RESUMEN
Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/fisiología , Mitocondrias/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Animales , Autofagia , Metabolismo Energético , Homeostasis , Humanos , Mitocondrias/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
In response to many apoptotic stimuli, oligomerization of Bax is essential for mitochondrial outer membrane permeabilization and the ensuing release of cytochrome c. These events are accompanied by mitochondrial fission that appears to require Drp1, a large GTPase of the dynamin superfamily. Loss of Drp1 leads to decreased cytochrome c release by a mechanism that is poorly understood. Here we show that Drp1 stimulates tBid-induced Bax oligomerization and cytochrome c release by promoting tethering and hemifusion of membranes in vitro. This function of Drp1 is independent of its GTPase activity and relies on arginine 247 and the presence of cardiolipin in membranes. In cells, overexpression of Drp1 R247A/E delays Bax oligomerization and cell death. Our findings uncover a function of Drp1 and provide insight into the mechanism of Bax oligomerization.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Cardiolipinas/metabolismo , Sistema Libre de Células , Dinaminas , Células HeLa , Humanos , Liposomas/metabolismo , Membranas Mitocondriales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , RatasRESUMEN
Recent advances in genome editing technologies have enabled the insertion of epitope tags at endogenous loci with relative efficiency. We describe an approach for investigation of protein interaction dynamics of the AMP-activated kinase complex AMPK using a catalytic subunit AMPKα2 (PRKAA2 gene) as the bait, based on CRISPR/Cas9-mediated genome editing coupled to stable isotope labeling in cell culture, multidimensional protein identification technology, and computational and statistical analyses. Furthermore, we directly compare this genetic epitope tagging approach to endogenous immunoprecipitations of the same gene under homologous conditions to assess differences in observed interactors. Additionally, we directly compared each enrichment strategy in the genetically modified cell-line with two separate endogenous antibodies. For each approach, we analyzed the interaction profiles of this protein complex under basal and activated states, and after implementing the same analytical, computational, and statistical analyses, we found that high-confidence protein interactors vary greatly with each method and between commercially available endogenous antibodies.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genómica/métodos , Mapeo de Interacción de Proteínas/métodos , Anticuerpos , Células Cultivadas , Cromatografía de Afinidad , Edición Génica , Células HEK293 , Humanos , Inmunoprecipitación , Marcaje Isotópico , Espectrometría de MasasRESUMEN
We aimed to evaluate the feasibility of neurochemical profiling of embryonic mouse brain developments in utero and to seek potential in vivo evidence of an energy shift in a mitochondrial pyruvate carrier 1 (MPC1) deficient mouse model. C57BL/6 embryonic mouse brains were studied in utero by anatomical MRI and short echo localized proton (1 H) MRS at 14.1 T. Two embryonic stages were studied, the energy shift (e.g., embryonic day 12.5-13, E12.5-13) and close to the birth (E17.5-18). In addition, embryonic brains devoid of MPC1 were studied at E12.5-13. The MRI provided sufficient anatomical contrasts for visualization of embryonic brain. Localized 1 H MRS offered abundant metabolites through the embryonic development from E12.5 and close to the birth, e.g., E17.5 and beyond. The abundant neurochemical information at E12.5 provided metabolic status and processes relating to cellular development at this stage, i.e., the energy shift from glycolysis to oxidative phosphorylation, evidenced by accumulation of lactate in E12.5-13 embryonic brain devoid of MPC1. The further evolution of the neurochemical profile of embryonic brains at E17.5-18 is consistent with cellular and metabolic processes towards the birth. Localized 1 H MRS study of embryonic brain development in utero is feasible, and longitudinal neurochemical profiling of embryonic brains offers valuable insight into early brain development.
Asunto(s)
Química Encefálica , Encéfalo/diagnóstico por imagen , Encéfalo/embriología , Embrión de Mamíferos/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Animales , Estudios de Factibilidad , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival.
