RESUMO
Pentatricopeptide (PPR) proteins bind RNA and are present in mitochondria and chloroplasts of Eukaryota. In fungi they are responsible for controlling mitochondrial genome expression, mainly on the posttranscriptional level. Candida albicans is a human opportunistic pathogen with a facultative anaerobic metabolism which, unlike the model yeast S. cerevisiae, possesses mitochondrially encoded respiratory Complex I (CI) subunits and does not tolerate loss of mtDNA. We characterized the function of 4 PPR proteins of C. albicans that lack orthologs in S. cerevisiae, and found that they are required for the expression of mitochondrially-encoded CI subunits. We demonstrated that these proteins localize to mitochondria and are essential to maintain the respiratory capacity of cells. Deletion of genes encoding these PPR proteins results in changes in steady state levels of mitochondrial RNAs and proteins. We demonstrated that C. albicans cells lacking CaPpr4, CaPpr11, and CaPpr13 proteins show no CI assembly, whereas the lack of CaPpr7p results in a decreased CI activity. CaPpr13p is required to maintain the bicistronic NAD4L-NAD5 mRNA, whereas the other three PPR proteins are likely involved in translation-related assembly of mitochondrially encoded CI subunits. In addition, we show that CaAep3p which is an ortholog of ScAep3p, performs the evolutionary conserved function of controlling expression of the ATP8-ATP6 mRNA. We also show that C. albicans cells lacking PPR proteins express a higher level of the inducible alternative oxidase (AOX2) which likely rescues respiratory defects and compensates for oxidative stress.
RESUMO
Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Ribonucleoproteínas , Edição de Genes/métodos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Leishmania/genética , Leishmania/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma/genética , Trypanosoma/metabolismo , TransfecçãoRESUMO
Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.
Assuntos
Glicerol Quinase/metabolismo , Glicerol/metabolismo , Hexoquinase/metabolismo , Microcorpos/enzimologia , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem CelularRESUMO
Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.
Assuntos
Glucose/metabolismo , Prolina/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma/efeitos dos fármacos , Moscas Tsé-Tsé/efeitos dos fármacos , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Insetos Vetores/parasitologia , Oxirredução/efeitos dos fármacos , Prolina/metabolismo , Interferência de RNA/fisiologia , Trypanosoma/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/tratamento farmacológico , Moscas Tsé-Tsé/parasitologiaRESUMO
Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzyme's biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (Fo). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a direct correlation between TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation, two-dimensional blue-native/SDS-PAGE, and pulse-chase experiments reveal that TMEM70 forms large oligomers that interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Linhagem Celular , Metabolismo Energético , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Microscopia Eletrônica , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Domínios Proteicos , Multimerização ProteicaRESUMO
The m.8993T>G mutation of the mitochondrial MT-ATP6 gene has been associated with numerous cases of neuropathy, ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome, which are diseases known to result from abnormalities affecting mitochondrial energy transduction. We previously reported that an equivalent point mutation severely compromised proton transport through the ATP synthase membrane domain (FO) in Saccharomyces cerevisiae and reduced the content of cytochrome c oxidase (Complex IV or COX) by 80%. Herein, we report that overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) considerably increases Complex IV abundance and tricarboxylic acid-mediated substrate-level phosphorylation of ADP coupled to conversion of α-ketoglutarate into succinate in m.8993T>G yeast. Consistently in m.8993T>G yeast cells, the retrograde signaling pathway was found to be strongly induced in order to preserve α-ketoglutarate production; when Odc1p was overexpressed, this stress pathway returned to an almost basal activity. Similar beneficial effects were induced by a partial uncoupling of the mitochondrial membrane with the proton ionophore, cyanide m-chlorophenyl hydrazone. This chemical considerably improved the glutamine-based, respiration-dependent growth of human cytoplasmic hybrid cells that are homoplasmic for the m.8993T>G mutation. These findings shed light on the interdependence between ATP synthase and Complex IV biogenesis, which could lay the groundwork for the creation of nutritional or metabolic interventions for attenuating the effects of mtDNA mutations.
Assuntos
Mitocôndrias/metabolismo , Miopatias Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Retinose Pigmentar/metabolismo , Trifosfato de Adenosina/metabolismo , Ataxia/genética , Deficiência de Citocromo-c Oxidase/genética , DNA Mitocondrial/genética , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Transporte de Íons , Doença de Leigh , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Retinose Pigmentar/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cardiolipin (CL) is a diglycerol phospholipid mostly found in mitochondria where it optimizes numerous processes, including oxidative phosphorylation (OXPHOS). To function properly, CL needs to be unsaturated, which requires the acyltransferase tafazzin. Loss-of-function mutations in this protein are responsible for Barth syndrome (BTHS), presumably because of a diminished OXPHOS capacity. Here, we show that overexpressing Odc1p, a conserved oxodicarboxylic acid carrier located in the mitochondrial inner membrane, fully restores oxidative phosphorylation in a yeast model (taz1Δ) of BTHS. The rescuing activity involves the recovery of normal expression of key components that sustain oxidative phosphorylation, including cytochrome c and electron transport chain complexes IV and III, which are strongly downregulated in taz1Δ yeast. Interestingly, overexpression of Odc1p was also shown previously to rescue yeast models of mitochondrial diseases caused by defects in the assembly of ATP synthase and by mutations in the MPV17 protein that result in hepatocerebral mitochondrial DNA depletion syndrome. These findings define the transport of oxodicarboxylic acids across the inner membrane as a potential therapeutic target for a large spectrum of mitochondrial diseases, including BTHS.
Assuntos
Síndrome de Barth/metabolismo , Síndrome de Barth/patologia , Transportadores de Ácidos Dicarboxílicos/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/biossíntese , Aerobiose , Eletroforese em Gel de Poliacrilamida , Metabolismo Energético/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Ácido Oleico/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.
Assuntos
Flagelos/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/química , Sequência de Aminoácidos , DNA de Protozoário/isolamento & purificação , Eletroporação , Flagelina/química , Leishmania/química , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Interferência de RNA , RNA de Protozoário/isolamento & purificação , Alinhamento de Sequência , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestruturaRESUMO
F1F0-ATP synthase is a key enzyme of the mitochondrial energetic metabolism responsible for the production of most cellular ATP in humans. Mayr et al. (2010) recently described a patient with a homozygote (Y12C) mutation in the nuclear gene ATP5E encoding the ε-subunit of ATP synthase. To better define how it affects ATP synthase, we have modeled this mutation in the yeast Saccharomyces cerevisiae. A yeast equivalent of this mutation (Y11C) had no significant effect on the growth of yeast on non-fermentable carbon sources (glycerol/ethanol or lactate), conditions under which the activity of the mitochondrial energy transducing system is absolutely essential. In addition, similar to what was observed in patient, this mutation in yeast has a minimal effect on the ATPase/synthase activities. On the contrary, this mutation which has been shown to have a strong impact on the assembly of the ATP synthase complex in humans, shows no significant impact on the assembly/stability of this complex in yeast, suggesting that biogenesis of this complex differs significantly.
RESUMO
In eukaryotic cells, oxidative phosphorylation involves multisubunit complexes of mixed genetic origin. Assembling these complexes requires an organelle-independent synchronizing system for the proper expression of nuclear and mitochondrial genes. Here we show that proper expression of the F1FO ATP synthase (complex V) depends on a cytosolic complex (AME) made of two aminoacyl-tRNA synthetases (cERS and cMRS) attached to an anchor protein, Arc1p. When yeast cells adapt to respiration the Snf1/4 glucose-sensing pathway inhibits ARC1 expression triggering simultaneous release of cERS and cMRS. Free cMRS and cERS relocate to the nucleus and mitochondria, respectively, to synchronize nuclear transcription and mitochondrial translation of ATP synthase genes. Strains releasing asynchronously the two aminoacyl-tRNA synthetases display aberrant expression of nuclear and mitochondrial genes encoding subunits of complex V resulting in severe defects of the oxidative phosphorylation mechanism. This work shows that the AME complex coordinates expression of enzymes that require intergenomic control.
Assuntos
ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Núcleo Celular/genética , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Mitocôndrias/genética , Complexos Multienzimáticos , Multimerização Proteica , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Ligação a RNA/fisiologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
When mitochondria become deenergized, futile ATP hydrolysis is prevented by reversible binding of an endogenous inhibitory peptide called IF1 to ATP synthase. Between initial IF1 binding and IF1 locking the enzyme experiences large conformational changes. While structural studies give access to analysis of the dead-end inhibited state, transient states have thus far not been described. Here, we studied both initial and final states by reporting, for the first time, the consequences of mutations of Saccharomyces cerevisiae ATP synthase on its inhibition by IF1. Kinetic studies allowed the identification of amino acids or motifs of the enzyme that are involved in recognition and/or locking of IF1 α-helical midpart. This led to an outline of IF1 binding process. In the recognition step, protruding parts of α and especially ß subunits grasp IF1, most likely by a few residues of its α-helical midpart. Locking IF1 within the αß interface involves additional residues of both subunits. Interactions of the α and ß subunits with the foot of the γ subunit might contribute to locking and stabilizing of the dead-end state.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/metabolismo , Peptídeos/metabolismo , Cinética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismoRESUMO
The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F1 and the proton translocation channel in F0 for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F0-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F0 subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F0.
