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J D Rochaix

Publications and source records attributed to J D Rochaix.

At least 19 recordsLinked to original sources
PubMed2001

Identification of an RNA-protein complex involved in chloroplast group II intron trans-splicing in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving trans-splicing of separate transcripts, encoded at three separate loci of the chloroplast genome. At least 14 nuclear loci and one chloroplast gene, tscA, are needed for this process. We have cloned Raa3, the first nuclear gene implicated in the splicing of intron 1. The predicted sequence of Raa3 consists of 1783 amino acids and shares a small region of homology with pyridoxamine 5'-phosphate oxidases. Raa3 is present in the soluble fraction of the chloroplast and is part of a large 1700 kDa complex, which also contains tscA RNA and the first psaA exon transcript. These partners, in association with other factors, form a chloroplast RNP particle that is required for the splicing of the first intron of psaA and which may be the counterpart of eukaryotic snRNPs involved in nuclear splicing.

Algal Proteins↗
PubMed2001

The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii.

The nuclear gene PsaD encodes an abundant chloroplast protein located on the stromal side of the Photosystem I complex. We have cloned and sequenced a genomic fragment containing the PsaD gene from the green alga Chlamydomonas reinhardtii. Sequence comparison with its cDNA revealed that the PsaD ORF contains no introns. Thus, the regulatory sequences required for high-level expression of PsaD must lie in the flanking promoter and untranslated regions. We used this genomic fragment to construct a vector that allows for high-level expression of endogenous and exogenous genes, as well as cDNAs that could not be expressed from existing vectors. It is also possible to use the PsaD transit sequence to target the expressed protein to the chloroplast compartment.

Animals↗
PubMed2001

Functional studies of Ycf3: its role in assembly of photosystem I and interactions with some of its subunits.

The Ycf3 protein is essential for the accumulation of the photosystem I (PSI) complex and acts at a post-translational level. The sequence of Ycf3 is conserved in cyanobacteria, algae, and plants and contains three tetratrico-peptide repeats (TPR). TPRs have been shown to function as sites for protein-protein interactions. The mutations Y95A/Y96A and Y142A/W143A in the second and third TPR repeats lead to a modest decrease of PSI, but they prevent photoautotrophic growth and cause enhanced light sensitivity even though the accumulated PSI complex is fully functional. This phenotype can be reversed under anaerobic conditions and appears to be the result of photooxidative damage. A temperature-sensitive ycf3 mutant, generated by random mutagenesis of a conserved region near the N-terminal end of Ycf3, was used in temperature-shift experiments to show that Ycf3 is required for PSI assembly but not for its stability. Immunoblot analysis of thylakoid membranes separated by two-dimensional gel electrophoresis and immunoprecipitations shows that Ycf3 interacts directly with the PSI subunits PsaA and PsaD, but not with subunits from other photosynthetic complexes. Thus, Ycf3 appears to act as a chaperone that interacts directly and specifically with at least two of the PSI subunits during assembly of the PSI complex.

Aerobiosis↗
PubMed2000

Characterization of Mbb1, a nucleus-encoded tetratricopeptide-like repeat protein required for expression of the chloroplast psbB/psbT/psbH gene cluster in Chlamydomonas reinhardtii.

Genetic analysis has revealed that the accumulation of several chloroplast mRNAs of the green alga Chlamydomonas reinhardtii requires specific nucleus-encoded functions. To gain insight into this process, we have cloned the nuclear gene encoding the Mbb1 factor by genomic rescue of a mutant specifically deficient in the accumulation of the mRNAs of the psbB/psbT/psbH chloroplast transcription unit. Mbb1 is a soluble protein in the stromal phase of the chloroplast. It consists of 662 amino acids with a putative chloroplast-transit peptide at its N-terminal end. A striking feature is the presence of 10 tandemly arranged tetratricopeptide-like repeats that account for half of the protein sequence and are thought to be involved in protein-protein interactions. The Mbb1 protein seems to have a homologue in higher plants and is part of a 300-kDa complex that is associated with RNA. This complex is most likely involved in psbB mRNA processing, stability, and/or translation.

Algal Proteins↗
PubMed2000

The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability.

