RESUMEN
Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.
Asunto(s)
Mitocondrias , Saccharomyces cerevisiae , Potenciales de la Membrana , Mitocondrias/metabolismo , Transporte Biológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function.
Asunto(s)
Daño del ADN , Escherichia coli/genética , Fumarato Hidratasa/metabolismo , Fumaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Enzimas AlkB , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ciclo del Ácido Cítrico , Roturas del ADN de Doble Cadena , ADN Bacteriano/genética , Fumarato Hidratasa/química , Genes BacterianosRESUMEN
The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.
RESUMEN
Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.
Asunto(s)
Genoma Fúngico , Biblioteca Genómica , Proteoma/genética , Saccharomyces cerevisiae/genética , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Lugares Marcados de SecuenciaRESUMEN
Fumarate hydratase (FH, fumarase), is a tricarboxylic acid cycle enzyme localized in the mitochondrial matrix. However, a common theme, conserved from yeast to human, is the existence of a large cytosolic population of FH. FH has been shown to function as a tumor suppressor gene and is now implicated in various diseases. We have previously indicated that the cytosolic echoform of FH has a role in the DNA damage response and specifically in the response to DNA double strand breaks. In fact, recently FH has been shown to be involved in histone demethylation. Therefore, it has become important to understand the underlying mechanism of FH dual subcellular location in human cells. We revealed that in human cells, in contrast to yeast, the FH gene encodes two gene products, one containing and one lacking the mitochondrial targeting sequence. On the basis of expression of endogenous wild-type FH and mutant FH cDNAs from plasmids, RT-PCR, RACE to determine the 5' termini of FH mRNAs, and mass spectrometry of FH products, we show that the mechanism of FH distribution is alternative transcription initiation from a broad promoter. This is contrary to the suggested mechanism for rat liver cells which had claimed alternative translation initiation.
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Empalme Alternativo/genética , Citosol/enzimología , Fumarato Hidratasa/metabolismo , Mitocondrias/enzimología , Sitio de Iniciación de la Transcripción , Transcripción Genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Técnicas de Cultivo de Célula , Fumarato Hidratasa/genética , Células HEK293 , Células HeLa , Humanos , Isoenzimas , Mitocondrias/genética , Plásmidos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN Mensajero/genéticaRESUMEN
One of the most severe forms of DNA damage is the double-strand break (DSB). Failure to properly repair the damage can cause mutation, gross chromosomal rearrangements and lead to the development of cancer. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are the main DSB repair pathways. Fumarase is a mitochondrial enzyme which functions in the tricarboxylic acid cycle. Intriguingly, the enzyme can be readily detected in the cytosolic compartment of all organisms examined, and we have shown that cytosolic fumarase participates in the DNA damage response towards DSBs. In human cells, fumarase was shown to be involved in NHEJ, but it is still unclear whether fumarase is also important for the HR pathway. Here we show that the depletion of cytosolic fumarase in yeast prolongs the presence of Mre11 at the DSBs, and decreases the kinetics of repair by the HR pathway. Overexpression of Sae2 endonuclease reduced the DSB sensitivity of the cytosolic fumarase depleted yeast, suggesting that Sae2 and fumarase functionally interact. Our results also suggest that Sae2 and cytosolic fumarase physically interact in vivo. Sae2 has been shown to be important for the DSB resection process, which is essential for the repair of DSBs by the HR pathway. Depletion of cytosolic fumarase inhibited DSB resection, while the overexpression of cytosolic fumarase or Sae2 restored resection. Together with our finding that cytosolic fumarase depletion reduces Sae2 cellular amounts, our results suggest that cytosolic fumarase is important for the DSB resection process by regulating Sae2 levels.
