RESUMEN
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.
RESUMEN
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.
Asunto(s)
Citoesqueleto de Actina , Adenosina Trifosfato , Proteínas de Arabidopsis , Arabidopsis , Diana Mecanicista del Complejo 1 de la Rapamicina , Fosfatidilinositol 3-Quinasas , Citoesqueleto de Actina/metabolismo , Actinas , Adenosina Trifosfato/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/fisiologíaRESUMEN
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.
Asunto(s)
Arabidopsis , Ferredoxinas , Ferredoxinas/genética , Ferredoxinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Filogenia , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismoRESUMEN
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.
Asunto(s)
Cloroplastos/metabolismo , Mutación del Sistema de Lectura/genética , Genes de Plantas , Nicotiana/genética , Oenothera/genética , Proteínas de Plantas/genética , Biosíntesis de Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cloroplastos/ultraestructura , ADN Complementario/genética , Escherichia coli/metabolismo , Genotipo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Péptidos/química , Péptidos/metabolismo , Fenotipo , Fotosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , ReproducciónRESUMEN
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.
Asunto(s)
Membranas Mitocondriales , Fosforilación Oxidativa , Adenosina Trifosfato/metabolismo , Animales , Mamíferos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Early stages of speciation in plants might involve genetic incompatibilities between plastid and nuclear genomes, leading to inter-lineage hybrid breakdown due to the disruption between co-adapted plastid and nuclear genes encoding subunits of the same plastid protein complexes. We tested this hypothesis in Silene nutans, a gynodioecious Caryophyllaceae, where four distinct genetic lineages exhibited strong reproductive isolation among each other, resulting in chlorotic or variegated hybrids. By sequencing the whole gene content of the four plastomes through gene capture, and a large part of the nuclear genes encoding plastid subunits from RNAseq data, we searched for non-synonymous substitutions fixed in each lineage on both genomes. Lineages of S. nutans exhibited a high level of dN/dS ratios for plastid and nuclear genes encoding most plastid complexes, with a strong pattern of coevolution for genes encoding the subunits of ribosome and cytochrome b6/f that could explain the chlorosis of hybrids. Overall, relaxation of selection due to past bottlenecks and positive selection have driven the diversity pattern observed in S. nutans plastid complexes, leading to plastid-nuclear incompatibilities. We discuss the possible role of gynodioecy in the evolutionary dynamics of the plastomes through linked selection.
Asunto(s)
Caryophyllaceae , Genoma de Plastidios , Silene , Caryophyllaceae/genética , Evolución Molecular , Filogenia , Plastidios/genética , Aislamiento Reproductivo , Silene/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocondrias/metabolismo , Orgánulos/metabolismo , Proteómica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Bases de Datos de Proteínas , Mitocondrias/genética , Biogénesis de Organelos , Orgánulos/genética , Proteoma/metabolismoRESUMEN
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.
Asunto(s)
Arabidopsis/enzimología , Complejo I de Transporte de Electrón/metabolismo , Arabidopsis/genética , Transporte de Electrón , Complejo I de Transporte de Electrón/genética , Evolución Molecular , Mitocondrias/metabolismo , ProteómicaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastidios/metabolismo , Plantones/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Fotosíntesis/genética , Fotosíntesis/fisiología , Plantones/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatografía en Gel/métodos , Metaboloma , Proteoma/metabolismo , Proteómica/métodos , Programas Informáticos , Unión Proteica , Proteoma/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Carbono/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Oxidación-Reducción , Oxidorreductasas/genética , Fotosíntesis/genética , Fotosíntesis/fisiología , Proteínas de Plantas/genéticaRESUMEN
The exchange of signals between cellular compartments coordinates development and differentiation, modulates metabolic pathways, and triggers responses to environmental conditions. The proposed central regulator of plastid-to-nucleus retrograde signaling, GENOMES UNCOUPLED1 (GUN1), is present at very low levels, which has hampered the discovery of its precise molecular function. Here, we show that the Arabidopsis (Arabidopsis thaliana) GUN1 protein accumulates to detectable levels only at very early stages of leaf development, where it functions in the regulation of chloroplast biogenesis. GUN1 mRNA is present at high levels in all tissues, but GUN1 protein undergoes rapid degradation (with an estimated half-life of â¼4 h) in all tissues where chloroplast biogenesis has been completed. The rapid turnover of GUN1 is controlled mainly by the chaperone ClpC1, suggesting degradation of GUN1 by the Clp protease. Degradation of GUN1 slows under stress conditions that alter retrograde signaling, thus ensuring that the plant has sufficient GUN1 protein. We also find that the pentatricopeptide repeat motifs of GUN1 are important determinants of GUN1 stability. Moreover, overexpression of GUN1 causes an early flowering phenotype, suggesting a function of GUN1 in developmental phase transitions beyond chloroplast biogenesis. Taken together, our results provide new insight into the regulation of GUN1 by proteolytic degradation, uncover its function in early chloroplast biogenesis, and suggest a role in developmental phase transitions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas de Unión al ADN/genética , Flores/genética , Regulación de la Expresión Génica de las Plantas , Semivida , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plastidios/genética , Plastidios/metabolismo , Biosíntesis de Proteínas , Estabilidad Proteica , Transducción de SeñalRESUMEN
While mitochondrial mutants of the respiratory machinery are rare and often lethal, cytoplasmic male sterility (CMS), a mitochondrially inherited trait that results in pollen abortion, is frequently encountered in wild populations. It generates a breeding system called gynodioecy. In Beta vulgaris ssp. maritima, a gynodioecious species, we found CMS-G to be widespread across the distribution range of the species. Despite the sequencing of the mitochondrial genome of CMS-G, the mitochondrial sterilizing factor causing CMS-G is still unknown. By characterizing biochemically CMS-G, we found that the expression of several mitochondrial proteins is altered in CMS-G plants. In particular, Cox1, a core subunit of the cytochrome c oxidase (complex IV), is larger but can still assemble into complex IV. However, the CMS-G-specific complex IV was only detected as a stabilized dimer. We did not observe any alteration of the affinity of complex IV for cytochrome c; however, in CMS-G, complex IV capacity is reduced. Our results show that CMS-G is maintained in many natural populations despite being associated with an atypical complex IV. We suggest that the modified complex IV could incur the associated cost predicted by theoretical models to maintain gynodioecy in wild populations.
