RESUMEN
Mitochondrial fatty acids synthesis (mtFAS) is a conserved metabolic pathway essential for mitochondrial respiration. The best characterized mtFAS product is the medium-chain fatty acid octanoate (C8) used as a substrate in the synthesis of lipoic acid (LA), a cofactor required by several mitochondrial enzyme complexes. In humans, mutations in the mtFAS component enoyl reductase MECR cause childhood-onset neurodegenerative disorder MEPAN. A complete deletion of Mecr in mice is embryonically lethal, while selective deletion of Mecr in cerebellar Purkinje cells causes neurodegeneration in these cells. A fundamental question in the research of mtFAS deficiency is if the defect is amenable to treatment by supplementation with known mtFAS products. Here we used the Purkinje-cell specific mtFAS deficiency neurodegeneration model mice to study if feeding the mice with a medium-chain triacylglycerol-rich formula supplemented with LA could slow down or prevent the neurodegeneration in Purkinje cell-specific Mecr KO mice. Feeding started at the age of 4 weeks and continued until the age of 9 months. The neurological status on the mice was assessed at the age of 3, 6, and 9 months with behavioral tests and the state of the Purkinje cell deterioration in the cerebellum was studied histologically. We showed that feeding the mice with medium chain triacylglycerols and LA affected fatty acid profiles in the cerebellum and plasma but did not prevent the development of neurodegeneration in these mice. Our results indicate that dietary supplementation with medium chain fatty acids and LA alone is not an efficient way to treat mtFAS disorders.
RESUMEN
Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory function. MtFAS generates the octanoic acid precursor for lipoic acid synthesis, but the role of longer fatty acid products has remained unclear. The structurally well-characterized component of mtFAS, human 2E-enoyl-ACP reductase (MECR) rescues respiratory growth and lipoylation defects of a Saccharomyces cerevisiae Δetr1 strain lacking native mtFAS enoyl reductase. To address the role of longer products of mtFAS, we employed in silico molecular simulations to design a MECR variant with a shortened substrate binding cavity. Our in vitro and in vivo analyses indicate that the MECR G165Q variant allows synthesis of octanoyl groups but not long chain fatty acids, confirming the validity of our computational approach to engineer substrate length specificity. Furthermore, our data imply that restoring lipoylation in mtFAS deficient yeast strains is not sufficient to support respiration and that long chain acyl-ACPs generated by mtFAS are required for mitochondrial function.
Asunto(s)
Mitocondrias , Oxidorreductasas , Humanos , Ácidos Grasos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Oxidorreductasas/metabolismo , Respiración , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Enoil-ACP Reductasa (NADH)RESUMEN
Om45 is a major protein of the yeast's outer mitochondrial membrane under respiratory conditions. However, the cellular role of the protein has remained obscure. Previously, deletion mutant phenotypes have not been found, and clear amino acid sequence similarities that would allow inferring its functional role are not available. In this work, we describe synthetic petite mutants of GEM1 and UGO1 that depend on the presence of OM45 for respiratory growth, as well as the identification of several multicopy suppressors of the synthetic petite phenotypes. In the analysis of our mutants, we demonstrate that Om45p and Gem1p have a collaborative role in the maintenance of mitochondrial morphology, cristae structure, and mitochondrial DNA maintenance. A group of multicopy suppressors rescuing the synthetic lethal phenotypes of the mutants on non-fermentable carbon sources additionally supports this result. Our results imply that the synthetic petite phenotypes we observed are due to the disturbance of the inner mitochondrial membrane and point to this mitochondrial sub-compartment as the main target of action of Om45p, Ugo1p, and the yeast Miro GTPase Gem1p.