Asunto(s)
Citosol/enzimología , Roturas del ADN de Doble Cadena , ADN de Hongos/metabolismo , ADN/metabolismo , Endonucleasas/metabolismo , Fumarato Hidratasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Reparación del ADN por Unión de Extremidades , Unión Proteica , Saccharomyces cerevisiae/enzimologíaRESUMEN
Mitochondrial encephalopathies are a heterogeneous group of disorders which generally carries a grave prognosis. Using exome sequencing, we identified a homozygous mutation, Pro-304-His in the IDH3A gene, in a patient suffering from infantile encephalopathy with peripheral and autonomic nervous system involvement. Mammalian isocitrate dehydrogenase (IDH) 3 is a heterotetramer of 2alfa, 1beta, and 1gamma subunits, and IDH3A encodes the alfa subunit of the mitochondrial NAD+-dependent IDH. Here we show that in contrast to wild-type human IDH3A, the human IDH3A which harbor the p.Pro304His mutation does not complement the yeast Δidh1/Δidh2 growth defect on ethanol-acetate. We therefore propose that homozygosity for the p.Pro304His mutation is deleterious for mitochondrial NAD+-specific IDH3A activity in human. IDH3A now joins the list of TCA cycle-related proteins, which includes ACO2, DLD, SLC25A19, FH, and succinate dehydrogenase subunits, all associated with neurological disorders.
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Encefalopatías/genética , Isocitrato Deshidrogenasa/genética , Mutación Missense , Factores de Edad , Sustitución de Aminoácidos , Histidina/genética , Homocigoto , Humanos , Recién Nacido , Enfermedades del Recién Nacido/genética , Isocitrato Deshidrogenasa/química , Masculino , Enfermedades Mitocondriales/genética , Prolina/genética , Subunidades de Proteína/genética , Índice de Severidad de la EnfermedadRESUMEN
Hsp60, an essential oligomeric molecular mitochondrial chaperone, has been subject to rigorous basic and clinical research. With yeast as a model system, we provide evidence for the ability of cytosolic yHsp60 to inhibit the yeast proteasome. (i) Following biological turnover of murine Bax (a proteasome substrate), we show that co-expression of cytosolic yHsp60 stabilizes Bax, enhances its association with mitochondria, and enhances its killing capacity. (ii) Expression of yHsp60 in the yeast cytosol (yHsp60c) inhibits degradation of a cytosolic protein ΔMTS-Aco1 tagged with the degron SL17 (a ubiquitin-proteasome substrate). (iii) Conditions under which Hsp60 accumulates in the cytosol (elevated Hsp60c or growth at 37 °C) correlate with reduced 20 S peptidase activity in proteasomes purified from cell extracts. (iv) Elevated yHsp60 in the cytosol correlate with accumulation of polyubiquitinated proteins. (v) According to 20 S proteasome pulldown experiments, Hsp60 is physically associated with proteasomes in extracts of cells expressing Hsp60c or grown at 37 °C. Even mutant Hsp60 proteins, lacking chaperone activity, were still capable of proteasome inhibition. The results support the hypothesis that localization of Hsp60 to the cytosol may modulate proteasome activity according to cell need.
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Chaperonina 60/metabolismo , Citosol/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Chaperonina 60/genética , Proteína 1 Reguladora de Hierro/metabolismo , Ratones , Mitocondrias/metabolismo , Unión Proteica , Transporte de Proteínas , Proteolisis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinación , Proteína X Asociada a bcl-2/metabolismoRESUMEN
In eukaryotic cells, identical proteins can be located in more than a single subcellular compartment, a phenomenon termed dual targeting. We hypothesized that dual-targeted proteins should be more evolutionary conserved than exclusive mitochondrial proteins, due to separate selective pressures administered by the different compartments to maintain the functions associated with the protein sequences. We employed codon usage bias, propensity for gene loss, phylogenetic relationships, conservation analysis at the DNA level, and gene expression, to test our hypothesis. Our findings indicate that, indeed, dual-targeted proteins are significantly more conserved than their exclusively targeted counterparts. We then used this trait of gene conservation, together with previously identified traits of dual-targeted proteins (such as protein net charge and mitochondrial targeting sequence strength) to 1) create, for the first time (due to addition of conservation parameters), a tool for the prediction of dual-targeted mitochondrial proteins based on protein and mRNA sequences, and 2) show that molecular mechanisms involving one versus two translation products are not correlated with specific dual-targeting parameters. Finally, we discuss what evolutionary pressure maintains protein dual targeting in eukaryotes and deduce, as we initially hypothesized, that it is the discrete functions of these proteins in the different subcellular compartments, regardless of their dual-targeting mechanism.