Asunto(s)
Beta vulgaris/genética , Citoplasma/genética , Complejo IV de Transporte de Electrones/genética , Infertilidad Vegetal/genética , Beta vulgaris/crecimiento & desarrollo , Genoma Mitocondrial/genética , Mitocondrias/enzimología , Mitocondrias/genética , Mutación , Polen/genéticaRESUMEN
Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co-factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF-deficient mutant plants, and we show that re-initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.
Asunto(s)
Nicotiana/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/metabolismo , Fotosíntesis/genética , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema I/genética , Proteínas de Plantas/genética , Tilacoides/metabolismo , Nicotiana/genéticaRESUMEN
Recent advances in gene function prediction rely on ensemble approaches that integrate results from multiple inference methods to produce superior predictions. Yet, these developments remain largely unexplored in plants. We have explored and compared two methods to integrate 10 gene co-function networks for Arabidopsis thaliana and demonstrate how the integration of these networks produces more accurate gene function predictions for a larger fraction of genes with unknown function. These predictions were used to identify genes involved in mitochondrial complex I formation, and for five of them, we confirmed the predictions experimentally. The ensemble predictions are provided as a user-friendly online database, EnsembleNet. The methods presented here demonstrate that ensemble gene function prediction is a powerful method to boost prediction performance, whereas the EnsembleNet database provides a cutting-edge community tool to guide experimentalists.
Asunto(s)
Arabidopsis/genética , Bases de Datos Genéticas , Complejo I de Transporte de Electrón/genética , Genes de Plantas , Programas Informáticos , Benchmarking , Ontología de Genes , Redes Reguladoras de Genes , Mutación/genéticaRESUMEN
RNA editing in plastids and mitochondria of flowering plants requires pentatricopeptide repeat proteins (PPR proteins) for site recognition and proteins of the multiple organellar RNA editing factor (MORF) family as cofactors. Two MORF proteins, MORF5 and MORF8, are dual-targeted to plastids and mitochondria; two are targeted to plastids, and five are targeted to mitochondria. Pulldown assays from Arabidopsis thaliana tissue culture extracts with the mitochondrial MORF1 and the plastid MORF2 proteins, respectively, both identify the dual-targeted MORF8 protein, showing that these complexes can assemble in the organelles. We have now determined the scope of potential interactions between the various MORF proteins by yeast two-hybrid, in vitro pulldown, and bimolecular fluorescence complementation assays. The resulting MORF-MORF interactome identifies specific heteromeric MORF protein interactions in plastids and in mitochondria. Heteromers are observed for MORF protein combinations affecting a common site, suggesting their functional relevance. Most MORF proteins also undergo homomeric interactions. Submolecular analysis of the MORF1 protein reveals that the MORF-MORF protein connections require the C-terminal region of the central conserved MORF box. This domain has no similarity to known protein modules and may form a novel surface for protein-protein interactions.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Mapas de Interacción de Proteínas , Estructura Terciaria de Proteína , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mitocondrias/química , Mitocondrias/genética , Mitocondrias/metabolismo , Plastidios/química , Plastidios/genética , Plastidios/metabolismo , Unión Proteica , Multimerización de Proteína , Edición de ARN/genéticaRESUMEN
L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.
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Arabidopsis/enzimología , Ácido Ascórbico/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Arabidopsis/genética , Transporte de Electrón , Complejo I de Transporte de Electrón/genética , Membranas Intracelulares/enzimología , Mitocondrias/enzimología , Modelos Biológicos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genéticaRESUMEN
Complex I (NADH:ubiquinone oxidoreductase) is central to cellular NAD(+) recycling and accounts for approximately 40% of mitochondrial ATP production. To understand how complex I function impacts respiration and plant development, we isolated Arabidopsis (Arabidopsis thaliana) lines that lack complex I activity due to the absence of the catalytic subunit NDUFV1 (for NADH:ubiquinone oxidoreductase flavoprotein1) and compared these plants with ndufs4 (for NADH:ubiquinone oxidoreductase Fe-S protein4) mutants possessing trace amounts of complex I. Unlike ndufs4 plants, ndufv1 lines were largely unable to establish seedlings in the absence of externally supplied sucrose. Measurements of mitochondrial respiration and ATP synthesis revealed that compared with ndufv1, the complex I amounts retained by ndufs4 did not increase mitochondrial respiration and oxidative phosphorylation capacities. No major differences were seen in the mitochondrial proteomes, cellular metabolomes, or transcriptomes between ndufv1 and ndufs4. The analysis of fluxes through the respiratory pathway revealed that in ndufv1, fluxes through glycolysis and the tricarboxylic acid cycle were dramatically increased compared with ndufs4, which showed near wild-type-like fluxes. This indicates that the strong growth defects seen for plants lacking complex I originate from a switch in the metabolic mode of mitochondria and an up-regulation of respiratory fluxes. Partial reversion of these phenotypes when traces of active complex I are present suggests that complex I is essential for plant development and likely acts as a negative regulator of respiratory fluxes.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Complejo I de Transporte de Electrón/genética , Mitocondrias/genética , Mutación , Adenosina Trifosfato/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/metabolismo , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Mitocondrias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Consumo de Oxígeno , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Regulación hacia ArribaRESUMEN
Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions.Mitochondria have emerged as an important organelle for sensing and coping with stress in addition to being the sites of important metabolic pathways. Here, responses to moderate light and drought stress were examined in different Arabidopsis (Arabidopsis thaliana) mutant plants lacking a functional alternative oxidase (alternative oxidase1a [aox1a]), those with reduced cytochrome electron transport chain capacity (T3/T7 bacteriophage-type RNA polymerase, mitochondrial, and plastidial [rpoTmp]), and double mutants impaired in both pathways (aox1a:rpoTmp). Under conditions considered optimal for growth, transcriptomes of aox1a and rpoTmp were distinct. Under adverse growth conditions, however, transcriptome changes in aox1a and rpoTmp displayed a highly significant overlap and were indicative of a common mitochondrial stress response and down-regulation of photosynthesis. This suggests that the role of mitochondria to support photosynthesis is provided through either the alternative pathway or the cytochrome pathway, and when either pathway is inhibited, such as under environmental stress, a common, dramatic, and succinct mitochondrial signal is activated to alter energy metabolism in both organelles. aox1a:rpoTmp double mutants grown under optimal conditions showed dramatic reductions in biomass production compared with aox1a and rpoTmp and a transcriptome that was distinct from aox1a or rpoTmp. Transcript data indicating activation of mitochondrial biogenesis in aox1a:rpoTmp were supported by a proteomic analysis of over 200 proteins. Under optimal conditions, aox1a:rpoTmp plants seemed to switch on many of the typical mitochondrial stress regulators. Under adverse conditions, aox1a:rpoTmp turned off these responses and displayed a biotic stress response. Taken together, these results highlight the diverse signaling pathways activated by the perturbation of mitochondrial function under different growth conditions.
Asunto(s)
Arabidopsis/metabolismo , Citocromos/fisiología , Transporte de Electrón/fisiología , Fenómenos Fisiológicos de las Plantas , Arabidopsis/fisiología , Respiración de la Célula/fisiología , Deshidratación/metabolismo , Perfilación de la Expresión Génica , Luz , Mitocondrias/metabolismo , Mitocondrias/fisiología , Transducción de Señal/fisiología , Estrés Fisiológico/fisiologíaRESUMEN
The assembly of respiratory complexes is a multistep process, requiring coordinate expression of mitochondrial and nuclear genes and cofactor biosynthesis. We functionally characterized the iron-sulfur protein required for NADH dehydrogenase (INDH) in the model plant Arabidopsis thaliana. An indh knockout mutant lacked complex I but had low levels of a 650-kD assembly intermediate, similar to mutations in the homologous NUBPL (nucleotide binding protein-like) in Homo sapiens. However, heterozygous indh/+ mutants displayed unusual phenotypes during gametogenesis and resembled mutants in mitochondrial translation more than mutants in complex I. Gradually increased expression of INDH in indh knockout plants revealed a significant delay in reassembly of complex I, suggesting an indirect role for INDH in the assembly process. Depletion of INDH protein was associated with decreased (35)S-Met labeling of translation products in isolated mitochondria, whereas the steady state levels of several mitochondrial transcripts were increased. Mitochondrially encoded proteins were differentially affected, with near normal levels of cytochrome c oxidase subunit2 and Nad7 but little Nad6 protein in the indh mutant. These data suggest that INDH has a primary role in mitochondrial translation that underlies its role in complex I assembly.