RESUMEN
In the green alga Chlamydomonas (Chlamydomonas r einhardtii), chloroplast gene expression is tightly regulated posttranscriptionally by gene-specific trans-acting protein factors. Here, we report the identification of the octotricopeptide repeat protein MTHI1, which is critical for the biogenesis of chloroplast ATP synthase oligomycin-sensitive chloroplast coupling factor. Unlike most trans-acting factors characterized so far in Chlamydomonas, which control the expression of a single gene, MTHI1 targets two distinct transcripts: it is required for the accumulation and translation of atpH mRNA, encoding a subunit of the selective proton channel, but it also enhances the translation of atpI mRNA, which encodes the other subunit of the channel. MTHI1 targets the 5' untranslated regions of both the atpH and atpI genes. Coimmunoprecipitation and small RNA sequencing revealed that MTHI1 binds specifically a sequence highly conserved among Chlorophyceae and the Ulvale clade of Ulvophyceae at the 5' end of triphosphorylated atpH mRNA. A very similar sequence, located â¼60 nucleotides upstream of the atpI initiation codon, was also found in some Chlorophyceae and Ulvale algae species and is essential for atpI mRNA translation in Chlamydomonas. Such a dual-targeted trans-acting factor provides a means to coregulate the expression of the two proton hemi-channels.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , ATPasas de Translocación de Protón de Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Subunidades de Proteína/genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Secuencia de Bases , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Genes Reporteros , Prueba de Complementación Genética , Mutación/genética , Fenotipo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Subunidades de Proteína/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In Chlamydomonas reinhardtii, regulation of chloroplast gene expression is mainly post-transcriptional. It requires nucleus-encoded trans-acting protein factors for maturation/stabilization (M factors) or translation (T factors) of specific target mRNAs. We used long- and small-RNA sequencing to generate a detailed map of the transcriptome. Clusters of sRNAs marked the 5' end of all mature mRNAs. Their absence in M-factor mutants reflects the protection of transcript 5' end by the cognate factor. Enzymatic removal of 5'-triphosphates allowed identifying those cosRNA that mark a transcription start site. We detected another class of sRNAs derived from low abundance transcripts, antisense to mRNAs. The formation of antisense sRNAs required the presence of the complementary mRNA and was stimulated when translation was inhibited by chloramphenicol or lincomycin. We propose that they derive from degradation of double-stranded RNAs generated by pairing of antisense and sense transcripts, a process normally hindered by the traveling of the ribosomes. In addition, chloramphenicol treatment, by freezing ribosomes on the mRNA, caused the accumulation of 32-34 nt ribosome-protected fragments. Using this 'in vivo ribosome footprinting', we identified the function and molecular target of two candidate trans-acting factors.
Asunto(s)
Chlamydomonas reinhardtii/genética , ARN del Cloroplasto/metabolismo , ARN Pequeño no Traducido/metabolismo , Transcriptoma , Chlamydomonas reinhardtii/crecimiento & desarrollo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Perfilación de la Expresión Génica , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Procesos Fototróficos , Proteínas de Plantas/metabolismo , Biosíntesis de Proteínas , ARN sin Sentido/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Análisis de Secuencia de ARN , Transcripción Genética/efectos de los fármacosRESUMEN
KEY MESSAGE: Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5' exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation. RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5' end maturation is thought to be achieved by the combined action of a 5' exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5' exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5' exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5' exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Cloroplastos/enzimología , Exorribonucleasas/metabolismo , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Exorribonucleasas/genética , ARN del Cloroplasto/genética , ARN del Cloroplasto/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/genética , Homología de Secuencia de AminoácidoRESUMEN
The biogenesis of the photosynthetic electron transfer chain in the thylakoid membranes requires the concerted expression of genes in the chloroplast and the nucleus. Chloroplast gene expression is subjected to anterograde control by a battery of nucleus-encoded proteins that are imported in the chloroplast, where they mostly intervene at posttranscriptional steps. Using a new genetic screen, we identify a nuclear mutant that is required for expression of the PsaA subunit of photosystem I (PSI) in the chloroplast of Chlamydomonas reinhardtii. This mutant is affected in the stability and translation of psaA messenger RNA. The corresponding gene, TRANSLATION OF psaA1 (TAA1), encodes a large protein with two domains that are thought to mediate RNA binding: an array of octatricopeptide repeats (OPR) and an RNA-binding domain abundant in apicomplexans (RAP) domain. We show that as expected for its function, TAA1 is localized in the chloroplast. It was previously shown that when mixotrophic cultures of C. reinhardtii (which use both photosynthesis and mitochondrial respiration for growth) are shifted to conditions of iron limitation, there is a strong decrease in the accumulation of PSI and that this is rapidly reversed when iron is resupplied. Under these conditions, TAA1 protein is also down-regulated through a posttranscriptional mechanism and rapidly reaccumulates when iron is restored. These observations reveal a concerted regulation of PSI and of TAA1 in response to iron availability.
Asunto(s)
Chlamydomonas reinhardtii/genética , Proteínas de Cloroplastos/metabolismo , Hierro/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Complejo de Proteína del Fotosistema I/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tilacoides/metabolismoRESUMEN
as1, for antenna size mutant 1, was obtained by insertion mutagenesis of the unicellular green alga Chlamydomonas reinhardtii. This strain has a low chlorophyll content, 8% with respect to the wild type, and displays a general reduction in thylakoid polypeptides. The mutant was found to carry an insertion into a homologous gene, prokaryotic arsenite transporter (ARSA), whose yeast and mammal counterparts were found to be involved in the targeting of tail-anchored (TA) proteins to cytosol-exposed membranes, essential for several cellular functions. Here we present the characterization in a photosynthetic organism of an insertion mutant in an ARSA-homolog gene. The ARSA1 protein was found to be localized in the cytosol, and yet its absence in as1 leads to a small chloroplast and a strongly decreased chlorophyll content per cell. ARSA1 appears to be required for optimal biogenesis of photosynthetic complexes because of its involvement in the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon (TOC) complex, which, in turn, catalyzes the import of nucleus-encoded precursor polypeptides into the chloroplast. Remarkably, the effect of the mutation appears to be restricted to biogenesis of chlorophyll-binding polypeptides and is not compensated by the other ARSA homolog encoded by the C. reinhardtii genome, implying a non-redundant function.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/genética , Arsenitos/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crecimiento & desarrollo , Chlamydomonas reinhardtii/ultraestructura , Clorofila/metabolismo , Mapeo Cromosómico , Citosol/metabolismo , Microscopía Electrónica de Transmisión , Mutagénesis Insercional , Mutación , Fotosíntesis , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes , Análisis de Secuencia de ADNRESUMEN
We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ' untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ' >3 ' degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.
Asunto(s)
Proteínas Algáceas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Chlamydomonas reinhardtii/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Regiones no Traducidas 5' , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Secuencia de Bases , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Fotosíntesis , Filogenia , Estabilidad del ARN , ARN de Algas/metabolismo , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Ribulosa-Bifosfato Carboxilasa/genéticaRESUMEN
After endosymbiosis, organelles lost most of their initial genome. Moreover, expression of the few remaining genes became tightly controlled by the nucleus through trans-acting protein factors that are required for post-transcriptional expression (maturation/stability or translation) of a single (or a few) specific organelle target mRNA(s). Here, we characterize the nucleus-encoded TDA1 factor, which is specifically required for translation of the chloroplast atpA transcript that encodes subunit α of ATP synthase in Chlamydomonas reinhardtii. The sequence of TDA1 contains eight copies of a degenerate 38-residue motif, that we named octotrico peptide repeat (OPR), which has been previously described in a few other trans-acting factors targeted to the C. reinhardtii chloroplast. Interestingly, a proportion of the untranslated atpA transcripts are sequestered into high-density, non-polysomic, ribonucleoprotein complexes. Our results suggest that TDA1 has a dual function: (i) trapping a subset of untranslated atpA transcripts into non-polysomic complexes, and (ii) translational activation of these transcripts. We discuss these results in light of our previous observation that only a proportion of atpA transcripts are translated at any given time in the chloroplast of C. reinhardtii.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , ATPasas de Translocación de Protón de Cloroplastos/genética , Cloroplastos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones no Traducidas 5' , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Núcleo Celular/genética , Chlamydomonas reinhardtii/genética , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Cloroplastos/genética , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Ribonucleoproteínas/metabolismoRESUMEN
Microalgae are prominent aquatic organisms, responsible for about half of the photosynthetic activity on Earth. Over the past two decades, breakthroughs in genomics and ecosystem biology, as well as the development of genetic resources in model species, have redrawn the boundaries of our knowledge on the relevance of these microbes in global ecosystems. However, considering their vast biodiversity and complex evolutionary history, our comprehension of algal biology remains limited. As algae rely on light, both as their main source of energy and for information about their environment, we focus here on photosynthesis, photoperception, and chloroplast biogenesis in the green alga Chlamydomonas reinhardtii and marine diatoms. We describe how the studies of light-driven processes are key to assessing functional biodiversity in evolutionary distant microalgae. We also emphasize that integration of laboratory and environmental studies, and dialogues between different scientific communities are both timely and essential to understand the life of phototrophs in complex ecosystems and to properly assess the consequences of environmental changes on aquatic environments globally.