Asunto(s)
Evolución Molecular , Mitocondrias/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , Modelos Genéticos , Filogenia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Degeneration of the cerebrum, cerebellum, and retina in infancy is part of the clinical spectrum of lysosomal storage disorders, mitochondrial respiratory chain defects, carbohydrate glycosylation defects, and infantile neuroaxonal dystrophy. We studied eight individuals from two unrelated families who presented at 2-6 months of age with truncal hypotonia and athetosis, seizure disorder, and ophthalmologic abnormalities. Their course was characterized by failure to acquire developmental milestones and culminated in profound psychomotor retardation and progressive visual loss, including optic nerve and retinal atrophy. Despite their debilitating state, the disease was compatible with survival of up to 18 years. Laboratory investigations were normal, but the oxidation of glutamate by muscle mitochondria was slightly reduced. Serial brain MRI displayed progressive, prominent cerebellar atrophy accompanied by thinning of the corpus callosum, dysmyelination, and frontal and temporal cortical atrophy. Homozygosity mapping followed by whole-exome sequencing disclosed a Ser112Arg mutation in ACO2, encoding mitochondrial aconitase, a component of the Krebs cycle. Specific aconitase activity in the individuals' lymphoblasts was severely reduced. Under restrictive conditions, the mutant human ACO2 failed to complement a yeast ACO1 deletion strain, whereas the wild-type human ACO2 succeeded, indicating that this mutation is pathogenic. Thus, a defect in mitochondrial aconitase is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. In the absence of noninvasive biomarkers, determination of the ACO2 sequence or of aconitase activity in lymphoblasts are warranted in similarly affected individuals, based on clinical and neuroradiologic grounds.
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Aconitato Hidratasa/genética , Cerebelo/anomalías , Mitocondrias/enzimología , Mutación , Enfermedades Neurodegenerativas/genética , Retina/anomalías , Adolescente , Atrofia/enzimología , Atrofia/genética , Cerebelo/enzimología , Niño , Preescolar , Exoma , Exones , Femenino , Genotipo , Ácido Glutámico/metabolismo , Heterocigoto , Homocigoto , Humanos , Lactante , Imagen por Resonancia Magnética/métodos , Masculino , Mitocondrias/genética , Enfermedades Neurodegenerativas/enzimología , Oxidación-Reducción , Polimorfismo de Nucleótido Simple , Retina/enzimologíaRESUMEN
Dual targeting is an important and abundant phenomenon. Indeed, we estimate that more than a third of the yeast mitochondrial proteome is dual localized. The enzyme fumarase is a highly conserved protein in all organisms with respect to its sequence, structure, and enzymatic activity. In eukaryotes, it is dual localized to the cytosol and mitochondria. In Saccharomyces cerevisiae, the dual localization of fumarase is achieved by the reverse translocation mechanism; all fumarase molecules harbor a mitochondrial targeting sequence (MTS), are targeted to mitochondria, begin their translocation, and are processed by mitochondrial processing peptidase in the matrix. A subset of these processed fumarase molecules in transit is then fully imported into the matrix, whereas the majority moves back into the cytosol by reverse translocation. The proposed driving force for fumarase distribution is protein folding during import. Here, we asked how reverse translocation could have evolved on a prokaryotic protein that had already acquired expression from the nuclear genome and a targeting sequence. To address this question, we used, as a model, the Escherichia coli FumC Class II fumarase, which is homologous to eukaryotic fumarases (â¼58% identity and â¼74% similarity to the yeast Fum1). Starting with an exclusively mitochondrial targeted FumC (attached to a strong MTS), we show that two randomly acquired mutations within the prokaryotic FumC sequence are sufficient to cause substantial dual targeting by reverse translocation. In fact, the unmutated MTS-FumC also has some ability to be dual targeted but only at low temperatures. Our results suggest that in this case, evolution of dual targeting by reverse translocation is based on naturally occurring and fortuitously conserved features of fumarase folding.