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ADN de Hongos , ADN Mitocondrial/metabolismo , GTP Fosfohidrolasas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Mutación , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Saccharomyces cerevisiae Rsm22 protein (Sc-Rsm22), encoded by the nuclear RSM22 (systematic name YKL155c) gene, is a distant homologue of Rsm22 from Trypanosoma brucei (Tb-Rsm22) and METTL17 from mouse (Mm-METTL17). All three proteins have been shown to be associated with mitochondrial gene expression, and Sc-Rsm22 has been documented to be essential for mitochondrial respiration. The Sc-Rsm22 protein comprises a polypeptide of molecular weight 72.2â kDa that is predicted to harbor an N-terminal mitochondrial targeting sequence. The precise physiological function of Rsm22-family proteins is unknown, and no structural information has been available for Sc-Rsm22 to date. In this study, Sc-Rsm22 was expressed and purified in monomeric and dimeric forms, their folding was confirmed by circular-dichroism analyses and their low-resolution structures were determined using a small-angle X-ray scattering (SAXS) approach. The solution structure of the monomeric form of Sc-Rsm22 revealed an elongated three-domain arrangement, which differs from the shape of Tb-Rsm22 in its complex with the mitochondrial small ribosomal subunit in T. brucei (PDB entry 6sg9). A bioinformatic analysis revealed that the core domain in the middle (Leu117-Asp462 in Sc-Rsm22) resembles the corresponding region in Tb-Rsm22, including a Rossmann-like methyltransferase fold followed by a zinc-finger-like structure. The latter structure is not present in this position in other methyltransferases and is therefore a unique structural motif for this family. The first half of the C-terminal domain is likely to form an OB-fold, which is typically found in RNA-binding proteins and is also seen in the Tb-Rsm22 structure. In contrast, the N-terminal domain of Sc-Rsm22 is predicted to be fully α-helical and shares no sequence similarity with other family members. Functional studies demonstrated that the monomeric variant of Sc-Rsm22 methylates mitochondrial tRNAs in vitro. These data suggest that Sc-Rsm22 is a new and unique member of the RNA methyltransferases that is important for mitochondrial protein synthesis.
Asunto(s)
Modelos Moleculares , Proteínas Ribosómicas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Elementos Estructurales de las ProteínasRESUMEN
BACKGROUND: Lipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate. RESULTS: We show that the requirement for mitochondrial fatty acid synthesis to provide substrates for lipoylation of the 2-ketoacid dehydrogenases can be bypassed by supplying the cells with free lipoic acid (LA) or octanoic acid (C8) and a mitochondrially targeted fatty acyl/lipoyl activating enzyme. We also provide evidence that the S. cerevisiae lipoyl transferase Lip3, in addition to transferring LA from the glycine cleavage system H protein to the pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGD) E2 subunits, can transfer this cofactor from the PDH complex to the KGD complex. In support of yeast as a model system for human metabolism, we demonstrate that the human octanoyl/lipoyl transferases can substitute for their counterparts in yeast to support respiratory growth and protein lipoylation. Like the wild-type yeast enzyme, the human lipoyl transferase LIPT1 responds to LA supplementation in the presence of the activating enzyme LplA. CONCLUSIONS: In the yeast model system, the eukaryotic lipoylation pathway can use free LA and C8 as substrates when fatty/lipoic acid activating enzymes are targeted to mitochondria. Lip3 LA transferase has a wider substrate specificity than previously recognized. We show that these features of the lipoylation mechanism in yeast are conserved in mammalian mitochondria. Our findings have important implications for the development of effective therapies for the treatment of LA or mtFAS deficiency-related disorders.
Asunto(s)
Lipoilación , Mitocondrias/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Fatty acids in mitochondria, in sensu stricto, arise either as ß-oxidation substrates imported via the carnitine shuttle or through de novo synthesis by the mitochondrial fatty acid synthesis (mtFAS) pathway. Defects in mtFAS or processes involved in the generation of the mtFAS product derivative lipoic acid (LA), including iron-sulfur cluster synthesis required for functional LA synthase, have emerged only recently as etiology for neurodegenerative disease. Intriguingly, mtFAS deficiencies very specifically affect CNS function, while LA synthesis and attachment defects have a pleiotropic presentation beyond neurodegeneration. Typical mtFAS defect presentations include optical atrophy, as well as basal ganglia defects associated with dystonia. The phenotype display of patients with mtFAS defects can resemble the presentation of disorders associated with coenzyme A (CoA) synthesis. A recent publication links these processes together based on the requirement of CoA for acyl carrier protein maturation. MtFAS defects, CoA synthesis- as well as Fe-S cluster-deficiencies share lack of LA as a common symptom.