Les microalgues, organismes aquatiques majeurs, sont responsables de la moitié de l'activité photosynthétique planétaire. La lumière représente pour les microalgues une source d'énergie ainsi que d'informations sur leur environnement. Ces 20 dernières années, les progrès en génomique et biologie des écosystèmes et la disponibilité de ressources génétiques pour de nouvelles espèces modèles ont permis d'apprécier leur importance dans les écosystèmes globaux. Néanmoins, du fait de leur grande diversité et de leur histoire évolutive complexe, notre compréhension de la biologie des microalgues reste limitée. Nous nous concentrons ici sur la photosynthèse, la photoperception, et la biogenèse des plastes chez l'algue verte Chlamydomonas reinhardtii et les diatomées marines. Nous décrivons comment l'étude des processus gouvernés par la lumière ouvre de nouvelles perspectives pour l'étude de la biodiversité fonctionnelle des microalgues. Nous soulignons combien seule l'intégration d'études en laboratoire et en contexte environnemental et le dialogue entre les communautés scientifiques concernées permettront de comprendre la vie de ces phototrophes dans des écosystèmes complexes, et d'évaluer correctement les conséquences des changements environnementaux sur les milieux aquatiques.
Asunto(s)
Chlamydomonas reinhardtii , Microalgas , Ecosistema , Fotosíntesis , Biodiversidad , Chlamydomonas reinhardtii/genéticaRESUMEN
Recent progress in understanding the Q-cycle mechanism of the bc(1) complex is reviewed. The data strongly support a mechanism in which the Q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe-2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q(o)-site, and the reduced iron-sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c(1) and liberate the H(+). When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O(2) is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b(L) to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b(L) to enhance the rate constant. The acceptor reactions at the Q(i)-site are still controversial, but likely involve a "two-electron gate" in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b(150) phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed. The mechanism discussed is applicable to a monomeric bc(1) complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b(L) hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.
Asunto(s)
Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/metabolismo , Sitios de Unión , Dimerización , Homeostasis , Cinética , Modelos Moleculares , Oxidación-Reducción , Conformación ProteicaRESUMEN
We recently characterized a novel heme biogenesis pathway required for heme c(i)' covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme c(i)' binding.
Asunto(s)
Proteínas Algáceas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cloroplastos/enzimología , Complejo de Citocromo b6f/metabolismo , Citocromos c/genética , Proteínas Algáceas/genética , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Chlamydomonas/enzimología , Chlamydomonas/genética , Cloroplastos/genética , Complejo de Citocromo b6f/genética , Citocromos c/metabolismo , Hemo/genética , Hemo/metabolismo , Mutagénesis Insercional , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
A salient feature of organelle gene expression is the requirement for nucleus-encoded factors that act posttranscriptionally in a gene-specific manner. A central issue is to understand whether these factors are merely constitutive or have a regulatory function. In the unicellular alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene-encoding cytochrome f, a major subunit of the cytochrome b(6)f complex, depends on two specific nucleus-encoded factors: MCA1, required for stable accumulation of the petA transcript, and TCA1, required for its translation. We cloned the TCA1 gene, encoding a pioneer protein, and transformed appropriate mutant strains with tagged versions of MCA1 and TCA1. In transformed strains expressing decreasing amounts of MCA1 or TCA1, the concentration of these factors proved limiting for petA mRNA accumulation and cytochrome f translation, respectively. This observation suggests that in exponentially growing cells, the abundance of MCA1 sets the pool of petA transcripts, some of which are TCA1-selected for an assembly-dependent translation of cytochrome f. We show that MCA1 is a short-lived protein. Its abundance varies rapidly with physiological conditions that deeply affect expression of the petA gene in vivo, for instance in aging cultures or upon changes in nitrogen availability. We observed similar but more limited changes in the abundance of TCA1. We conclude that in conditions where de novo biogenesis of cytochrome b(6)f complexes is not required, a rapid drop in MCA1 exhausts the pool of petA transcripts, and the progressive loss of TCA1 further prevents translation of cytochrome f.
Asunto(s)
Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Biosíntesis de Proteínas/genética , Estabilidad del ARN/genética , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Animales , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Citocromos f/genética , Citocromos f/metabolismo , Regulación hacia Abajo , Vectores Genéticos/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Nitrógeno/farmacología , ARN Mensajero/genética , Sensibilidad y EspecificidadRESUMEN
Oxygenic photosynthesis is an important bioenergetic process that maintains the Earth's atmosphere and allows carbon fixation. A critical enzyme in this process, the cytochrome b(6)f complex, differs from other protein complexes of the same family by an unusual covalently attached cofactor chemically defined as a c' heme. We have identified a set of pioneer proteins that carry the biogenesis of this c' heme and started their characterization. They are encoded by the genomes of all organisms performing oxygenic photosynthesis, whatever their phylogenetic distances. These proteins are thus among the few that distinguish photosynthetic cells evolving oxygen from other types of living cells.
Asunto(s)
Proteínas Algáceas/genética , Chlamydomonas reinhardtii/fisiología , Complejo de Citocromo b6f/metabolismo , Hemo/metabolismo , Complejos Multiproteicos/biosíntesis , Fotosíntesis/fisiología , Filogenia , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Prueba de Complementación Genética , Funciones de Verosimilitud , Mediciones Luminiscentes , Modelos Genéticos , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Análisis de Secuencia de ADNRESUMEN
Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.