts are predicted to be highly disordered. The amino acid compositions of the inserts are highly depleted in hydrophobic residues and enriched in polar residues. An indel in the Saccharomyces cerevisiae Sth1 protein, the catalytic subunit of the RSC (Remodel the Structure of Chromatin) complex is predicted to be disordered until it forms a ß-strand upon interaction. This interaction performs a vital role in RSC-mediated transcriptional regulation, thereby expanding protein function.

A forward genetic screening approach identified orf19.2500 as a gene controlling Candida albicans biofilm dispersal and biofilm detachment. Three-dimensional (3D) protein modeling and bioinformatics revealed that orf19.2500 is a conserved mitochondrial protein, structurally similar to, but functionally diverged from, the squalene/phytoene synthases family. The C. albicans orf19.2500 is distinguished by 3 evolutionarily acquired stretches of amino acid inserts, absent from all other eukaryotes except a small number of ascomycete fungi. Biochemical assays showed that orf19.2500 is required for the assembly and activity of the NADH ubiquinone oxidoreductase Complex I (CI) of the respiratory electron transport chain (ETC) and was thereby named NDU1. NDU1 is essential for respiration and growth on alternative carbon sources, important for immune evasion, required for virulence in a mouse model of hematogenously disseminated candidiasis, and for potentiating resistance to antifungal drugs. Our study is the first report on a protein that sets the Candida-like fungi phylogenetically apart from all other eukaryotes, based solely on evolutionary “gain” of new amino acid inserts that are also the functional hub of the protein.

The NADH:ubiquinone oxidoreductase (complex I) is the largest member of the mitochondrial respiratory chain. Its FMN cofactor accepts two electrons from NADH and transfers them to ubiquinone via a chain of iron-sulphur centers. A central core of 14 highly conserved subunits can couple electron transfer to proton translocation. The mammalian enzyme has an additional ~30 accessory subunits. Complex I has important bioenergetic and metabolic functions and is a known source of reactive oxygen species; these functions link it to a number of hereditary and degenerative diseases. For many complex I deficiencies, the primary defect is not in a subunit-encoding gene, but rather in an assembly factor or chaperone that participates in the biogenesis of newly synthesized complex I from individual subunits and cofactors. NDUFAF6 encodes a complex I assembly factor and mutations result in complex I deficiency, Leigh syndrome or Acadian variant Fanconi syndrome. Human NDUFAF6 is a mitochondria-targeted 333-amino acid protein belonging to the family of squalene and phytoene synthases. Sequence and structural information suggests that NDUFAF6 likely has enzymatic activity, but one that has evolved considerable differences from canonical squalene and phytoene synthases. Most but not all metazoans have an NDUFAF6 ortholog, indicating that in some organisms, complex I biogenesis does not require this protein. NDUFAF6 is a peripheral membrane protein and predictions identify a conserved C-terminal attachment site that have implications for substrate access.

Complex I (NADH:ubiquinone oxidoreductase) is the major entry point for electrons into the respiratory chains of bacteria and mitochondria. Mammalian complex I is composed of 45 subunits and harbors FMN and iron–sulfur cluster cofactors. A heterogeneous disease profile is associated with complex I deficiency. In a large fraction of complex I deficiencies, the primary defect is not in any of the genes encoding a subunit. The proper assembly and function of complex I require the participation of at least 12 assembly factors or chaperones. FOXRED1 encodes a complex I-specific assembly factor and mutations in this gene result in complex I deficiency, infantile onset encephalomyopathy and Leigh syndrome. The human FOXRED1 protein is a mitochondria-targeted 486-amino acid FAD-dependent oxidoreductase. It is most closely related to N-methyl amino acid dehydrogenases. FOXRED1 orthologs are present in archaea, bacteria and eukaryotes. Fungal FOXRED1 orthologs were likely acquired from alphaproteobacteria by horizontal gene transfer. The phylogenetic profile of FOXRED1 orthologs does not parallel the phylogenetic profile of complex I, strongly suggesting that, at least in some organisms, FOXRED1 has a function unrelated to complex I. The only large clade where all members investigated contain both FOXRED1 and complex I is the metazoans. Some bacterial FOXRED1 genes are present in metabolic operons related to amino acid metabolism. FOXRED1 phylogenetic distribution and gene organization suggest a metabolic role for FOXRED1 in complex I biogenesis should be considered.

FOXRED1 mutations result in complex I (NADH:ubiquinone oxidoreductase) deficiencies and Leigh syndrome (subacute necrotizing encephalomyelopathy). FOXRED1 is a mitochondrial flavoprotein related to N-methyl amino acid dehydrogenases. How is FOXRED1 required for the biogenesis of complex I? I present a hypothesis that suggests FOXRED1 catalytic activity as a sarcosine oxidase protects the developing fetus from oxidative stress during pregnancy. Loss of FOXRED1, coupled with protein, choline and/or folate-deficient diets results in the depletion of glutathione, the dysregulation of nitric oxide metabolism and the peroxynitrite-mediated inactivation of complex I.

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.

The application of metabolomics to human and animal model systems is poised to provide great insight into our understanding of disease etiology and the metabolic changes that are associated with these conditions. However, metabolomic studies have also revealed that there is significant, inherent biological variation in human samples and even in samples from animal model systems where the animals are housed under carefully controlled conditions. This inherent biological variability is an important consideration for all metabolomics analyses. In this study, we examined the biological variation in (1)H NMR-based metabolic profiling of two model systems, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Using relative standard deviations (RSD) as a measure of variability, our results reveal that both model systems have significant amounts of biological variation. The C. elegans metabolome possesses greater metabolic variance with average RSD values of 29 and 39%, depending on the food source that was used. The S. cerevisiae exometabolome RSD values ranged from 8% to 12% for the four strains examined. We also determined whether biological variation occurs between pairs of phenotypically identical yeast strains. Multivariate statistical analysis allowed us to discriminate between pair members based on their metabolic phenotypes. Our results highlight the variability of the metabolome that exists even for less complex model systems cultured under defined conditions. We also highlight the efficacy of metabolic profiling for defining these subtle metabolic alterations.

Aging-associated muscle insulin resistance has been hypothesized to be due to decreased mitochondrial function, secondary to cumulative free radical damage, leading to increased intramyocellular lipid content. To directly test this hypothesis, we examined both in vivo and in vitro mitochondrial function, intramyocellular lipid content, and insulin action in lean healthy mice with targeted overexpression of the human catalase gene to mitochondria (MCAT mice). Here, we show that MCAT mice are protected from age-induced decrease in muscle mitochondrial function (∼30%), energy metabolism (∼7%), and lipid-induced muscle insulin resistance. This protection from age-induced reduction in mitochondrial function was associated with reduced mitochondrial oxidative damage, preserved mitochondrial respiration and muscle ATP synthesis, and AMP-activated protein kinase-induced mitochondrial biogenesis. Taken together, these data suggest that the preserved mitochondrial function maintained by reducing mitochondrial oxidative damage may prevent age-associated whole-body energy imbalance and muscle insulin resistance.

New antifungal agents are needed to treat life-threatening fungal infections, particularly with the development of resistance. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a time-dependent drug exposure assay that allows us to rapidly investigate the mechanism of surface-active antifungal drug action. The assay uses a multidrug pump-deficient strain of Saccharomyces cerevisiae and the potentiometric dye 3,3'-dipropylthiacarbocyanine iodide [diS-C₃(3)] and can assess whether cells are depolarized, hyperpolarized, or permeabilized by drug exposure. In this work, we investigated the mechanisms of action of five surface-active compounds: SDS, nystatin, amphotericin B, octenidine dihydrochloride, and benzalkonium chloride. The diS-C₃(3) time-dependent drug exposure assay can be used to identify the mechanisms of action of a wide range of drugs. It is a fast and cost-effective method for screening drugs to determine their lowest effective concentrations.

Metabolomics is a powerful method of examining the intricate connections between mutations, metabolism, and disease. Metabolic footprinting examines the extracellular metabolome or exometabolome. We employed NMR-based metabolic footprinting and multivariate statistical analysis to examine a yeast model of mitochondrial dysfunction. Succinate dehydrogenase (SDH) is a component of both the tricarboxylic acid cycle and the mitochondrial respiratory chain. Mutations in the human SDH are linked to a variety of cancers or neurodegenerative disorders, highlighting the genotype/phenotype complexity associated with SDH dysfunction. To gain insight into the underlying global metabolic consequences of SDH dysfunction, we examined the metabolic footprints of SDH3 and SDH4 mutants. We identified and quantified 36 metabolites in the exometabolome. Our results indicate that SDH mutations cause significant alterations to several areas of yeast metabolism. Multivariate statistical analysis allowed us to discriminate between the different metabotypes of individual mutants, including mutants that were phenotypically indistinguishable. Metabotypes were highly correlated to mutant growth yields, suggesting that the characterization of metabotypes offers a rapid means of investigating the phenotype of a new mutation. Our study provides novel insight into the metabolic effects of SDH dysfunction and highlights the effectiveness of metabolic footprinting for examining complex disorders, such as mitochondrial diseases.

Prohibitins in eukaryotes consist of two subunits (PHB1 and PHB2) that together form a high molecular weight complex in the mitochondrial inner membrane. The evolutionary conservation and the ubiquitous expression in mammalian tissues of the prohibitin complex suggest an important function among eukaryotes. The PHB complex has been shown to play a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes in the yeast Saccharomyces cerevisiae. We have used Caenorhabditis elegans as model system to study the role of the PHB complex during development of a multicellular organism. We demonstrate that prohibitins in C. elegans form a high molecular weight complex in the mitochondrial inner membrane similar to that of yeast and humans. By using RNA-mediated gene inactivation, we show that PHB proteins are essential during embryonic development and are required for somatic and germline differentiation in the larval gonad. We further demonstrate that a deficiency in PHB proteins results in altered mitochondrial biogenesis in body wall muscle cells. This paper reports a strong loss of function phenotype for prohibitin gene inactivation in a multi-cellular organism and shows for the first time that prohibitins serve an essential role in mitochondrial function during organismal development.

The mitochondrial succinate dehydrogenase (SDH) is an iron–sulfur flavoenzyme linking the Krebs cycle and the mitochondrial respiratory chain. Mutations in the human SDHB, SDHC and SDHD genes are responsible for the development of paraganglioma and pheochromocytoma, tumors of the head and neck or the adrenal medulla, respectively. In recent years, SDH has become recognized as a source of reactive oxygen species, which may contribute to tumorigenesis. We have developed a Caenorhabditis elegans model to investigate the molecular and catalytic effects of mutations in the sdhb-1 gene, which encodes the SDH iron–sulfur subunit. We created mutations in Pro211; this residue is located near the site of ubiquinone reduction and is conserved in human SDHB (Pro197), where it is associated with tumorigenesis. Mutant phenotypes ranged from relatively benign to lethal and were characterized by hypersensitivity to oxidative stress, a shortened life span, impaired respiration and overproduction of superoxide. Our data suggest that the SDH ubiquinone-binding site can become a source of superoxide and that the pathological consequences of SDH mutations can be mitigated with antioxidants, such as ascorbate and N-acetyl-l-cysteine. Our work leads to a better understanding of the relationship between genotype and phenotype in respiratory chain mutations and of the mechanisms of aging and tumorigenesis.

Energy production via oxidative phosphorylation generates a mitochondrial membrane potential (ΔΨm) across the inner membrane. In this work, we show that a lower ΔΨm is associated with increased lifespan in Caenorhabditis elegans. The long-lived mutants daf-2(e1370), age-1(hx546), clk-1(qm30), isp-1(qm150) and eat-2(ad465) all have a lower ΔΨm than wild type animals. The lower ΔΨm of daf-2(e1370) is daf-16 dependent, indicating that the insulin-like signaling pathway not only regulates lifespan but also mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease ΔΨm. Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates ΔΨm. We conclude that longevity pathways converge on the mitochondria and lead to a decreased ΔΨm. Our results are consistent with the ‘uncoupling to survive’ hypothesis, which states that dissipation of the ΔΨm will extend lifespan.

Mitochondria are essential organelles with central roles in diverse cellular processes such as apoptosis, energy production via oxidative phosphorylation, ion homeostasis, and the synthesis of heme, lipid, amino acids, and iron-sulfur clusters. Defects in the mitochondrial respiratory chain lead to or are associated with a wide variety of diseases in humans. The nematode Caenorhabditis elegans provides a powerful genetic and developmental model system for reproducing deleterious mutations causing mitochondrial dysfunction and investigating their metabolic consequences and their mechanisms of pathology. In this chapter, we describe the growth of C. elegans in liquid culture, the isolation of crude and purified mitochondria, and polarographic and histochemical approaches for measuring mitochondrial respiratory chain function.

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron–sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (QP) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the QP site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.

The mitochondrial respiratory chain plays a crucial role in cellular and organismal health. In addition to being the major source of energy for most cells, mitochondrial respiratory chain function regulates or modulates redox and metabolite homeostasis, apoptosis and the generation of reactive oxygen species. In order to measure the relative in vivo mitochondrial membrane potential of different strains of the nematode, Caenorhabditis elegans, we have developed a fluorescence assay using the cationic, lipophilic carbocyanine dye, diS-C(3)(3). We demonstrate that two complex I-deficient mutants have significantly lower mitochondrial membrane potentials in vivo than wild type animals. Our fluorescence assay will enable us to better dissect and understand the complex phenotypic consequences of mitochondrial dysfunction.

The mitochondrial succinate dehydrogenase (SDH) is an essential component of the electron transport chain and of the tricarboxylic acid cycle. Also known as complex II, this tetrameric enzyme catalyzes the oxidation of succinate to fumarate and reduces ubiquinone. Mutations in the human SDHB, SDHC, and SDHD genes are tumorigenic, leading to the development of several types of tumors, including paraganglioma and pheochromocytoma. The mechanisms linking SDH mutations to oncogenesis are still unclear. In this work, we used the yeast SDH to investigate the molecular and catalytic effects of tumorigenic or related mutations. We mutated Arg(47) of the Sdh3p subunit to Cys, Glu, and Lys and Asp(88) of the Sdh4p subunit to Asn, Glu, and Lys. Both Arg(47) and Asp(88) are conserved residues, and Arg(47) is a known site of cancer causing mutations in humans. All of the mutants examined have reduced ubiquinone reductase activities. The SDH3 R47K, SDH4 D88E, and SDH4 D88N mutants are sensitive to hyperoxia and paraquat and have elevated rates of superoxide production in vitro and in vivo. We also observed the accumulation and secretion of succinate. Succinate can inhibit prolyl hydroxylase enzymes, which initiate a proliferative response through the activation of hypoxia-inducible factor 1alpha. We suggest that SDH mutations can promote tumor formation by contributing to both reactive oxygen species production and to a proliferative response normally induced by hypoxia via the accumulation of succinate.

The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron-sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S. cerevisiae strain expressing a mutant enzyme lacking the two heme axial ligands, Sdh3p His-106 and Sdh4p Cys-78. The mutant enzyme was characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction and for its heme b content. Replacement of both Sdh3p His-106 and Sdh4p Cys-78 with alanine residues leads to an undetectable level of cytochrome b(562). Although enzyme assembly is slightly impaired, the apocytochrome SDH retains a significant ability to reduce quinone. The enzyme has a reduced affinity for quinone and its catalytic efficiency is reduced by an order of magnitude. To better understand the effects of the mutations, we employed atomistic molecular dynamic simulations to investigate the enzyme's structure and stability in the absence of heme. Our results strongly suggest that heme is not required for electron transport from succinate to quinone nor is it necessary for assembly of the S. cerevisiae SDH.

The mitochondrial genome is vital for Caenorhabditis elegans metabolism, physiology, and development. The C. elegans mitochondrial DNA is typical of animal mitochondrial genomes in its size and gene content. It is 13,794 nucleotides in length and encodes 36 genes: 2 ribosomal RNAs, 22 transfer RNAs, and 12 protein subunits of the mitochondrial respiratory chain. Although it represents only a small number of genes, an elaborate cellular machinery comprised of over 200 nuclear genes is needed to replicate, transcribe, and maintain the mitochondrial chromosome and to assemble the translation machinery needed to express this dozen proteins. Mitochondrial genetics is peculiar and complex because mitochondrial DNA is maternally inherited and can be present at tens to tens of thousands of copies per cell. The mitochondrial genome content of the developing nematode is developmentally regulated; it increases about 30-fold between the L1 and the adult stages and blocking the increase leads to larval arrest. Energy metabolism is also intimately linked to aging and lifespan determination. The nematode model system offers numerous advantages for understanding the full importance and scope of the mitochondrial genome in animal life.