The psbD mRNA, which encodes the D2 reaction center polypeptide of photosystem II, is one of the most abundant chloroplast mRNAs. We have used genomic complementation to isolate the nuclear Nac2 gene, which is required for the stable accumulation of the psbD mRNA in Chlamydomonas reinhardtii. Nac2 encodes a hydrophilic polypeptide of 1385 amino acids with nine tetratricopeptide-like repeats (TPRs) in its C-terminal half. Cell fractionation studies indicate that the Nac2 protein is localized in the stromal compartment of the chloroplast. It is part of a high molecular weight complex that is associated with non-polysomal RNA. Change of a conserved alanine residue of the fourth TPR motif by site-directed mutagenesis leads to aggregation of Nac2 protein and completely abrogates its function, indicating that this TPR is important for proper folding of the protein and for psbD mRNA stability, processing and/or translation.

Algal Proteins↗
PubMed2000

Limitation in electron transfer in photosystem I donor side mutants of Chlamydomonas reinhardtii. Lethal photo-oxidative damage in high light is overcome in a suppressor strain deficient in the assembly of the light harvesting complex.

Strains of Chlamydomonas reinhardtii lacking the PsaF gene or containing the mutation K23Q within the N-terminal part of PsaF are sensitive to high light (>400 microE m(-2) s(-1)) under aerobic conditions. In vitro experiments indicate that the sensitivity to high light of the isolated photosystem I (PSI) complex from wild type and from PsaF mutants is similar. In vivo measurements of photochemical quenching and oxygen evolution show that impairment of the donor side of PSI in the PsaF mutants leads to a diminished linear electron transfer and/or a decrease of photosystem II (PSII) activity in high light. Thermoluminescence measurements indicate that the PSII reaction center is directly affected under photo-oxidative stress when the rate of electron transfer becomes limiting in the PsaF-deficient strain and in the PsaF mutant K23Q. We have isolated a high light-resistant PsaF-deficient suppressor strain that has a high chlorophyll a/b ratio and is affected in the assembly of light-harvesting complex. These results indicate that under high light a functionally intact donor side of PSI is essential for protection of C. reinhardtii against photo-oxidative damage when the photosystems are properly connected to their light-harvesting antennae.

Animals↗
PubMed2000

Stability determinants in the chloroplast psbB/T/H mRNAs of Chlamydomonas reinhardtii.

The chloroplast gene psbB encodes the chlorophyll-a binding protein P5 (CP47), one of the core subunits of photosystem II (PSII). The psbB mRNA and the downstream psbT and psbH transcripts fail to accumulate in the Chlamydomonas reinhardtii nuclear mutant 222E affected in the Mbb1 gene (Monod et al. 1992, Mol. Gen. Genet. 231, 449-459). By introducing chimeric genes consisting of sequences from psbB and the reporter gene aadA into the chloroplast, the target site of Mbb1 was mapped in the psbB 5' untranslated region (UTR). Primer extension analysis indicates that the psbB RNA exists in a less abundant long form and a more abundant short form, with 5' ends at positions -147 and -35 relative to the AUG initiation codon, respectively. The longer transcript is present both in the wild type (WT) and 222E mutant, but the shorter one accumulates only in the WT. Two putative stem-loop structures in the longer 5' UTR can be deleted individually without affecting psbB mRNA accumulation. Insertion of a poly G cassette in the long leader stabilizes a chimeric psbB transcript in the 222E mutant, suggesting the involvement of a 5'-3' exonuclease. We also show that psbH and psbT are transcribed from the upstream psbB gene promoter, and that the psbH mRNA has its own target sequence for Mbb1 function. We discuss the role of this nucleus-encoded factor, required for specific chloroplast gene expression, in the assembly of the multi-protein PSII complex.

5' Untranslated Regions↗
PubMed1999

A factor related to pseudouridine synthases is required for chloroplast group II intron trans-splicing in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving two steps of trans-splicing that remove two group II introns and give rise to the mature mRNA. The products of at least 14 nuclear genes and one chloroplast gene (tscA) are necessary for this process. We have cloned Maa2, one of the nuclear genes involved in trans-splicing of the second intron. Maa2 encodes a protein with similarity to conserved domains of pseudouridine synthases, but mutagenesis of putative catalytic residues showed that this activity may not be required for trans-splicing of psaA RNA. Although it is not clear whether the pseudouridine synthase activity has been maintained in Maa2, it is possible that this enzyme was recruited during evolution as an RNA chaperone for folding or stabilizing the psaA intron. The Maa2 protein appears to be associated through ionic interactions with a low density membrane system in the chloroplast that also contains RNA-binding proteins involved in translation.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.

Animals↗
PubMed1999

Site-directed mutagenesis of the PsaC subunit of photosystem I. F(b) is the cluster interacting with soluble ferredoxin.

