Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas (original) (raw)
Related papers
Photosystem Biogenesis Is Localized to the Translation Zone in the Chloroplast of Chlamydomonas
The Plant Cell, 2019
Intracellular processes can be localized for efficiency or regulation. For example, localized mRNA translation by chloroplastic ribosomes occurs in the biogenesis of PSII, one of the two photosystems of the photosynthetic electron transport chain in the chloroplasts of plants and algae. The biogenesis of PSI and PSII requires the synthesis and assembly of their constituent polypeptide subunits, pigments, and cofactors. Although these biosynthetic pathways are well characterized, less is known about when and where they occur in developing chloroplasts. Here, we used fluorescence microscopy in the unicellular alga Chlamydomonas reinhardtii to reveal spatiotemporal organization in photosystem biogenesis. We focused on translation by chloroplastic ribosomes and chlorophyll biosynthesis in two developmental contexts of active photosystem biogenesis: (1) growth of the mature chloroplast and (2) greening of a nonphotosynthetic chloroplast. The results reveal that a translation zone is the primary location of the biogenesis of PSI and PSII. This discretely localized region within the chloroplast contrasts with the distributions of photosystems throughout this organelle and, therefore, is likely a hub where anabolic pathways converge for photosystem biogenesis.
A Nuclear-Encoded Function Essential for Translation of the Chloroplast psaB mRNA in Chlamydomonas
Plant Cell, 1997
We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, that contains a mutation at the TA67 (for translation of psa& mRNA) nuclear locus. Pulse labeling of chloroplast proteins revealed that the synthesis of the two photosystem I reaction center polypeptides PSAA and PSAB was undetectable in this mutant. The mRNA levels of these proteins were only moderately reduced, suggesting that the primary defect occurs at a step during or after translation. We constructed chimeric genes consisting of the psaA and psa6 5' untranslated region (5' UTR) fused to the aminoglycoside adenyltransferase (aadA) coding sequence, which confers spectinomycin resistance. Insertion of these genes into the chloroplast genome through biolistic transformation and analysis of their expression in the TA67 mutant nuclear background revealed that the psa6 (but not the psaA) 5' UTR is the target of the wild-type TAB7 function. This suggests that TA67 is required for the initiation of psa6 mRNA translation. The dependence of PSAA synthesis or accumulation on PSAB synthesis is strongly suggested by the identification of a suppressor mutation within the psa6 5' UTR. The suppressor specifically restores the synthesis of both proteins in the presence of the tab7-Fl5 mutation. The location of the suppressor mutation within a putative base-paired region near the psa6 initiation codon suggests a role for TA67 in the activation of translation of the psa6 mRNA. Zumbrunn, G., Schneider, M., and Rochaix, J.-D. (1989). A simple particle gun for DNA-mediated cell transformation. Technique I, 204-21 6.
Plant Journal, 2005
Biosynthesis of chloroplast proteins is to a large extent regulated post-transcriptionally, and a number of nuclear-encoded genes have been identified that are required for translation or stability of specific chloroplast mRNAs. A nuclear mutant of Chlamydomonas reinhardtii, hf261, deficient in the translation of the psbA mRNA, has reduced association of the psbA mRNA with ribosomes and is deficient in binding of the chloroplast localized poly (A) binding protein (cPAB1) to the psbA mRNA. Cloning of the hf261 locus and complementation of hf261 using a wt genomic clone has identified a novel gene, Tba1, for translational affector of psbA. Strains complemented with the wt Tba1 gene restore the ability of the psbA mRNA to associate with ribosomes, and restores RNA binding activity of cPAB1 for the psbA mRNA. Analysis of the Tba1 gene identified a protein with significant homology to oxidoreductases. The effect of Tba1 expression on the RNA binding activity of cPAB1, and on the association of psbA mRNA with ribosomes, implies that Tba1 functions as a redox regulator of cPAB1 RNA binding activity to indirectly promote psbA mRNA translation initiation. A model of chloroplast translation incorporating Tba1 and other members of the psbA mRNA binding complex is presented.
Molecular and Cellular Biology, 1994
In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded functi...
The EMBO Journal, 1997
The chloroplast genes ycf3 and ycf4 from the green alga Chlamydomonas reinhardtii have been characterized. The deduced amino acid sequences of Ycf4 (197 residues) and Ycf3 (172 residues) display 41-52% and 64-78% sequence identity, respectively, with their homologues from algae, land plants and cyanobacteria. In C.reinhardtii, ycf4 and ycf3 are co-transcribed as members of the rps9-ycf4-ycf3-rps18 polycistronic transcriptional unit into RNAs of 8.0 kb and 3.0 kb corresponding to the entire unit and to rps9-ycf4-ycf3, respectively. Using biolistic transformation, ycf4 and ycf3 were disrupted with a chloroplast selectable marker cassette. Transformants lacking ycf4 or ycf3 were unable to grow photoautotrophically and were deficient in photosystem I activity. Western blot analysis showed that the photosystem I (PSI) complex does not accumulate stably in thylakoid membranes of these transformants. Ycf4 and Ycf3 were localized on thylakoid membranes but not stably associated with the PSI complex and accumulated to wild-type levels in mutants lacking PSI. RNA blot hybridizations showed that transcripts of psaA, psaB and psaC accumulate normally in these mutants and use of chimeric reporter genes revealed that Ycf3 is not required for initiation of translation of psaA and psaB mRNA. Our results indicate that Ycf3 and Ycf4 are required for stable accumulation of the PSI complex.
Plant Cell, 2006
The biogenesis of photosystem II, one of the major photosynthetic protein complexes, involves a cascade of assemblygoverned regulation of translation of its major chloroplast-encoded subunits. In Chlamydomonas reinhardtii, the presence of the reaction center subunit D2 is required for the expression of the other reaction center subunit D1, while the presence of D1 is required for the expression of the core antenna subunit apoCP47. Using chimeric genes expressed in the chloroplast, we demonstrate that the decreased synthesis of D1 or apoCP47 in the absence of protein assembly is due to a genuine downregulation of translation. This regulation is mediated by the 59 untranslated region of the corresponding mRNA and originates from negative feedback exerted by the unassembled D1 or apoCP47 polypeptide. However, autoregulation of translation of subunit D1 is not implicated in the recovery from photoinhibition, which involves an increased translation of psbA mRNA in response to the degradation of photodamaged D1. De novo synthesis and repair of photosystem II complexes are independently controlled.
The Journal of Cell Biology, 2002
Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38–40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA.
2005
Biosynthesis of chloroplast proteins is to a large extent regulated post-transcriptionally, and a number of nuclear-encoded genes have been identified that are required for translation or stability of specific chloroplast mRNAs. A nuclear mutant of Chlamydomonas reinhardtii, hf261, deficient in the translation of the psbA mRNA, has reduced association of the psbA mRNA with ribosomes and is deficient in binding of the chloroplast localized poly (A) binding protein (cPAB1) to the psbA mRNA. Cloning of the hf261 locus and complementation of hf261 using a wt genomic clone has identified a novel gene, Tba1, for translational affector of psbA. Strains complemented with the wt Tba1 gene restore the ability of the psbA mRNA to associate with ribosomes, and restores RNA binding activity of cPAB1 for the psbA mRNA. Analysis of the Tba1 gene identified a protein with significant homology to oxidoreductases. The effect of Tba1 expression on the RNA binding activity of cPAB1, and on the association of psbA mRNA with ribosomes, implies that Tba1 functions as a redox regulator of cPAB1 RNA binding activity to indirectly promote psbA mRNA translation initiation. A model of chloroplast translation incorporating Tba1 and other members of the psbA mRNA binding complex is presented.
cis- and trans-Acting Determinants for Translation of psbD mRNA in Chlamydomonas reinhardtii
Molecular and Cellular Biology, 2000
Chloroplast translation is mediated by nucleus-encoded factors that interact with distinct cis-acting RNA elements. A U-rich sequence within the 5 untranslated region of the psbD mRNA has previously been shown to be required for its translation in Chlamydomonas reinhardtii. By using UV cross-linking assays, we have identified a 40-kDa RNA binding protein, which binds to the wild-type psbD leader, but is unable to recognize a nonfunctional leader mutant lacking the U-rich motif. RNA binding is restored in a chloroplast cis-acting suppressor. The functions of several site-directed psbD leader mutants were analyzed with transgenic C. reinhardtii chloroplasts and the in vitro RNA binding assay. A clear correlation between photosynthetic activity and the capability to bind RNA by the 40-kDa protein was observed. Furthermore, the data obtained suggest that the poly(U) region serves as a molecular spacer between two previously characterized cis-acting elements, which are involved in RNA stabilization and translation. RNA-protein complex formation depends on the nuclear Nac2 gene product that is part of a protein complex required for the stabilization of the psbD mRNA. The sedimentation properties of the 40-kDa RNA binding protein suggest that it interacts directly with this Nac2 complex and, as a result, links processes of chloroplast RNA metabolism and translation.
The Plant Journal, 2012
Genetic analysis of mutants deficient in the biosynthesis of the photosystem I complex has revealed several nucleus-encoded factors that act at different post-transcriptional steps of chloroplast gene expression. Here we have identified and characterized the gene affected in the tab 1-F15 mutant, which is specifically deficient in the translation of the photosystem I reaction center protein PsaB as the result of a single nucleotide deletion. This gene encodes Tab 1, a 1287 amino acid protein that contains 10 tandem 38-40 amino acid degenerate repeats of the PPPEW/OPR (octatricopeptide repeat) family, first described for the chloroplast translation factor Tbc2. These repeats are involved in the binding of Tab 1 to the 5¢-untranslated region of the psaB mRNA based on gel mobility shift assays. Tab 1 is part of a large family of proteins in Chlamydomonas that are also found in several bacteria and protozoans, but are rare in land plants.