Asunto(s)
Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Transducción de Señal/fisiología , Animales , Coenzima A/biosíntesis , Coenzima A/genética , Ácidos Grasos/genética , Humanos , Mitocondrias/genética , Enfermedades Neurodegenerativas/genéticaRESUMEN
Acyl carrier protein (ACP) is a principal partner in the cytosolic and mitochondrial fatty acid synthesis (FAS) pathways. The active form holo-ACP serves as FAS platform, using its 4'-phosphopantetheine group to present covalently attached FAS intermediates to the enzymes responsible for the acyl chain elongation process. Mitochondrial unacylated holo-ACP is a component of mammalian mitoribosomes, and acylated ACP species participate as interaction partners in several ACP-LYRM (leucine-tyrosine-arginine motif)-protein heterodimers that act either as assembly factors or subunits of the electron transport chain and Fe-S cluster assembly complexes. Moreover, octanoyl-ACP provides the C8 backbone for endogenous lipoic acid synthesis. Accumulating evidence suggests that mtFAS-generated acyl-ACPs act as signaling molecules in an intramitochondrial metabolic state sensing circuit, coordinating mitochondrial acetyl-CoA levels with mitochondrial respiration, Fe-S cluster biogenesis and protein lipoylation.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Mitocondrias/metabolismo , Acetilcoenzima A/metabolismo , Proteína Transportadora de Acilo/genética , Secuencia de Aminoácidos , Animales , Humanos , Alineación de SecuenciaRESUMEN
Utilization of non-AUG alternative translation start sites is most common in bacteria and viruses, but it has been also reported in other organisms. This phenomenon increases proteome complexity by allowing expression of multiple protein isoforms from a single gene. In Saccharomyces cerevisiae, a few described cases concern proteins that are translated from upstream near-cognate start codons as N-terminally extended variants that localize to mitochondria. Using bioinformatics tools, we provide compelling evidence that in yeast the potential for producing alternative protein isoforms by non-AUG translation initiation is much more prevalent than previously anticipated and may apply to as many as a few thousand proteins. Several hundreds of candidates are predicted to gain a mitochondrial targeting signal (MTS), generating an unrecognized pool of mitochondrial proteins. We confirmed mitochondrial localization of a subset of proteins previously not identified as mitochondrial, whose standard forms do not carry an MTS. Our data highlight the potential of non-canonical translation initiation in expanding the capacity of the mitochondrial proteome and possibly also other cellular features.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Codón Iniciador/metabolismo , Biología Computacional , Prueba de Complementación Genética , Humanos , Mitocondrias/genética , Iniciación de la Cadena Peptídica Traduccional , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
17ß-Hydroxysteroid dehydrogenases (HSD17B) catalyze the oxidation/reduction of 17ß-hydroxy/keto group in position C17 in C18- and C19 steroids. Most HSD17Bs are also catalytically active with substrates other than steroids. A subset of these enzymes is able to process thioesters of carboxylic acids. This group of enzymes includes HSD17B4, HSD17B8, HSD17B10 and HSD17B12, which execute reactions in intermediary metabolism, participating in peroxisomal ß-oxidation of fatty acids, mitochondrial oxidation of 3R-hydroxyacyl-groups, breakdown of isoleucine and fatty acid chain elongation in endoplasmic reticulum. Divergent substrate acceptance capabilities exemplify acquirement of catalytic site adaptiveness during evolution. As an additional common feature these HSD17Bs are multifunctional enzymes that arose either via gene fusions (HSD17B4) or are incorporated as subunits into multifunctional protein complexes (HSD17B8 and HSD17B10). Crystal structures of HSD17B4, HSD17B8 and HSD17B10 give insight into their structure-function relationships. Thus far, deficiencies of HSD17B4 and HSD17B10 have been assigned to inborn errors in humans, underlining their significance as enzymes of metabolism.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Ésteres/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/química , Animales , Enfermedad , Ácidos Grasos Insaturados/metabolismo , Humanos , Mitocondrias/metabolismo , ARN/metabolismoRESUMEN
There has been a growing interest toward mitochondrial fatty acid synthesis (mtFAS) since the recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration), which is caused by mutations in the mitochondrial enoyl-CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS function in neural tissue, here, we generated a mouse line with a PC-specific knock-out (KO) of Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation, and reduced respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties â¼7 months of age and ataxic symptoms were evident from 8-9 months of age on. Our data show that impairment of mtFAS results in functional and ultrastructural changes in mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia with a distinct and well trackable cellular phenotype. This mouse model will allow the future investigation of the feasibility of metabolite supplementation approaches toward the prevention of neurodegeneration due to dysfunctional mtFAS.SIGNIFICANCE STATEMENT We have recently reported a novel neurodegenerative disorder in humans termed MEPAN (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al., 2016). The cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding mitochondrial 2-enoyl-CoA/ACP reductase. The report presented here describes the analysis of the first mouse model suffering from mtFAS-defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in Purkinje cells. Our work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and provides a test bed for future treatment approaches.