The two [4Fe-4S] clusters F(A) and F(B) are the terminal electron acceptors of photosystem I (PSI) that are bound by the stromal subunit PsaC. Soluble ferredoxin (Fd) binds to PSI via electrostatic interactions and is reduced by the outermost iron-sulfur cluster of PsaC. We have generated six site-directed mutants of the green alga Chlamydomonas reinhardtii in which residues located close to the iron-sulfur clusters of PsaC are changed. The acidic residues Asp(9) and Glu(46), which are located one residue upstream of the first cysteine liganding cluster F(B) and F(A), respectively, were changed to a neutral or a basic amino acid. Although Fd reduction is not affected by the E46Q and E46K mutations, a slight increase of Fd affinity (from 1.3- to 2-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA(2) triple mutant (V49I/K52T/R53Q), modification of residues located next to the F(A) cluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster F(A) are affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the F(B) cluster in the FB(1) triple mutant (I12V/T15K/Q16R) results in a 60-fold increase of Fd affinity as measured by flash absorption spectroscopy and a larger amount of PsaC-Fd cross-linking product. The first-order kinetics are similar to wild type kinetics (two phases with t((1)/(2)) of <1 and approximately 4.5 microseconds) for all mutants except FB(1), where Fd reduction is almost monophasic with t((1)/(2)) < 1 microseconds. These data indicate that F(B) is the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.

Animals↗
PubMed1999

A large fraction of PsaF is nonfunctional in photosystem I complexes lacking the PsaJ subunit.

PsaJ is a small hydrophobic subunit of the photosystem I complex (PSI) whose function is not yet fully understood. Here we describe mutants of the green alga Chlamydomonas reinhardtii, in which the psaJ chloroplast gene has been inactivated either in a wild-type or in a PsaF-deficient nuclear background. Cells lacking one or both subunits grow photoautotrophically and contain normal levels of PSI. Flash-absorption spectroscopy performed with isolated PSI particles isolated from the PsaJ-deficient strain indicates that only 30% of the PSI complexes oxidize plastocyanin (Pc) or cytochrome c6 (Cyt c6) with kinetics identical to wild type, whereas the remaining 70% follow slow kinetics similar to those observed with PsaF-deficient PSI complexes. This feature is not due to partial loss of PsaF, as the PsaJ-less PSI complex contains normal levels of the PsaF subunit. The N-terminal domain of PsaF can be cross-linked to Pc and Cyt c6 indicating that in the absence of PsaJ, this domain is exposed in the lumenal space. Therefore, the decreased amount of functional PsaF revealed by the electron-transfer measurements is best explained by a displacement of the N-terminal domain of PsaF which is known to provide the docking site for Pc and Cyt c6. We propose that one function of PsaJ is to maintain PsaF in a proper orientation which allows fast electron transfer from soluble donor proteins to P700(+).

Amino Acid Sequence↗
PubMed1999

Photosystem I is indispensable for photoautotrophic growth, CO2 fixation, and H2 photoproduction in Chlamydomonas reinhardtii.

Certain Chlamydomonas reinhardtii mutants deficient in photosystem I due to defects in psaA mRNA maturation have been reported to be capable of CO2 fixation, H2 photoevolution, and photoautotrophic growth (Greenbaum, E., Lee, J. W., Tevault, C. V., Blankinship, S. L. , and Mets, L. J. (1995) Nature 376, 438-441 and Lee, J. W., Tevault, C. V., Owens, T. G.; Greenbaum, E. (1996) Science 273, 364-367). We have generated deletions of photosystem I core subunits in both wild type and these mutant strains and have analyzed their abilities to grow photoautotrophically, to fix CO2, and to photoevolve O2 or H2 (using mass spectrometry) as well as their photosystem I content (using immunological and spectroscopic analyses). We find no instance of a strain that can perform photosynthesis in the absence of photosystem I. The F8 strain harbored a small amount of photosystem I, and it could fix CO2 and grow slowly, but it lost these abilities after deletion of either psaA or psaC; these activities could be restored to the F8-psaADelta mutant by reintroduction of psaA. We observed limited O2 photoevolution in mutants lacking photosystem I; use of 18O2 indicated that this O2 evolution is coupled to O2 uptake (i.e. respiration) rather than CO2 fixation or H2 evolution. We conclude that the reported instances of CO2 fixation, H2 photoevolution, and photoautotrophic growth of photosystem I-deficient mutants result from the presence of unrecognized photosystem I.