Asunto(s)
Proteínas de Transporte de Anión/genética , Ciclo del Ácido Cítrico/genética , Dieta Cetogénica , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Animales , Proteínas de Transporte de Anión/deficiencia , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Genes Letales , Glucosa/metabolismo , Glutamina/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Transportadores de Ácidos Monocarboxílicos , Embarazo , Ácido Pirúvico/metabolismoRESUMEN
Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin-related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress-induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L-OPA1, MFN1, and the mitochondrial inner membrane protein SLP-2. In the absence of SLP-2, L-OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro-survival response against stress.
Asunto(s)
Fibroblastos/fisiología , Proteínas de la Membrana/fisiología , Mitocondrias/fisiología , Estrés Fisiológico , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Dactinomicina/toxicidad , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , GTP Fosfohidrolasas/fisiología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Rayos UltravioletaRESUMEN
BACKGROUND: The mitochondrial pyruvate carrier (MPC) is a protein complex composed of two subunits, MPC1 and MPC2. This carrier is at the interface between glycolysis and mitochondrial metabolism and plays an essential role in hepatic glucose production. METHODS: Here we describe an in vitro screen for small molecule inhibitors of the MPC using a strain of Lactococcus lactis that has been engineered to co-express the two subunits of the human MPC and is able to import exogenous 14C-pyruvate. We then tested the top candidates for potential antidiabetic effects through the repression of gluconeogenesis. RESULTS: By screening the Prestwick compound library of 1'200 drugs approved by the Food and Drug Administration for inhibitors of pyruvate uptake, twelve hit molecules were identified. In a secondary screen, the most potent inhibitors were found to inhibit pyruvate-driven oxygen consumption in mouse C2C12 muscle cells. Assessment of gluconeogenesis showed that Zaprinast, as well as the established MPC inhibitor UK5099, inhibited in vitro and in vivo hepatic glucose production. However, when tested acutely in mice without the administration of gluconeogenic substrates, MPC inhibitors raised blood glucose levels, pointing to liver-independent effects. Furthermore, chronic treatment with Zaprinast failed to correct hyperglycemia in both lean and obese diabetic mouse models. CONCLUSIONS: New MPC inhibitors have been identified, showing inhibitory effects on hepatic glucose production. GENERAL SIGNIFICANCE: For potential antidiabetic applications, MPC inhibitors should target the liver without undesired inhibition of mitochondrial pyruvate metabolism in the skeletal muscles or pancreatic beta-cells in order to avoid dual effects on glycemia.
Asunto(s)
Diabetes Mellitus , Glucosa , Estados Unidos , Humanos , Ratones , Animales , Glucosa/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/farmacología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Hígado/metabolismo , Diabetes Mellitus/metabolismo , Hipoglucemiantes/farmacología , Piruvatos/metabolismo , Piruvatos/farmacologíaRESUMEN
During the early stages of Alzheimer's disease (AD) in both mouse models and human patients, soluble forms of Amyloid-ß 1-42 oligomers (Aß42o) trigger loss of excitatory synapses (synaptotoxicity) in cortical and hippocampal pyramidal neurons (PNs) prior to the formation of insoluble amyloid plaques. In a transgenic AD mouse model, we observed a spatially restricted structural remodeling of mitochondria in the apical tufts of CA1 PNs dendrites corresponding to the dendritic domain where the earliest synaptic loss is detected in vivo. We also observed AMPK over-activation as well as increased fragmentation and loss of mitochondrial biomass in Ngn2-induced neurons derived from a new APPSwe/Swe knockin human ES cell line. We demonstrate that Aß42o-dependent over-activation of the CAMKK2-AMPK kinase dyad mediates synaptic loss through coordinated phosphorylation of MFF-dependent mitochondrial fission and ULK2-dependent mitophagy. Our results uncover a unifying stress-response pathway causally linking Aß42o-dependent structural remodeling of dendritic mitochondria to synaptic loss.
