RESUMO
Photosystem I (PSI) forms a large macromolecular complex of â¼580 kDa that resides in the thylakoid membrane and mediates photosynthetic electron transfer. PSI is composed of eighteen protein subunits and nearly two hundred co-factors. The assembly of the complex in thylakoid membranes requires high spatial and temporal coordination, and is critically dependent on a sophisticated assembly machinery. Here, we report and characterize CO-EXPRESSED WITH PSI ASSEMBLY1 (CEPA1), a PSI assembly factor in Arabidopsis (Arabidopsis thaliana). The CEPA1 gene was identified bioinformatically as being co-expressed with known PSI assembly factors. Disruption of the CEPA1 gene leads to a pale phenotype and retarded plant development but does not entirely abolish photoautotrophy. Biophysical and biochemical analyses revealed that the phenotype is caused by a specific defect in PSI accumulation. We further show that CEPA1 acts at the post-translational level and co-localizes with PSI in non-appressed thylakoid membranes. In native gels, CEPA1 co-migrates with thylakoid protein complexes, including putative PSI assembly intermediates. Finally, protein-protein interaction assays suggest cooperation of CEPA1 with the PSI assembly factor PHOTOSYSTEM I ASSEMBLY3 PSA3. Together, our data support an important but non-essential role of CEPA1 in PSI assembly.
RESUMO
Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.
Assuntos
Edição de Genes , Genes Mitocondriais , Melhoramento Vegetal , Plantas , Mitocôndrias/genética , Genoma de PlantaRESUMO
In plant cells, translation occurs in three compartments: the cytosol, the plastids and the mitochondria. While the structures of the (prokaryotic-type) ribosomes in plastids and mitochondria are well characterized, high-resolution structures of the eukaryotic 80S ribosomes in the cytosol have been lacking. Here the structure of translating tobacco (Nicotiana tabacum) 80S ribosomes was solved by cryo-electron microscopy with a global resolution of 2.2 Å. The ribosome structure includes two tRNAs, decoded mRNA and the nascent peptide chain, thus providing insights into the molecular underpinnings of the cytosolic translation process in plants. The map displays conserved and plant-specific rRNA modifications and the positions of numerous ionic cofactors, and it uncovers the role of monovalent ions in the decoding centre. The model of the plant 80S ribosome enables broad phylogenetic comparisons that reveal commonalities and differences in the ribosomes of plants and those of other eukaryotes, thus putting our knowledge about eukaryotic translation on a firmer footing.
Assuntos
RNA Ribossômico , Ribossomos , Citosol , RNA Ribossômico/química , Microscopia Crioeletrônica , Filogenia , Modelos Moleculares , Ribossomos/química , Plantas/genética , Nicotiana/genéticaRESUMO
In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of 5 multiprotein complexes (complexes I-V). Analyses of the OXPHOS system by native gel electrophoresis have revealed an organization of OXPHOS complexes into supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mitochondrial ferredoxin-like, mFDX-like). This protein was previously found to be part of the bridge domain linking the matrix and membrane arms of the complex I. Phylogenetic analysis suggested that the Arabidopsis (Arabidopsis thaliana) mFDX-like evolved from classical mitochondrial ferredoxins (mFDXs) but lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster, supposedly essential for the electron transfer function of FDXs. Accordingly, our biochemical study showed that AtmFDX-like does not bind an Fe-S cluster and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9-based strategy. These lines did not show any abnormal phenotype under standard growth conditions. However, the characterization of the OXPHOS system demonstrated that mFDX-like is important for the assembly of complex I and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III2 to form supercomplexes.