Animals↗
PubMed1999

Insertion of the N-terminal part of PsaF from Chlamydomonas reinhardtii into photosystem I from Synechococcus elongatus enables efficient binding of algal plastocyanin and cytochrome c6.

A strain of the cyanobacterium Synechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene. The transformants express a new PsaF subunit similar in size to PsaF from C. reinhardtii that is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700(+) reduction by C. reinhardtii cytochrome c6 or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI. The corresponding optimum second-order rate constants (k2 = 4.0 and 1.7 x 10(7) M1 s1 for cytochrome c6 and plastocyanin) are similar to those of PSI from C. reinhardtii. The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI from S. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c6, a fast first-order kinetic component (t(1)/(2) = 4 microseconds) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochrome c6. This first-order phase is characteristic for P700(+) reduction by cytochrome c6 or plastocyanin in algae and higher plants. However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild type S. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c6 or plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c6 or plastocyanin.

Amino Acid Sequence↗
PubMed1999

The argininosuccinate lyase gene of Chlamydomonas reinhardtii: cloning of the cDNA and its characterization as a selectable shuttle marker.

We describe the cDNA sequence for ARG7, the gene that encodes argininosuccinate lyase--a selectable nuclear marker--in Chlamydomonas reinhardtii. The 5' end of the cDNA contains one more exon and the organisation of the mRNA is different from that predicted from the genomic sequence. When expressed under the control of the endogenous RbcS2 promoter, the 2.22-kb cDNA complements the arg7 mutation as well as the genomic DNA. A linear cDNA fragment lacking promoter sequences is also able to complement, suggesting that it could be used in promoter-trapping experiments. Despite the presence of a sequence encoding a potential chloroplast transit peptide in the cDNA the protein is not targeted to the chloroplast, nor can it complement the arg7 mutation when expressed there. By inserting a T7 bacteriophage promoter into the plasmid, a version of the cDNA which is able to complement both the C. reinhardtii arg7 mutant and the Escherichia coli argH mutant has been created. This modified Arg7 cDNA provides two advantages over the genomic DNA currently in use for gene tagging: it is shorter (6.2 kb versus 11.9 kb for pARG7.8phi3), and the selectable marker used in C. reinhardtii is the same as that used in E. coli, making plasmid rescue of the tag much more likely to succeed.

Algal Proteins↗
PubMed1999

Lys35 of PsaC is required for the efficient photoreduction of flavodoxin by photosystem I from Chlamydomonas reinhardtii.

The photoreduction of the oxidized and the semiquinone form of flavodoxin from Synechocystis sp. PCC 6803 by the photosystem I (PSI) of wild-type Chlamydomonas reinhardtii and the mutant strains Lys35Asp, Lys35Glu and Lys35Arg was analysed by flash-absorption spectroscopy to investigate the role of residue Lys35 of the PSI subunit PsaC in flavodoxin reduction. For PSI preparations from C. reinhardtii the reduction of oxidized flavodoxin was monoexponential and approached limiting electron transfer rates similar to those of cyanobacterial PSI from the wild-type and the Lys35Arg mutant. For PSI from the Lys35Glu mutant, however, a approximately 2.5-fold smaller value was determined. The photoreduction of flavodoxin semiquinone by PSI from C. reinhardtii lacked fast first-order kinetic components and, in contrast with PSI from cyanobacteria, displayed only a single concentration-dependent phase. From this phase, second-order rate constants were calculated for wild-type PSI and PSI from the Lys35Arg mutant which were comparable to those of PSI from cyanobacteria. For PSI from the Lys35Glu and the Lys35Asp mutants the derived second-order rate constants were 19 and 10 times smaller. Thus, the inversion of charge at position 35 of PsaC negatively affects the rate of electron transfer to both forms of flavodoxin, whereas PSI complexes that retain a positive charge at this position show wild-type kinetics. However, the positive charge at this position of PsaC is not essential for flavodoxin photoreduction as the number of flavodoxin molecules reduced per PSI was similar for all of the PSI complexes investigated. In addition, chemical cross-linking assays showed that the binary cross-linking product between flavodoxin and PsaC of PSI from wild-type C. reinhardtii was not formed with PSI complexes from the Lys13Asp and Lys35Glu mutants. This indicates that Lys35 of PsaC is probably essential for the chemical cross-link between PsaC and flavodoxin. Taken together, these experiments show that Lys35 of PsaC plays a strikingly similar role in the electron transfer from PSI to both ferredoxin and flavodoxin.

Animals↗