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Cerebelo/metabolismo , Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Degeneración Nerviosa/metabolismo , Animales , Animales Recién Nacidos , Cerebelo/patología , Ácidos Grasos/genética , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genéticaRESUMEN
Mitochondrial fatty acid synthesis (mtFAS) is a highly conserved pathway essential for mitochondrial biogenesis. The mtFAS process is required for mitochondrial respiratory chain assembly and function, synthesis of the lipoic acid cofactor indispensable for the function of several mitochondrial enzyme complexes and essential for embryonic development in mice. Mutations in human mtFAS have been reported to lead to neurodegenerative disease. The source of malonyl-CoA for mtFAS in mammals has remained unclear. We report the identification of a conserved vertebrate mitochondrial isoform of ACC1 expressed from an ACACA transcript splicing variant. A specific knockdown (KD) of the corresponding transcript in mouse cells, or CRISPR/Cas9-mediated inactivation of the putative mitochondrial targeting sequence in human cells, leads to decreased lipoylation and mitochondrial fragmentation. Simultaneous KD of ACSF3, encoding a mitochondrial malonyl-CoA synthetase previously implicated in the mtFAS process, resulted in almost complete ablation of protein lipoylation, indicating that these enzymes have a redundant function in mtFAS. The discovery of a mitochondrial isoform of ACC1 required for lipoic acid synthesis has intriguing consequences for our understanding of mitochondrial disorders, metabolic regulation of mitochondrial biogenesis and cancer.
Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Coenzima A Ligasas/metabolismo , Malonil Coenzima A/metabolismo , Mitocondrias/patología , Acetil-CoA Carboxilasa/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Coenzima A Ligasas/genética , Secuencia Conservada , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas , Malonil Coenzima A/genética , Ratones , Mitocondrias/enzimología , ARN Interferente Pequeño , Ácido TiócticoRESUMEN
Mitochondrial fatty acid synthesis (mtFAS) is an underappreciated but highly conserved metabolic process, indispensable for mitochondrial respiration. It was recently reported that dysfunction of mtFAS causes childhood onset of dystonia and optic atrophy in humans (MEPAN). To study the role of mtFAS in mammals, we generated three different mouse lines with modifications of the Mecr gene, encoding mitochondrial enoyl-CoA/ACP reductase (Mecr). A knock-out-first type mutation, relying on insertion of a strong transcriptional terminator between the first two exons of Mecr, displayed embryonic lethality over a broad window of time and due to a variety of causes. Complete removal of exon 2 or replacing endogenous Mecr by its functional prokaryotic analogue fabI (Mecrtm(fabI)) led to more consistent lethality phenotypes and revealed a hypoplastic placenta. Analyses of several mitochondrial parameters indicate that mitochondrial capacity for aerobic metabolism is reduced upon disrupting mtFAS function. Further analysis of the synthetic Mecrtm(fabI) models disclosed defects in development of placental trophoblasts consistent with disturbed peroxisome proliferator-activated receptor signalling. We conclude that disrupted mtFAS leads to deficiency in mitochondrial respiration, which lies at the root of the observed pantropic effects on embryonic and placental development in these mouse models.
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Enoil-ACP Reductasa (NADH)/genética , Enoil-ACP Reductasa (NADH)/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Animales , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos/metabolismo , Femenino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Oxidorreductasas/metabolismo , Placenta , Placentación/genética , Placentación/fisiología , EmbarazoRESUMEN
Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the "classic" cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both ß-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.