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Escherichia coli/genética , Fumarato Hidratasa/genética , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Ciclo del Ácido Cítrico/genética , Citosol/enzimología , Escherichia coli/enzimología , Mitocondrias , Células Procariotas/enzimología , Pliegue de Proteína , Saccharomyces cerevisiae/enzimologíaRESUMEN
BACKGROUND: Agenesis of corpus callosum has been associated with several defects of the mitochondrial respiratory chain and the citric acid cycle. We now report the results of the biochemical and molecular studies of a patient with severe neurodevelopmental disease manifesting by agenesis of corpus callosum and optic nerve hypoplasia. METHODS AND RESULTS: A mitochondrial disease was suspected in this patient based on the prominent excretion of 2-hydroxyglutaric acid and Krebs cycle intermediates in urine and the finding of increased reactive oxygen species content and decreased mitochondrial membrane potential in her fibroblasts. Whole exome sequencing disclosed compound heterozygosity for two pathogenic variants in the SLC25A1 gene, encoding the mitochondrial citrate transporter. These variants, G130D and R282H, segregated in the family and were extremely rare in controls. The mutated residues were highly conserved throughout evolution and in silico modeling investigations indicated that the mutations would have a deleterious effect on protein function, affecting either substrate binding to the transporter or its translocation mechanism. These predictions were validated by the observation that a yeast strain harbouring the mutations at equivalent positions in the orthologous protein exhibited a growth defect under stress conditions and by the loss of activity of citrate transport by the mutated proteins reconstituted into liposomes. CONCLUSIONS: We report for the first time a patient with a mitochondrial citrate carrier deficiency. Our data support a role for citric acid cycle defects in agenesis of corpus callosum as already reported in patients with aconitase or fumarate hydratase deficiency.
Asunto(s)
Agenesia del Cuerpo Calloso/genética , Proteínas de Transporte de Anión/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Nervio Óptico/patología , Adolescente , Agenesia del Cuerpo Calloso/patología , Proteínas de Transporte de Anión/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Femenino , Humanos , Enfermedades Mitocondriales , Proteínas Mitocondriales/metabolismo , Mutación , Nervio Óptico/metabolismo , Transportadores de Anión OrgánicoRESUMEN
Almost all mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol as precursor proteins. Signals in the amino acid sequence of these precursors ensure their targeting and translocation into mitochondria. However, in many cases, only a certain fraction of a specific protein is transported into mitochondria, while the rest either remains in the cytosol or undergoes reverse translocation to the cytosol, and can populate other cellular compartments. This phenomenon is called dual localization which can be instigated by different mechanisms. These include alternative start or stop codons, differential transcripts, and ambiguous or competing targeting sequences. In many cases, dual localization might serve as an economic strategy to reduce the number of required genes; for example, when the same groups of enzymes are required both in mitochondria and chloroplasts or both in mitochondria and the nucleus/cytoplasm. Such cases frequently employ ambiguous targeting sequences to distribute proteins between both organelles. However, alternative localizations can also be used for signaling, for example when non-imported precursors serve as mitophagy signals or when they represent transcription factors in the nucleus to induce the mitochondrial unfolded stress response. This review provides an overview regarding the mechanisms and the physiological consequences of dual targeting.
RESUMEN
Mitochondria are dynamic, intracellular organelles with a separate genome originating from prokaryotes. They perform numerous functions essential for cellular metabolism and energy production. Mitochondrial-derived vesicles (MDVs) are single or double membrane-enclosed vesicles, formed and released from the mitochondrial sub-compartments into the cytosol, in response to various triggers. MDVs interact with other organelles such as lysosomes and peroxisomes or may be incorporated and excreted via extracellular vesicles (EVs). MDVs selectively incorporate diverse protein and lipid cargoes and are involved in various functions such as mitochondrial quality control, immunomodulation, energy complementation, and compartmentalization and transport. This review aims to provide a summary of the current knowledge of MDVs biogenesis, release, cargoes, and roles.
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Primary microcephaly of postnatal onset is a feature of many neurological disorders, mostly associated with mental retardation, seizures, and spasticity, and it typically carries a grave prognosis. Five infants from four unrelated families of Caucasus Jewish origin presented soon after birth with spasticity, epilepsy, and profound psychomotor retardation. Head circumference percentiles declined, and brain MRI disclosed marked cereberal and cerebellar atrophy with severe myelination defect. A search for a common homozygous region revealed a 2.28 Mb genomic segment on chromosome 11 that encompassed 16 protein-coding genes. A missense mutation in one of them, MED17, segregated with the disease state in the families and was carried by four of 79 anonymous Caucasus Jews. A corresponding mutation in the homologous S.cerevisiae gene SRB4 inactivated the protein, according to complementation assays. Screening of MED17 in additional patients with similar clinical and radiologic findings revealed four more patients, all homozygous for the p.L371P mutation and all originating from Caucasus Jewish families. We conclude that the p. L371P mutation in MED17 is a founder mutation in the Caucasus Jewish community and that homozygosity for this mutation is associated with infantile cerebral and cerebellar atrophy with poor myelination.
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Atrofia/genética , Encéfalo/patología , Cerebelo/patología , Complejo Mediador/genética , Mutación , Cromosomas Humanos Par 11 , Efecto Fundador , Homocigoto , Humanos , Lactante , Judíos/genética , Microcefalia/genética , Enfermedades del Sistema Nervioso/genéticaRESUMEN
In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation.
Asunto(s)
Núcleo Celular/metabolismo , Citosol/metabolismo , Daño del ADN , Fumarato Hidratasa/metabolismo , Isoenzimas/metabolismo , Mitocondrias/enzimología , Fumarato Hidratasa/genética , Fumaratos/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isoenzimas/genética , Neoplasias Renales/enzimología , Neoplasias Renales/genética , Leiomiomatosis/enzimología , Leiomiomatosis/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Dual localization or dual targeting refers to the phenomenon by which identical, or almost identical, proteins are localized to two (or more) separate compartments of the cell. From previous work in the field, we had estimated that a third of the mitochondrial proteome is dual-targeted to extra-mitochondrial locations and suggested that this abundant dual targeting presents an evolutionary advantage. Here, we set out to study how many additional proteins whose main activity is outside mitochondria are also localized, albeit at low levels, to mitochondria (eclipsed). To do this, we employed two complementary approaches utilizing the α-complementation assay in yeast to uncover the extent of such an eclipsed distribution: one systematic and unbiased and the other based on mitochondrial targeting signal (MTS) predictions. Using these approaches, we suggest 280 new eclipsed distributed protein candidates. Interestingly, these proteins are enriched for distinctive properties compared to their exclusively mitochondrial-targeted counterparts. We focus on one unexpected eclipsed protein family of the Triose-phosphate DeHydrogenases (TDH) and prove that, indeed, their eclipsed distribution in mitochondria is important for mitochondrial activity. Our work provides a paradigm of deliberate eclipsed mitochondrial localization, targeting and function, and should expand our understanding of mitochondrial function in health and disease.
Asunto(s)
Proteínas Mitocondriales , Saccharomyces cerevisiae , Proteínas Mitocondriales/metabolismo , Saccharomyces cerevisiae/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Proteoma/metabolismoRESUMEN
Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins.
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Proteínas de Unión al ADN/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Hierro/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Mitocondriales/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Azufre/metabolismo , Factores de Transcripción/genéticaRESUMEN
One solution found in evolution to increase the number of cellular functions, without increasing the number of genes, is distribution of single gene products to more than one cellular compartment. It is well documented that in eukaryotic cells, molecules of one protein can be located in several subcellular locations, a phenomenon termed dual targeting, dual localization, or dual distribution. The differently localized proteins are coined in this review "echoforms" indicating repetitious forms of the same protein (echo in Greek denotes repetition) distinctly placed in the cell. This term replaces the term to "isoproteins" or "isoenzymes" which are reserved for proteins with the same activity but different amino acid sequences. Echoforms are identical or nearly identical, even though, as referred to in this review may, in some cases, surprisingly have a totally different function in the different compartments. With regard to mitochondria, our operational definition of dual targeted proteins refers to situations in which one of the echoforms is translocated through/into a mitochondrial membrane. In this review we ask how, when and why mitochondrial proteins are dual localized in the cell. We describe mechanisms of dual targeting of proteins between mitochondria and other compartments of the eukaryotic cell. In particular, we have paid attention to situations in which dual localization is regulated in time, location or function. In addition, we have attempted to provide a broader view concerning the phenomenon of dual localization of proteins by looking at mechanisms that are beyond our simple definition of dual targeting. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.