Assuntos
Arabidopsis , Ferredoxinas , Ferredoxinas/genética , Ferredoxinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Filogenia , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismoRESUMO
Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component, RAPTOR1B, or inhibition of TOR activity by specific inhibitors, displayed reduced sensitivity to actin cytoskeleton disruptors compared to their controls. Consistently, actin filament dynamics, but not organization, were suppressed in TORC1-impaired cells. Subcellular localization analysis and quantification of ATP concentration demonstrated that RAPTOR1B localized at cytoplasm and mitochondria and that ATP levels were significantly reduced in TORC1-impaired plants. Further pharmacologic experiments showed that the inhibition of mitochondrial functions led to phenotypes mimicking those observed in raptor1b mutants at the level of both plant growth and actin dynamics. Exogenous feeding of adenine could partially restore ATP levels and actin dynamics in TORC1-deficient plants. Thus, these data support an important role for TORC1 in coordinating ATP homeostasis and actin dynamics in plant cells.
Assuntos
Citoesqueleto de Actina , Trifosfato de Adenosina , Proteínas de Arabidopsis , Arabidopsis , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosfatidilinositol 3-Quinases , Citoesqueleto de Actina/metabolismo , Actinas , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologiaRESUMO
One of the key functions of mitochondria is the production of ATP to support cellular metabolism and growth. The last step of mitochondrial ATP synthesis is performed by the oxidative phosphorylation (OXPHOS) system, an ensemble of protein complexes embedded in the inner mitochondrial membrane. In the last 25 yr, many structures of OXPHOS complexes and supercomplexes have been resolved in yeast, mammals, and bacteria. However, structures of plant OXPHOS enzymes only became available very recently. In this review, we highlight the plant-specific features revealed by the recent structures and discuss how they advance our understanding of the function and assembly of plant OXPHOS complexes. We also propose new hypotheses to be tested and discuss older findings to be re-evaluated. Further biochemical and structural work on the plant OXPHOS system will lead to a deeper understanding of plant respiration and its regulation, with significant agricultural, environmental, and societal implications.
Assuntos
Membranas Mitocondriais , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Animais , Mamíferos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.
Assuntos
Genoma Mitocondrial , DNA de Plantas/genética , Genoma de Planta , Mutagênese , Mutação PuntualRESUMO
Patients undergoing lung resection are often active or former smokers who have chronic disease related to tobacco (coronary artery disease, chronic obstructive bronchitis, arteritis of the inferior limbs ). Those co-morbidities increase the operative risk for surgery in which morbi-mortality is relevant. On top of this, we are witnessing an increasing number of non-small cell lung cancers in an aging population that might show signs of frailty. The challenge for the surgeon is to identify early those co-morbid and/or frail patients by using predictive scores like Thoracoscore, mFI (modified frailty index) or MSK-F1 (Memorial Sloan-Kettering Frailty Index). Screening for those high-risk patients implies adapting surgical management through a multidisciplinary approach. That is the objective of co-managment, which allows geriatricians and surgeons to collaborate, or enhanced recovery after surgery which is more accessible to all group ages. The objective of this article is to review the management of co-morbid patients in thoracic surgery, by detailing certain predictive scores available and the multidisciplinary approaches developed to treat the patients screened.
Assuntos
Fragilidade , Cirurgia Torácica , Procedimentos Cirúrgicos Torácicos , Idoso , Fragilidade/complicações , Fragilidade/diagnóstico , Fragilidade/epidemiologia , Humanos , Complicações Pós-Operatórias/epidemiologia , Estudos Retrospectivos , Medição de RiscoRESUMO
The survival benefit derived from sorafenib treatment for patients with hepatocellular carcinoma (HCC) is modest due to acquired resistance. Targeting cancer stem cells (CSC) is a possible way to reverse drug resistance, however, inhibitors that specifically target liver CSCs are limited. In this study, we established two sorafenib-resistant, patient-derived tumor xenografts (PDX) that mimicked development of acquired resistance to sorafenib in patients with HCC. RNA-sequencing analysis of sorafenib-resistant PDXs and their corresponding mock controls identified EPH receptor B2 (EPHB2) as the most significantly upregulated kinase. EPHB2 expression increased stepwise from normal liver tissue to fibrotic liver tissue to HCC tissue and correlated with poor prognosis. Endogenous EPHB2 knockout showed attenuation of tumor development in mice. EPHB2 regulated the traits of liver CSCs; similarly, sorted EPHB2High HCC cells were endowed with enhanced CSC properties when compared with their EPHB2-Low counterparts. Mechanistically, EPHB2 regulated cancer stemness and drug resistance by driving the SRC/AKT/GSK3ß/ß-catenin signaling cascade, and EPHB2 expression was regulated by TCF1 via promoter activation, forming a positive Wnt/ß-catenin feedback loop. Intravenous administration of rAAV-8-shEPHB2 suppressed HCC tumor growth and significantly sensitized HCC cells to sorafenib in an NRAS/AKT-driven HCC immunocompetent mouse model. Targeting a positive feedback loop involving the EPHB2/ß-catenin axis may be a possible therapeutic strategy to combat acquired drug resistance in HCC. SIGNIFICANCE: This study identifies a EPHB2/ß-catenin/TCF1 positive feedback loop that augments cancer stemness and sorafenib resistance in HCC, revealing a targetable axis to combat acquired drug resistance in HCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3229/F1.large.jpg.