Assuntos
Mitocôndrias/metabolismo , Subunidades Proteicas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Engenharia Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Genótipo , Mitocôndrias/genética , Mutação , Fosforilação Oxidativa , Fenótipo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Termodinâmica , Proteína Inibidora de ATPaseRESUMO
During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein.
Assuntos
Evolução Molecular , Flagelos/genética , Leishmania/genética , Proteínas de Membrana/genética , Filogenia , Sequência de Bases , Biologia Computacional , Flagelos/metabolismo , Flagelos/ultraestrutura , Leishmania/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Análise de Sequência de DNARESUMO
Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.
Assuntos
Núcleo Celular/genética , Proteínas Fúngicas/genética , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Podospora/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Evolução Biológica , Núcleo Celular/enzimologia , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Mitocondriais , Genoma Mitocondrial , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação Oxidativa , Podospora/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , TransgenesRESUMO
In yeast, the two main F(O) proton-translocating subunits of the ATP synthase (subunits 6/a and 9/c) are encoded by mitochondrial DNA (mtDNA). Unfortunately, mutations that inactivate the F(O) typically result in loss of mtDNA under the form of ρ(-)/ρ(0) cells. Thus, we have designed a novel genetic strategy to circumvent this problem. It exploits previous findings that a null mutation in the nuclear ATP16 gene encoding ATP synthase subunit δ results in massive and lethal F(O)-mediated protons leaks across the inner mitochondrial membrane. Mutations that inactivate the F(O) can thus, in these conditions, be selected positively as cell viability rescuing events. A first set of seven mutants was analyzed and all showed, as expected, very severe F(O) deficiencies. Two mutants carried nuclear mutations in known genes (AEP1, AEP2) required for subunit c expression. The five other mutations were located in mtDNA. Of these, three affect synthesis or stability of subunit a transcripts and the two last consisted in a single amino acid replacement in subunit c. One of the subunit c mutations is particularly interesting. It consists in an alanine to valine change at position 60 of subunit c adjacent to the essential glutamate of subunit c (at position 59) that interacts with the essential arginine 186 of subunit a. The properties of this mutant suggest that the contact zone between subunit a and the ten subunits c-ring structure only involves critical transient interactions confined to the region where protons are exchanged between the subunit a and the c-ring.
Assuntos
Mutação , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Domínio Catalítico , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Complexo de Golgi/metabolismo , Leishmania/química , Proteínas de Membrana/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Membrana/genética , Mutação , Transporte Proteico , Proteínas de ProtozoáriosRESUMO
NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F(1) coupled to F(0)-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.
Assuntos
Ataxia/enzimologia , Doença de Leigh/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Biológicos , Retinose Pigmentar/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/genética , Substituição de Aminoácidos , Ataxia/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidrólise , Mitocôndrias/enzimologia , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação de Sentido Incorreto , Consumo de Oxigênio/genética , Prótons , Retinose Pigmentar/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of rho-/rho0 petites issued from large deletions in mtDNA could be restricted to 20-30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.
Assuntos
Genes Mitocondriais/genética , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Forma Celular , Citocromos c/metabolismo , DNA Mitocondrial/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Genótipo , Hidrólise , Microscopia Eletrônica , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Oxirredutases/metabolismo , Oxigênio/metabolismo , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genética , Transaminases/genética , Transaminases/metabolismo , Transcrição Gênica/genéticaRESUMO
Within the mitochondrial F(1)F(0)-ATP synthase, the nucleus-encoded delta-F(1) subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the delta subunit gene (Deltadelta) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% rho(-)/rho degrees petites (i.e. cells missing a large portion (>50%) of the mtDNA (rho(-)) or totally devoid of mtDNA (rho degrees )). Previous work has suggested that the absence of complete mtDNA (rho(+)) in Deltadelta yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8(m)) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Deltadelta yeast. We further show that combined with a nuclear mutation (Deltaatp4) preventing the ATP synthase proton channel assembly, a lack of delta subunit fails to destabilize the mtDNA, and rho(+) Deltadelta cells become viable. We conclude that Deltadelta yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the rho(-)/rho degrees mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae.