RESUMEN
The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.
Asunto(s)
Cloroplastos/genética , Proteínas de Plantas/genética , Empalme del ARN , Setaria (Planta)/genética , Zea mays/genética , Proteínas de Cloroplastos/genética , Intrones , Mutación , Proteínas de Plantas/metabolismo , Biosíntesis de Proteínas , Dominios Proteicos , ARN de TransferenciaRESUMEN
In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Complejos Multiproteicos/genética , Proteínas de Plantas/genética , Compartimento Celular , Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Complejos Multiproteicos/metabolismo , Proteínas de Plantas/metabolismo , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Chloroplasts import thousands of nucleus-encoded preproteins synthesized in the cytosol through the TOC and TIC translocons on the outer and inner envelope membranes, respectively. Preprotein translocation across the inner membrane requires ATP; however, the import motor has remained unclear. Here, we report that a 2-MD heteromeric AAA-ATPase complex associates with the TIC complex and functions as the import motor, directly interacting with various translocating preproteins. This 2-MD complex consists of a protein encoded by the previously enigmatic chloroplast gene ycf2 and five related nuclear-encoded FtsH-like proteins, namely, FtsHi1, FtsHi2, FtsHi4, FtsHi5, and FtsH12. These components are each essential for plant viability and retain the AAA-type ATPase domain, but only FtsH12 contains the zinc binding active site generally conserved among FtsH-type metalloproteases. Furthermore, even the FtsH12 zinc binding site is dispensable for its essential function. Phylogenetic analyses suggest that all AAA-type members of the Ycf2/FtsHi complex including Ycf2 evolved from the chloroplast-encoded membrane-bound AAA-protease FtsH of the ancestral endosymbiont. The Ycf2/FtsHi complex also contains an NAD-malate dehydrogenase, a proposed key enzyme for ATP production in chloroplasts in darkness or in nonphotosynthetic plastids. These findings advance our understanding of this ATP-driven protein translocation system that is unique to the green lineage of photosynthetic eukaryotes.
Asunto(s)
Proteínas de Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Adenosina Trifosfato/metabolismo , Cloroplastos/metabolismo , Malato Deshidrogenasa/metabolismo , Transporte de ProteínasRESUMEN
Whereas the plastid caseinolytic peptidase (Clp) P protease system is essential for plant development, substrates and substrate selection mechanisms are unknown. Bacterial ClpS is involved in N-degron substrate selection and delivery to the ClpAP protease. Through phylogenetic analysis, we show that all angiosperms contain ClpS1 and some species also contain ClpS1-like protein(s). In silico analysis suggests that ClpS1 is the functional homolog of bacterial ClpS. We show that Arabidopsis thaliana ClpS1 interacts with plastid ClpC1,2 chaperones. The Arabidopsis ClpS1 null mutant (clps1) lacks a visible phenotype, and no genetic interactions with ClpC/D chaperone or ClpPR core mutants were observed. However, clps1, but not clpc1-1, has increased sensitivity to the translational elongation inhibitor chloramphenicol suggesting a link between translational capacity and ClpS1. Moreover, ClpS1 was upregulated in clpc1-1, and quantitative proteomics of clps1, clpc1, and clps1 clpc1 showed specific molecular phenotypes attributed to loss of ClpC1 or ClpS1. In particular, clps1 showed alteration of the tetrapyrrole pathway. Affinity purification identified eight candidate ClpS1 substrates, including plastid DNA repair proteins and Glu tRNA reductase, which is a control point for tetrapyrrole synthesis. ClpS1 interaction with five substrates strictly depended on two conserved ClpS1 residues involved in N-degron recognition. ClpS1 function, substrates, and substrate recognition mechanisms are discussed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Endopeptidasa Clp/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Western Blotting , Cloroplastos/genética , Electroforesis en Gel de Poliacrilamida , Endopeptidasa Clp/clasificación , Endopeptidasa Clp/genética , Isoenzimas/clasificación , Isoenzimas/genética , Modelos Genéticos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Filogenia , Plantas Modificadas Genéticamente , Unión Proteica , Mapeo de Interacción de Proteínas , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5- to 10-fold up-regulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.
Asunto(s)
Arabidopsis/genética , Cloroplastos/enzimología , ARN Helicasas DEAD-box/metabolismo , Intrones/genética , Empalme del ARN/genética , Ribosomas/metabolismo , Zea mays/enzimología , Secuencia de Aminoácidos , Arabidopsis/embriología , Arabidopsis/enzimología , Cloroplastos/genética , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ADN Bacteriano/genética , Endopeptidasa Clp/metabolismo , Regulación de la Expresión Génica de las Plantas , Inmunoprecipitación , Datos de Secuencia Molecular , Mutagénesis Insercional/genética , Mutación/genética , Especificidad de Órganos/genética , Fotosíntesis , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN de Planta/metabolismo , Homología de Secuencia de Aminoácido , Zea mays/genéticaRESUMEN
Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.
Asunto(s)
Cloroplastos/metabolismo , Orgánulos/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Plastidios/genética , Proteoma/metabolismo , Zea mays/citología , Reparación del ADN/fisiología , Replicación del ADN , Regulación de la Expresión Génica de las Plantas , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Orgánulos/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Plastidios/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/química , Proteoma/genética , Edición de ARN , Empalme del ARN , Ribonucleasas/química , Ribosomas/genética , Ribosomas/metabolismo , Zea mays/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
C(4) grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C(4) photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C(4) differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C(4) specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.
Asunto(s)
Carbono/metabolismo , Diferenciación Celular/fisiología , Hojas de la Planta , Proteómica/métodos , Zea mays , Pared Celular/metabolismo , Cloroplastos/metabolismo , Bases de Datos de Proteínas , Genes de Plantas , Homeostasis , Células del Mesófilo/citología , Células del Mesófilo/fisiología , Mitocondrias/metabolismo , Familia de Multigenes , Oxidación-Reducción , Fotosíntesis/fisiología , Hojas de la Planta/citología , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Terpenos/metabolismo , Zea mays/anatomía & histología , Zea mays/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimología , Endopeptidasa Clp/metabolismo , Mutación , Proteómica/métodos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Western Blotting , Electroforesis en Gel de Poliacrilamida , Endopeptidasa Clp/química , Endopeptidasa Clp/genética , Retroalimentación Fisiológica , Regulación de la Expresión Génica de las Plantas , Homeostasis , Espectrometría de Masas/métodos , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Pliegue de Proteína , Plantones/genética , Plantones/metabolismoRESUMEN
The insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane of the chloroplast is cpSRP-dependent, and requires the stromal components cpSRP54 and cpSRP43, the membrane-bound SRP receptor cpFtsY and the integral membrane protein Alb3. Previous studies demonstrated that the Arabidopsis mutant lacking both cpSRP54 and cpSRP43 had pale yellow leaves, but was viable, whereas the mutants lacking Alb3 exhibit an albino phenotype that is more severe and seedling lethality. We previously showed that a maize mutant lacking cpFtsY had a pale yellow-green phenotype and was seedling lethal. To compare the in vivo requirements of cpFtsY and Alb3 in thylakoid biogenesis in greater detail, we isolated Arabidopsis null mutants of cpftsY, and performed biochemical comparisons with the Arabidopsis alb3 mutant. Both cpftsY and alb3 null mutants were seedling lethal on a synthetic medium lacking sucrose, whereas on a medium supplemented with sucrose, they were able to grow to later developmental stages, but were mostly infertile. cpftsY mutant plants had yellow leaves in which the levels of LHCPs were reduced to 10-33% compared with wild type. In contrast, alb3 had yellowish white leaves, and the LHCP levels were less than or equal to 10% of those of wild type. Intriguingly, whereas accumulation of the Sec and Tat machineries were normal in both mutants, the Sec pathway substrate Cyt f was more severely decreased in the cpftsY mutant than in alb3, which may indicate a functional link between cpFtsY and Sec translocation machinery. These results suggest that cpFtsY and Alb3 have essentially similar, but slightly distinct, contributions to thylakoid biogenesis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Complejos de Proteína Captadores de Luz/metabolismo , Proteínas de la Membrana/metabolismo , Tilacoides/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Clorofila/metabolismo , Proteínas de Cloroplastos , ADN Bacteriano/genética , ADN de Plantas/genética , Perfilación de la Expresión Génica , Genes de Plantas , Complejos de Proteína Captadores de Luz/genética , Proteínas de la Membrana/genética , Mutagénesis Insercional , Mutación , Fenotipo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.
Asunto(s)
Cloroplastos/genética , Intrones , Proteínas de Plantas/metabolismo , Empalme del ARN , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Mitocondrias , NADH Deshidrogenasa/genética , Oryza/metabolismo , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Zea mays/metabolismoRESUMEN
The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.
Asunto(s)
ADN de Cloroplastos/genética , Intrones/genética , Proteínas de Plantas/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , ADN de Cloroplastos/metabolismo , Inmunoprecipitación , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa , Unión Proteica , ARN de Planta/genética , Proteínas de Unión al ARN/genética , Zea mays/genética , Zea mays/metabolismoRESUMEN
The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.
Asunto(s)
Evolución Molecular , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/clasificación , Ribosomas/química , Secuencias de Aminoácidos , Núcleo Celular/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , ARN/química , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismoRESUMEN
Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Cloroplastos/genética , Intrones , Empalme del ARN , Arabidopsis/anatomía & histología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Genoma de Planta , Mutagénesis Insercional , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Factores de Empalme de ARN , ARN Mensajero/metabolismo , Plantones/anatomía & histología , Plantones/genética , Plantones/metabolismo , Especificidad de la Especie , Zea mays/genética , Zea mays/metabolismoAsunto(s)
Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Transporte de Proteínas/genética , Tilacoides/metabolismo , Proteínas de Arabidopsis/fisiología , Proteínas de Cloroplastos , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Plantas/fisiología , Pliegue de Proteína , Partícula de Reconocimiento de Señal/fisiologíaRESUMEN
A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a "leaky" allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.