Assuntos
Carcinoma Hepatocelular/patologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Receptor EphB2/metabolismo , Sorafenibe/farmacologia , beta Catenina/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Receptor EphB2/genética , Células Tumorais Cultivadas , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genéticaRESUMO
The mitochondrial ATP synthase is producing most of the energy required to support eucaryotic life. It is located in the mitochondrial inner-membrane and couples the dissipation of the proton gradient produced by the electron transfer chain with ATP production. It is composed of two domains, the F1 domain located in the matrix and the FO domain embedded in the inner membrane. The mitochondrial ATP synthase belongs to the F-type ATP synthase family together with bacterial and chloroplastic enzymes. The composition of the mitochondrial ATP synthase is well conserved across species, except in plants where several subunits found in opisthokonts were not identified and additional, plant-specific, subunits were found. The assembly of the F-type ATP synthase has been extensively studied in bacteria, yeast and mammals. The overall assembly pattern is conserved but species-specific steps have been identified. In plant, little is known about the assembly of the mitochondrial ATP synthase. We have mined our previously published complexome profiling dataset in order to identity assembly steps of the ATP synthase in the reference plant Arabidopsis thaliana. Several assembly intermediates were identified and we propose a model for the assembly pathway of the ATP synthase of plant mitochondria. In addition, combining complexome profiling with homology searches, we found that the previously described plant-specific subunits are actually present in other organisms. Overall, our work show that the subunit composition and the assembly pathway of the plant mitochondria ATP synthase are mostly conserved with other mitochondrial enzymes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteoma/análiseRESUMO
Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the ß-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.
Assuntos
Cloroplastos/metabolismo , Mutação da Fase de Leitura/genética , Genes de Plantas , Nicotiana/genética , Oenothera/genética , Proteínas de Plantas/genética , Biossíntese de Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Cloroplastos/ultraestrutura , DNA Complementar/genética , Escherichia coli/metabolismo , Genótipo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Fotossíntese , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , ReproduçãoRESUMO
Plant mitochondrial genomes are renowned for their structural complexity, extreme variation in size and mutation rates, and ability to incorporate foreign DNA. Parasitic flowering plants are no exception, and the close association between parasite and host may even enhance the likelihood of horizontal gene transfer (HGT) between them. Recent studies on mistletoes (Viscum) have revealed that these parasites have lost an exceptional number of mitochondrial genes, including all complex I genes of the respiratory chain. At the same time, an altered respiratory pathway has been demonstrated. Here we review the current understanding of mitochondrial evolution in parasitic plants with a special emphasis on HGT to and from parasite mitochondrial genomes, as well as the uniquely altered mitochondria in Viscum and related plants.
Assuntos
Genoma Mitocondrial , Magnoliopsida/genética , Mitocôndrias/genética , Evolução Molecular , Transferência Genética Horizontal , Variação Genética , FilogeniaRESUMO
BACKGROUND: It is now well documented that moonlight affects the life cycle of invertebrates, birds, reptiles, and mammals. The lunisolar tide is also well-known to alter plant growth and development. However, although plants are known to be very photosensitive, few studies have been undertaken to explore the effect of moonlight on plant physiology. RESULTS: Here for the first time we report a massive transcriptional modification in Coffea arabica genes under full moonlight conditions, particularly at full moon zenith and 3 h later. Among the 3387 deregulated genes found in our study, the main core clock genes were affected. CONCLUSIONS: Moonlight also negatively influenced many genes involved in photosynthesis, chlorophyll biosynthesis and chloroplast machinery at the end of the night, suggesting that the full moon has a negative effect on primary photosynthetic machinery at dawn. Moreover, full moonlight promotes the transcription of major rhythmic redox genes and many heat shock proteins, suggesting that moonlight is perceived as stress. We confirmed this huge impact of weak light (less than 6 lx) on the transcription of circadian clock genes in controlled conditions mimicking full moonlight.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Coffea/fisiologia , Luz , Lua , Coffea/genética , Fotossíntese/genéticaRESUMO
Mitochondria host vital cellular functions, including oxidative phosphorylation and co-factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity-based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage-Dependent Anion Channel 1 to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Organelas/metabolismo , Proteômica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Bases de Dados de Proteínas , Mitocôndrias/genética , Biogênese de Organelas , Organelas/genética , Proteoma/metabolismoRESUMO
The alternative oxidase (AOX) constitutes a nonphosphorylating pathway of electron transport in the mitochondrial respiratory chain that provides flexibility to energy and carbon primary metabolism. Its activity is regulated in vitro by the mitochondrial thioredoxin (TRX) system which reduces conserved cysteines residues of AOX. However, in vivo evidence for redox regulation of the AOX activity is still scarce. In the present study, the redox state, protein levels and in vivo activity of the AOX in parallel to photosynthetic parameters were determined in Arabidopsis knockout mutants lacking mitochondrial trxo1 under moderate (ML) and high light (HL) conditions, known to induce in vivo AOX activity. In addition, 13C- and 14C-labeling experiments together with metabolite profiling were performed to better understand the metabolic coordination between energy and carbon metabolism in the trxo1 mutants. Our results show that the in vivo AOX activity is higher in the trxo1 mutants at ML while the AOX redox state is apparently unaltered. These results suggest that mitochondrial thiol redox systems are responsible for maintaining AOX in its reduced form rather than regulating its activity in vivo. Moreover, the negative regulation of the tricarboxylic acid cycle by the TRX system is coordinated with the increased input of electrons into the AOX pathway. Under HL conditions, while AOX and photosynthesis displayed similar patterns in the mutants, photorespiration is restricted at the level of glycine decarboxylation most likely as a consequence of redox imbalance.
Assuntos
Arabidopsis/metabolismo , Carbono/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Oxirredução , Oxirredutases/genética , Fotossíntese/genética , Fotossíntese/fisiologia , Proteínas de Plantas/genéticaRESUMO
Retrograde signals emanate from the DNA-containing cell organelles (plastids and mitochondria) and control the expression of a large number of nuclear genes in response to environmental and developmental cues. Previous studies on retrograde signaling have mainly analyzed the regulation of nuclear gene expression at the transcript level. To determine the contribution of translational and posttranslational regulation to plastid retrograde signaling, we combined label-free proteomics with transcriptomic analysis of Arabidopsis (Arabidopsis thaliana) seedlings and studied their response to interference with the plastid gene expression pathway of retrograde signaling. By comparing the proteomes of the genomes uncoupled1 (gun1) and gun5 mutants with the wild type, we show that GUN1 is critical in the maintenance of plastid protein homeostasis (proteostasis) when plastid translation is blocked. Combining transcriptomic and proteomic analyses of the wild type and gun1, we identified 181 highly translationally or posttranslationally regulated (HiToP) genes. We demonstrate that HiToP photosynthesis-associated nuclear genes (PhANGs) are largely regulated by translational repression, while HiToP ribosomal protein genes are regulated posttranslationally, likely at the level of protein stability without the involvement of GUN1. Our findings suggest distinct posttranscriptional control mechanisms of nuclear gene expression in response to plastid-derived retrograde signals. They also reveal a role for GUN1 in the translational regulation of several PhANGs and highlight extensive posttranslational regulation that does not necessitate GUN1. This study advances our understanding of the molecular mechanisms underlying intracellular communication and provides new insight into cellular responses to impaired plastid protein biosynthesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastídeos/metabolismo , Plântula/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Fotossíntese/genética , Fotossíntese/fisiologia , Plântula/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Communication between organelles and the nucleus is essential for fitness and survival. Retrograde signals are cues emitted from the organelles to regulate nuclear gene expression. GENOMES UNCOUPLED1 (GUN1), a protein of unknown function, has emerged as a central integrator, participating in multiple retrograde signalling pathways that collectively regulate the nuclear transcriptome. Here, we show that GUN1 regulates chloroplast protein import through interaction with the import-related chaperone cpHSC70-1. We demonstrated that overaccumulation of unimported precursor proteins (preproteins) in the cytosol causes a GUN phenotype in the wild-type background and enhances the GUN phenotype of the gun1 mutant. Furthermore, we identified the cytosolic HSP90 chaperone complex, induced by overaccumulated preproteins, as a central regulator of photosynthetic gene expression that determines the expression of the GUN phenotype. Taken together, our results suggest a model in which protein import capacity, folding stress and the cytosolic HSP90 complex control retrograde communication.
Assuntos
Proteínas de Arabidopsis/fisiologia , Proteínas de Ligação a DNA/fisiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transdução de Sinais/fisiologia , TranscriptomaRESUMO
Plant mitochondria play a major role during respiration by producing the ATP required for metabolism and growth. ATP is produced during oxidative phosphorylation (OXPHOS), a metabolic pathway coupling electron transfer with ADP phosphorylation via the formation and release of a proton gradient across the inner mitochondrial membrane. The OXPHOS system is composed of large, multiprotein complexes coordinating metal-containing cofactors for the transfer of electrons. In this review, we summarize the current state of knowledge about assembly of the OXPHOS complexes in land plants. We present the different steps involved in the formation of functional complexes and the regulatory mechanisms controlling the assembly pathways. Because several assembly steps have been found to be ancestral in plants-compared with those described in fungal and animal models-we discuss the evolutionary dynamics that lead to the conservation of ancestral pathways in land plant mitochondria.
Assuntos
Embriófitas , Fosforilação Oxidativa , Animais , Respiração Celular , Transporte de Elétrons , MitocôndriasRESUMO
All present-day mitochondria originate from a single endosymbiotic event that gave rise to the last eukaryotic common ancestor more than a billion years ago. However, to date, many aspects of mitochondrial evolution have remained unresolved. Comparative genomics and proteomics have revealed a complex evolutionary origin for many mitochondrial components. To understand the evolution of the respiratory chain, we have examined both the components and the mechanisms of the assembly pathway of complex I. Complex I represents the first enzyme in the respiratory chain, and complex I deficiencies have dramatic consequences in both animals and plants. The complex is located in the mitochondrial inner membrane and possesses two arms: one embedded in the inner membrane and one protruding in the matrix. Here, we describe the assembly pathway of complex I in the model plant Arabidopsis thaliana. Using a proteomics approach called complexome profiling, we have resolved the different steps in the assembly process in plants. We propose a model for the stepwise assembly of complex I, including every subunit. We then compare this pathway with the corresponding pathway in humans and find that complex I assembly in plants follows a different, and likely ancestral, pathway compared with the one in humans. We show that the main evolutionary changes in complex I structure and assembly in humans occurred at the level of the membrane arm, whereas the matrix arm remained rather conserved.
Assuntos
Arabidopsis/enzimologia , Complexo I de Transporte de Elétrons/metabolismo , Arabidopsis/genética , Transporte de Elétrons , Complexo I de Transporte de Elétrons/genética , Evolução Molecular , Mitocôndrias/metabolismo , ProteômicaRESUMO
Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein-Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein-small molecule and of protein-protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein-protein (PPIs) and protein-metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr-Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.