Asunto(s)
Enfermedad de Alzheimer , Mitofagia , Proteínas Quinasas Activadas por AMP/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Dinámicas Mitocondriales , Fragmentos de Péptidos , Sinapsis/metabolismoRESUMEN
Age-related muscle dysfunction and sarcopenia are major causes of physical incapacitation in older adults and currently lack viable treatment strategies. Here we find that sphingolipids accumulate in mouse skeletal muscle upon aging and that both genetic and pharmacological inhibition of sphingolipid synthesis prevent age-related decline in muscle mass while enhancing strength and exercise capacity. Inhibition of sphingolipid synthesis confers increased myogenic potential and promotes protein synthesis. Within the sphingolipid pathway, we show that accumulation of dihydroceramides is the culprit disturbing myofibrillar homeostasis. The relevance of sphingolipid pathways in human aging is demonstrated in two cohorts, the UK Biobank and Helsinki Birth Cohort Study in which gene expression-reducing variants of SPTLC1 and DEGS1 are associated with improved and reduced fitness of older individuals, respectively. These findings identify sphingolipid synthesis inhibition as an attractive therapeutic strategy for age-related sarcopenia and co-occurring pathologies.
Asunto(s)
Sarcopenia , Animales , Ratones , Humanos , Anciano , Sarcopenia/prevención & control , Músculo Esquelético/metabolismo , Esfingolípidos/metabolismo , Estudios de Cohortes , Envejecimiento/genéticaRESUMEN
OBJECTIVES: The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined. METHODS: We studied an isogenic daptomycin-susceptible (DAP(S)) and daptomycin-resistant (DAP(R)) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques. RESULTS: Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAP(R) isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT-PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT-PCR of this same gene cadre from two distinct isogenic DAP(S)/DAP(R) clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAP(R) phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid. CONCLUSIONS: Compatible with previous in vitro observations, in vivo-acquired DAP(R) in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.
Asunto(s)
Antibacterianos/farmacología , Daptomicina/farmacología , Farmacorresistencia Bacteriana , Perfilación de la Expresión Génica , Proteoma/análisis , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Endocarditis Bacteriana/microbiología , Humanos , Análisis por Micromatrices , Hibridación de Ácido Nucleico , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Mitochondrial morphology is regulated by continuous fusion and fission events that are essential for maintaining a normal mitochondrial function. If the last years have witnessed major discoveries in the characterization of the fission and fusion machineries, little is known about the physiological role of mitochondrial dynamics. In this review we report the results showing evidences of relationships between mitochondrial dynamics and cellular metabolism, autophagy or apoptosis. We discuss how different mitochondrial alterations observed in cancer cells could be linked to unbalanced mitochondrial fission or fusion events and how this could impinge on key essential cellular processes, thereby contributing to tumorigenesis.
Asunto(s)
Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/fisiología , Autofagia/fisiología , Ciclo Celular/fisiología , Metabolismo Energético/fisiología , HumanosRESUMEN
Mitochondria are essential organelles of all eukaryotic cells that play a key role in several physiological processes and are involved in the pathology of many diseases. These organelles form a highly dynamic network, which results from continuous fusion and fission processes. Importance of these processes is underlined by inherited human diseases caused by mutations in two mitochondrial pro-fusion genes: Charcot-Marie-Tooth disease, caused by mutations in Mitofusin 2 gene and ADOA due to mutations in OPA1. During apoptosis, the mitochondrial network is disintegrated and the outer mitochondrial membrane permeabilized, which results in the release of several apoptogenic proteins, including cytochrome c. Although modulating mitochondrial fusion and fission machineries has been reported to influence the apoptotic response to various stimuli, it is still unclear whether fission is absolutely required for apoptosis. In this review, we present the latest progress in the field of mitochondrial dynamics with a particular emphasis on its implication in apoptosis and in diseases.
Asunto(s)
Apoptosis , Mitocondrias/metabolismo , Enfermedad de Charcot-Marie-Tooth/metabolismo , GTP Fosfohidrolasas/genética , Humanos , Proteínas de la Membrana/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Modelos Biológicos , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/metabolismoRESUMEN
Throughout the animal kingdom, mitochondria are the only organelles that retain their own genome and the transcription and translation machineries that are all essential for energy harvesting. Mitochondria have developed a complex communication network, allowing them to stay in tune with cellular needs and nuclear transcriptional programs and to alleviate mitochondrial dysfunction. Here, we review recent findings on the wide array of mechanisms that contribute to these mitocellular communication networks, spanning from well-studied messenger molecules to mitonuclear genetic interactions. Based on these observations and developments, we advocate a broad and inclusive view on mitocellular interactions, which can have profound impacts on physiological, pathological, and evolutionary processes.
Asunto(s)
Enfermedad , Mitocondrias/metabolismo , Estrés Fisiológico , Animales , Comunicación Celular , HumanosRESUMEN
AMPK, a conserved sensor of low cellular energy, can either repress or promote tumor growth depending on the context. However, no studies have examined AMPK function in autochthonous genetic mouse models of epithelial cancer. Here, we examine the role of AMPK in murine KrasG12D-mediated non-small-cell lung cancer (NSCLC), a cancer type in humans that harbors frequent inactivating mutations in the LKB1 tumor suppressor-the predominant upstream activating kinase of AMPK and 12 related kinases. Unlike LKB1 deletion, AMPK deletion in KrasG12D lung tumors did not accelerate lung tumor growth. Moreover, deletion of AMPK in KrasG12D p53f/f tumors reduced lung tumor burden. We identified a critical role for AMPK in regulating lysosomal gene expression through the Tfe3 transcription factor, which was required to support NSCLC growth. Thus, AMPK supports the growth of KrasG12D-dependent lung cancer through the induction of lysosomes, highlighting an unrecognized liability of NSCLC.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Línea Celular Tumoral , Mutación con Pérdida de Función , RatonesRESUMEN
The AMP-activated protein kinase (AMPK) is a highly conserved master regulator of metabolism, whose activation has been proposed to be therapeutically beneficial for the treatment of several metabolic diseases, including nonalcoholic fatty liver disease (NAFLD). NAFLD, characterized by excessive accumulation of hepatic lipids, is the most common chronic liver disease and a major risk factor for development of nonalcoholic steatohepatitis, type 2 diabetes, and other metabolic conditions. To assess the therapeutic potential of AMPK activation, we have generated a genetically engineered mouse model, termed iAMPKCA, where AMPK can be inducibly activated in vivo in mice in a spatially and temporally restricted manner. Using this model, we show that liver-specific AMPK activation reprograms lipid metabolism, reduces liver steatosis, decreases expression of inflammation and fibrosis genes, and leads to significant therapeutic benefits in the context of diet-induced obesity. These findings further support AMPK as a target for the prevention and treatment of NAFLD.
Asunto(s)
Proteínas Quinasas Activadas por AMP/uso terapéutico , Dieta Alta en Grasa/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/etiología , Obesidad/etiología , Proteínas Quinasas Activadas por AMP/farmacología , Animales , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/genéticaRESUMEN
Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA-linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Metabolismo Energético , Mitocondrias/fisiología , Dinámicas Mitocondriales , Estrés Fisiológico , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Adenosina Monofosfato/metabolismo , Secuencias de Aminoácidos , Línea Celular Tumoral , Citoplasma/enzimología , Dactinomicina/análogos & derivados , Dactinomicina/farmacología , Dinaminas , Activación Enzimática , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Rotenona/farmacologíaRESUMEN
Calcium enters mitochondria through a dedicated channel referred to as the mitochondrial calcium uniporter (MCU), whose molecular identity has long remained elusive. Since the discovery of the gene encoding the MCU protein two years ago, researchers have awaited the generation of a mouse lacking the MCU. These mice are fully viable and show defects limited to performance of high-energy-demanding exercises. Strikingly, no protection against necrosis is observed following ischaemia-reperfusion in the heart.
Asunto(s)
Canales de Calcio/genética , Calcio/fisiología , Mitocondrias Musculares/metabolismo , Animales , Femenino , MasculinoRESUMEN
The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Proproteína Convertasa 1/metabolismo , Ácido Pirúvico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Transporte Biológico , Vías Biosintéticas , Medios de Cultivo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Leucina/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Datos de Secuencia Molecular , Transportadores de Ácidos Monocarboxílicos , Proproteína Convertasa 1/química , Proproteína Convertasa 1/genética , Proproteína Convertasa 2 , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ácido Tióctico/biosíntesis , Ácido Tióctico/metabolismo , Valina/metabolismoRESUMEN
Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.