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Ácidos Grasos/metabolismo , Mitocondrias/metabolismo , Mitocondrias/fisiología , Proteína Transportadora de Acilo/metabolismo , Animales , Respiración de la Célula/fisiología , Humanos , Metabolismo de los Lípidos/fisiología , Lipogénesis/fisiología , Oxidación-ReducciónRESUMEN
Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285∗), c.247_250del (p.Asn83Hisfs∗4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.
Asunto(s)
Trastornos Distónicos/genética , Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Mutación , Atrofia Óptica/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Ganglios Basales/metabolismo , Células Cultivadas , Niño , Preescolar , Femenino , Fibroblastos , Prueba de Complementación Genética , Humanos , Lactante , Masculino , Enfermedades Mitocondriales/genética , Modelos Moleculares , Mutación Missense/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Linaje , Sitios de Empalme de ARN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) Δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP(+) oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.
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Ferredoxina-NADP Reductasa/metabolismo , Hemoglobinas/metabolismo , Óxido Nítrico/farmacología , Populus/enzimología , Cloroplastos/metabolismo , Citosol/metabolismo , Ferredoxina-NADP Reductasa/genética , Genes Reporteros , Mitocondrias/metabolismo , Mutación , Oxigenasas/genética , Oxigenasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/genética , Proteómica , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADNRESUMEN
Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.
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ADN Mitocondrial/genética , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Proteínas de la Membrana/genética , Células 3T3 , Alelos , Secuencia de Aminoácidos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Haplotipos , Linfocitos/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Transporte de ProteínasRESUMEN
The Saccharomyces cerevisiae genome encodes two sequence related acetyl-CoA carboxylases, the cytosolic Acc1p and the mitochondrial Hfa1p, required for respiratory function. Several aspects of expression of the HFA1 gene and its evolutionary origin have remained unclear. Here, we determined the HFA1 transcription initiation sites by 5' RACE analysis. Using a novel "Stop codon scanning" approach, we mapped the location of the HFA1 translation initiation site to an upstream AUU codon at position -372 relative to the annotated start codon. This upstream initiation leads to production of a mitochondrial targeting sequence preceding the ACC domains of the protein. In silico analyses of fungal ACC genes revealed conserved "cryptic" upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δhfa1 baker's yeast mutant phenotype rescue studies using the protoploid Kluyveromyces lactis ACC confirmed functionality of the cryptic upstream mitochondrial targeting signal. These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in S. cerevisiae have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms. Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5' upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.
Asunto(s)
Acetil-CoA Carboxilasa/biosíntesis , Acetil-CoA Carboxilasa/genética , Evolución Molecular , Regulación Fúngica de la Expresión Génica , Mitocondrias/genética , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Regiones no Traducidas 5'/genética , Secuencia de Bases , Codón Iniciador/genética , Kluyveromyces/genética , Mitocondrias/enzimología , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Transcripción GenéticaRESUMEN
Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory growth in yeast and mammalian embryonic survival. The human 3-ketoacyl-acyl carrier protein (ACP) reductase (KAR) of mtFAS is a heterotetrameric α2ß2-assembly composed of 17ß-hydroxysteroid dehydrogenase type-8 (HSD17B8, α-subunit) and carbonyl reductase type-4 (CBR4, ß-subunit). Here we provide a structural explanation for the stability of the heterotetramer from the crystal structure with NAD(+) and NADP(+) bound to the HSD17B8 and CBR4 subunits, respectively, and show that the catalytic activity of the NADPH- and ACP-dependent CBR4 subunit is crucial for a functional HsKAR. Therefore, mtFAS is NADPH- and ACP dependent, employing the 3R-hydroxyacyl-ACP intermediate. HSD17B8 assists in the formation of the competent HsKAR assembly. The intrinsic NAD(+)- and CoA-dependent activity of the HSD17B8 subunit on the 3R-hydroxyacyl-CoA intermediates may indicate a role for this subunit in routing 3R-hydroxyacyl-CoA esters, potentially arising from the metabolism of unsaturated fatty acids, into the mitochondrial ß-oxidation pathway.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Ácidos Grasos/biosíntesis , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Oxidorreductasas/metabolismo , Estructura Cuaternaria de Proteína , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/metabolismo , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Pruebas de Enzimas , Humanos , Mycobacterium tuberculosis/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LAâ per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell.