Respiratory Deficient Phenotype Research Articles

A yeast suppressor screen links Coa4 to the mitochondrial copper delivery pathway for cytochrome c oxidase.

Cytochrome c oxidase (CcO) is a multimeric copper-containing enzyme of the mitochondrial respiratory chain that powers cellular energy production. The two core subunits of cytochrome c oxidase, Cox1 and Cox2, harbor the catalytic CuB and CuA sites, respectively. Biogenesis of each copper site occurs separately and requires multiple proteins that constitute the mitochondrial copper delivery pathway. Currently, the identity of all the members of the pathway is not known, though several evolutionarily conserved twin CX9C motif-containing proteins have been implicated in this process. Here, we performed a targeted yeast suppressor screen that placed Coa4, a twin CX9C motif-containing protein, in the copper delivery pathway to the Cox1 subunit. Specifically, we show that overexpression of Cox11, a copper metallochaperone required for the formation of CuB site, can restore Cox1 abundance, cytochrome c oxidase assembly, and mitochondrial respiration in coa4Δ cells. This rescue is dependent on the copper-coordinating cysteines of Cox11. The abundance of Coa4 and Cox11 in mitochondria is reciprocally regulated, further linking Coa4 to the CuB site biogenesis. Additionally, we find that coa4Δ cells have reduced levels of copper and exogenous copper supplementation can partially ameliorate its respiratory-deficient phenotype, a finding that connects Coa4 to cellular copper homeostasis. Finally, we demonstrate that human COA4 can replace the function of yeast Coa4 indicating its evolutionarily conserved role. Our work provides genetic evidences for the role of Coa4 in the copper delivery pathway to the CuB site of cytochrome c oxidase.

Electron Transfer Coupled to Conformational Dynamics in Cell Respiration.

Cellular respiration is a fundamental process required for energy production in many organisms. The terminal electron transfer complex in mitochondrial and many bacterial respiratory chains is cytochrome c oxidase (CcO). This converts the energy released in the cytochrome c/oxygen redox reaction into a transmembrane proton electrochemical gradient that is used subsequently to power ATP synthesis. Despite detailed knowledge of electron and proton transfer paths, a central question remains as to whether the coupling between electron and proton transfer in mammalian mitochondrial forms of CcO is mechanistically equivalent to its bacterial counterparts. Here, we focus on the conserved span between H376 and G384 of transmembrane helix (TMH) X of subunit I. This conformationally-dynamic section has been suggested to link the redox activity with the putative H pathway of proton transfer in mammalian CcO. The two helix X mutants, Val380Met (V380M) and Gly384Asp (G384D), generated in the genetically-tractable yeast CcO, resulted in a respiratory-deficient phenotype caused by the inhibition of intra-protein electron transfer and CcO turnover. Molecular aspects of these variants were studied by long timescale atomistic molecular dynamics simulations performed on wild-type and mutant bovine and yeast CcOs. We identified redox- and mutation-state dependent conformational changes in this span of TMH X of bovine and yeast CcOs which strongly suggests that this dynamic module plays a key role in optimizing intra-protein electron transfers.

Intragenic suppressor mutations of the COQ8 protein kinase homolog restore coenzyme Q biosynthesis and function in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family.

5′ processing of Saccharomyces cerevisiae mitochondrial tRNAs requires expression of multiple genes

Mitochondrial tRNAs are processed at their 5’ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2.The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.

Cardiolipin is a key determinant for mtDNA stability and segregation during mitochondrial stress

Mitochondria play a key role in adaptation during stressing situations. Cardiolipin, the main anionic phospholipid in mitochondrial membranes, is expected to be a determinant in this adaptive mechanism since it modulates the activity of most membrane proteins. Here, we used Saccharomyces cerevisiae subjected to conditions that affect mitochondrial metabolism as a model to determine the possible role of cardiolipin in stress adaptation. Interestingly, we found that thermal stress promotes a 30% increase in the cardiolipin content and modifies the physical state of mitochondrial membranes. These changes have effects on mtDNA stability, adapting cells to thermal stress. Conversely, this effect is cardiolipin-dependent since a cardiolipin synthase-null mutant strain is unable to adapt to thermal stress as observed by a 60% increase of cells lacking mtDNA (ρ0). Interestingly, we found that the loss of cardiolipin specifically affects the segregation of mtDNA to daughter cells, leading to a respiratory deficient phenotype after replication. We also provide evidence that mtDNA physically interacts with cardiolipin both in S. cerevisiae and in mammalian mitochondria. Overall, our results demonstrate that the mitochondrial lipid cardiolipin is a key determinant in the maintenance of mtDNA stability and segregation.

Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient

The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (gethanolg(-1)biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82gg(-1)h(-1)). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80gL(-1)) and the RHO2-3RD strain was able to tolerate up to 1.5gL(-1) 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22%, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.

The Putative GTPase Encoded by MTG3 Functions in a Novel Pathway for Regulating Assembly of the Small Subunit of Yeast Mitochondrial Ribosomes

Very little is known about biogenesis of mitochondrial ribosomes. The GTPases encoded by the nuclear MTG1 and MTG2 genes of Saccharomyces cerevisiae have been reported to play a role in assembly of the ribosomal 54 S subunit. In the present study biochemical screens of a collection of respiratory deficient yeast mutants have enabled us to identify a third gene essential for expression of mitochondrial ribosomes. This gene codes for a member of the YqeH family of GTPases, which we have named MTG3 in keeping with the earlier convention. Mutations in MTG3 cause the accumulation of the 15 S rRNA precursor, previously shown to have an 80-nucleotide 5' extension. Sucrose gradient sedimentation of mitochondrial ribosomes from temperature-sensitive mtg3 mutants grown at the permissive and restrictive temperatures, combined with immunobloting with subunit-specific antibodies, indicate that Mtg3p is required for assembly of the 30 S but not 54 S ribosomal subunit. The respiratory deficient growth phenotype of an mtg3 null mutant is partially rescued by overexpression of the Mrpl4p constituent located at the peptide exit site of the 54 S subunit. The rescue is accompanied by an increase in processed 15 S rRNA. This suggests that Mtg3p and Mrpl4p jointly regulate assembly of the small subunit by modulating processing of the 15 S rRNA precursor.

Evaluation of SCO1 deletion on Saccharomyces cerevisiae metabolism through a proteomic approach

The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.

Mitochondrial Dysfunction As a Potential Source of Reactive Oxygen Species in Cellular Models of Shwachman-Diamond Syndrome

Abstract 1343Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome linked to defects in ribosome synthesis. The heterogeneous array of clinical findings associated with this disease state most commonly includes exocrine pancreas insufficiency, neutropenia, and metaphyseal chondroplasia. Patients also show a predisposition for progression to myelodysplastic syndromes and acute myelogenous leukemia. Mutations in the gene SBDS are known to be responsible for most cases of SDS. Initial studies of the yeast ortholog of SBDS, Sdo1, revealed that this family of proteins is involved in the maturation of 60S subunits. Other studies have suggested that SBDS is a multifunctional protein affecting processes other than ribosome synthesis. Most recently it has been shown that reactive oxygen species are dysregulated in TF-1 erythroleukemic cells depleted of SBDS leading to increased cell death (Pediatr Blood Cancer. 2010 Dec 1;55(6): 1138–44). In an effort to elucidate potential sources of increased reactive oxygen species we investigated mitochondrial function in yeast and human models of SDS. Yeast cells lacking Sdo1 fail to grow on media containing only respiratory carbon sources, indicative of a defect in mitochondrial energy metabolism. Related studies in human TF-1 cells revealed that cells depleted of SBDS exhibit reduced oxygen consumption relative to controls. Given that the largest producer of reactive oxygen species is the mitochondrial electron transport chain, perturbation of respiratory function in cells depleted of SBDS family members could be a potential source of elevated reactive oxygen species. To investigate the potential molecular mechanisms underlying these respiratory deficient phenotypes we carried out a proteomic analysis comparing yeast cells depleted of Sdo1 with controls. Our data reveal that cells lacking Sdo1 overexpress Por1, an ortholog of human VDAC1. VDAC1 is a voltage dependent anion channel of the mitochondrial outer membrane that is thought to be an essential component of the mitochondrial permeability pore. Both over and under expression of VDAC1 have been shown to disrupt mitochondrial function and lead to enhanced apoptosis. Current efforts are focused on possible changes in VDAC1 expression and the role they play in the respiratory deficient phenotype in human SDS models. These studies continue to shed further insight into the molecular mechanisms underlying SDS pathophysiology. Disclosures:No relevant conflicts of interest to declare.

Mutations in the Dimer Interface of Dihydrolipoamide Dehydrogenase Promote Site-specific Oxidative Damages in Yeast and Human Cells

Dihydrolipoamide dehydrogenase (DLD) is a multifunctional protein well characterized as the E3 component of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes. Previously, conditions predicted to destabilize the DLD dimer revealed that DLD could also function as a diaphorase and serine protease. However, the relevance of these cryptic activities remained undefined. We analyzed human DLD mutations linked to strikingly different clinical phenotypes, including E340K, D444V, R447G, and R460G in the dimer interface domain that are responsible for severe multisystem disorders of infancy and G194C in the NAD(+)-binding domain that is typically associated with milder presentations. In vitro, all of these mutations decreased to various degrees dihydrolipoamide dehydrogenase activity, whereas dimer interface mutations also enhanced proteolytic and/or diaphorase activity. Human DLD proteins carrying each individual mutation complemented fully the respiratory-deficient phenotype of yeast cells lacking endogenous DLD even when residual dihydrolipoamide dehydrogenase activity was as low as 21% of controls. However, under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. As in yeast, lipoic acid cofactor was damaged in human D444V-homozygous fibroblasts after exposure to oxidative stress. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and thus contribute to the clinical severity of DLD mutations.

Improvement in ethanol production using respiratory deficient phenotype of a wild type yeast Saccharomyces cerevisiae ITV-01

An osmotolerant low pH resistant wild type Saccharomyces cerevisiae ITV-01 yeast was employed to obtain respiratory deficient (RD) strains in order to improve ethanol production. The respiratory deficient phenotype was induced by exposure to chemical agents such as ethidium bromide, rhodamine, and ethanol. Isolation with physical agents such as temperature and UV radiation (254 nm) exposures was evaluated. 104 strains were tested. Selection criterion was based on increasing ethanol yield. All RD strains had higher ethanol yields (from 0.41 to 0.50 gg −1, corresponding 82–98% theoretical yields) than the wild type yeast (0.40 gg −1). Temperature and ethanol mutant agents caused a reversible phenotype in every case. Screening showed that S. cerevisiae ITV-01 RD-B14 (lacking cytochrome c), obtained using bromide ethidium, had the highest alcoholic fermentation efficiency (95%) and 97% viability under the tested conditions (150 gL −1 glucose, pH 3.5). The respiratory deficient mutation is of interest as a tool for improving ethanol production.

Identifying Novel Factors Involved In Yeast Mitochondrial Protein Synthesis

The goal of our work is to identify protein factors involved in the initiation of mitochondrial translation using yeast genetics. FMT1 encodes the S. cerevisiae methionyl‐tRNA formyltransferase, which catalyzes the formylation of mitochondrial initiator Met‐tRNA. A genetic screen was performed with an fmt1 deletion strain to generate synthetic petite strains – strains containing mutations that in combination with the fmt1 deletion lead to the loss of respiratory growth. One mutant strain, s.p. 72–1, was transformed with a yeast genomic DNA library to identify open reading frames that complement the respiratory‐deficient phenotype. A region of chromosome XVI, including the LPE10 gene, was found to rescue growth on non‐fermentable carbon sources. Sequencing of DNA from the mutant strain revealed that the LPE10 gene, encoding a mitochondrial inner membrane magnesium transporter, contained a point mutation leading to a premature stop codon. This mutation likely causes the synthetic petite phenotype with the deletion of fmt1. Indeed, an lpe10 fmt1 double mutant has a more severe respiratory growth defect than the single lpe10 deletion strain. Deletion of the MIS1 gene, also required for the formylation of mitochondrial initiator tRNA, also leads to the petite phenotype in combination with the lpe10 deletion. This work is supported by HHMI and NSF grants to the Freshman Research Initiative at UT‐Austin.

G-Domain Dimerization Orchestrates the tRNA Wobble Modification Reaction in the MnmE/GidA Complex

MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation between MnmE and GidA involves large conformational changes that induce G-domain dimerization of MmnE and that GidA co-stimulates GTP hydrolysis on MnmE. Starting from a structural model of the complex, we identify interface mutations disrupting complex formation or communication. Although GidA does not directly contact the G-domains, conformational changes in MnmE, induced by G-domain dimerization in the triphosphate state, regulate the affinity for GidA. We developed a tRNA modification assay and demonstrate for the first time in vitro that the MnmE/GidA complex catalyzes incorporation of glycine into tRNA. An intact MnmE/GidA complex rather than their sequential action is crucial for in vitro modification. Since only GTP, but not GDP or non-hydrolyzable GTP analogs, drives the MnmE/GidA-catalyzed modification reaction, we conclude that GTP hydrolysis is essential for activity. We finally show that an active GTPase, an intact MnmE/GidA communication, and dimerization of G-domains are necessary for in vivo functioning since mutations disrupting either result in a respiratory deficient phenotype in yeast.

Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae

The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm 5s 2U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (P S) or 15S rRNA C1409G allele (P R) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNA Lys, tRNA Glu, tRNA Gln, tRNA Leu, tRNA Gly, tRNA Arg and tRNA Phe were decreased significantly while those of tRNA Met and tRNA His were not affected in the mto1 strains carrying the P S allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNA Lys, tRNA Met and tRNA Phe. Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNA Lys, tRNA Leu, tRNA Arg but almost no effect in those of tRNA His. In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype.

Role of Lon1 protease in post‐germinative growth and maintenance of mitochondrial function in Arabidopsis thaliana

Maintenance of protein quality control and turnover is essential for cellular homeostasis. In plant organelles this biological process is predominantly performed by ATP-dependent proteases. Here, a genetic screen was performed that led to the identification of Arabidopsis thaliana Lon1 protease mutants that exhibit a post-embryonic growth retardation phenotype. Translational fusion to yellow fluorescent protein revealed AtLon1 subcellular localization in plant mitochondria, and the AtLon1 gene could complement the respiratory-deficient phenotype of the yeast PIM1 gene homolog. AtLon1 is highly expressed in rapidly growing plant organs of embryonic origin, including cotyledons and primary roots, and in inflorescences, which have increased mitochondria numbers per cell to fulfill their high energy requirements. In lon1 mutants, the expression of both mitochondrial and nuclear genes encoding respiratory proteins was normal. However, mitochondria isolated from lon1 mutants had a lower capacity for respiration of succinate and cytochrome c via complexes II and IV, respectively. Furthermore, the activity of key enzymes of the tricarboxylic acid (TCA) cycle was significantly reduced. Additionally, mitochondria in lon1 mutants had an aberrant morphology. These results shed light on the developmental mechanisms of selective proteolysis in plant mitochondria and suggest a critical role for AtLon1 protease in organelle biogenesis and seedling establishment.

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc 1 complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036–36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome ( ARG8 m ). Subsequently replacing ARG8 m with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit −). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.

The 3-hydroxyacyl-ACP dehydratase of mitochondrial fatty acid synthesis in Trypanosoma brucei

The trypanosomatid parasite Trypanosoma brucei synthesizes fatty acids in the mitochondrion using the type II fatty acid synthesis (FAS) machinery. When mitochondrial FAS was characterized in T. brucei, all of the enzymatic components were identified based on their homology to yeast mitochondrial FAS enzymes, except for 3-hydroxyacyl-ACP dehydratase. Here we describe the characterization of T. brucei mitochondrial 3-hydroxyacyl-ACP dehydratase (TbHTD2), which was identified by its similarity to the human mitochondrial dehydratase. TbHTD2 can rescue the respiratory deficient phenotype of the yeast knock-out strain and restore the lipoic acid content, is localized in the mitochondrion and exhibits hydratase 2 activity.

An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing

In bacteria, functionally related gene products are often encoded by a common transcript. Such polycistronic transcripts are rare in eukaryotes. Here we isolated several clones from human cDNA libraries, which rescued the respiratory-deficient phenotype of a yeast mitochondrial 3-hydroxyacyl thioester dehydratase 2 (htd2) mutant strain. All complementing cDNAs were derived from the RPP14 transcript previously described to encode the RPP14 subunit of the human ribonuclease P (RNase P) complex. We identified a second, 3' open reading frame (ORF) on the RPP14 transcript encoding a protein showing similarity to known dehydratases and hydratase 2 enzymes. The protein was localized in mitochondria, and the recombinant enzyme exhibited (3R)-specific hydratase 2 activity. Based on our results, we named the protein human 3-hydroxyacyl-thioester dehydratase 2 (HsHTD2), which is involved in mitochondrial fatty acid synthesis. The bicistronic arrangement of RPP14 and HsHTD2, as well as the general exon structure of the gene, is conserved in vertebrates from fish to humans, indicating a genetic link conserved for 400 million years between RNA processing and mitochondrial fatty acid synthesis.

Heterologous complementation of theKlaacnull mutation ofKluyveromyces lactisby theSaccharomyces cerevisiae AAC3gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.

Mutations in MTO2 Related to tRNA Modification Impair Mitochondrial Gene Expression and Protein Synthesis in the Presence of a Paromomycin Resistance Mutation in Mitochondrial 15 S rRNA

Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA.