J.-d. Rochaix - Profile on Academia.edu (original) (raw)

Papers by J.-d. Rochaix

Proceedings in Life Sciences, 1984

The EMBO Journal, 1994

We have engineered and analyzed a chloroplast mutant of Chlamydomonas reinhardtii that lacks ycf8... more We have engineered and analyzed a chloroplast mutant of Chlamydomonas reinhardtii that lacks ycf8, the chloroplast open reading frame 8, which is highly conserved in location and predicted amino acid sequence in land plants and C.reinhardtii. The ycf8 sequence was replaced with the aadA cassette which confers resistance to spectinomycin when expressed in the chloroplast. Although the mutant is able to grow phototrophically, photosystem II function and cell growth are impaired under stress conditions such as high light intensity and diminished chloroplast protein synthesis induced by spectinomycin. Use of an antibody generated against the ycf8 product has revealed that this hydrophobic polypeptide is associated with photosystem II, based on its severely reduced levels in various photosystem II- deficient mutants and on its copurification with photosystem II. This protein, therefore, appears to be (i) a novel photosystem II subunit and (ii) required for maintaining optimal photosystem II activity under adverse growth conditions. Key words: chloroplast open reading frame/photosystem 11/stress A small chloroplast ORF located downstream of, and co- transcribed with, psbB has been found in Chlamydomonas reinhardtii (Monod et al., 1992). This ORF, designated ycJ8 for 'hypothetical chloroplast open reading frame 8', is evolutionarily conserved from C. reinhardtii throughout all higher plants examined and it could encode a polypeptide between 31 and 38 amino acids. While the organization of most protein-encoding chloroplast genes in C.reinhardtii differs from that found in higher plants, a striking exception is provided by the psbB region in which the relative arrangement of psbB, ycJ8, psbN and psbH is conserved (Monod et al., 1992; Johnson and Schmidt, 1993). However, in contrast to higher plants where petB and petD are part of the psbB operon, these genes are located elsewhere on the chloroplast genome of C. reinhardtii (Biischlen et al., 1991). All homologs ofycJ8 encode a negatively charged glutamic acid as the second residue, a hydrophobic central region and several positively charged amino acids in the C-terminal region (Monod et al., 1992). This ORF, therefore, has the potential to encode a small membrane-spanning polypeptide. Furthermore, since ycJ8 is co-transcribed with one of the principal polypeptides of the PSII complex, it has been tempting to speculate that the ycJ8 product is associated with, and of functional importance to, this photosynthetic complex. Here we demonstrate that ycJ8 is expressed as part of the psbB-psbH operon and that its product is specifically associated with the PSII complex and, thus, represents a new photosystem II subunit. We also show that inactivation of ycJ8 puts the PSIH complex at a disadvantage under certain stress conditions, such as high light and reduced chloroplast protein synthesis induced by spectinomycin. This is the first identification of a small PSIH subunit that appears to be essential for maintaining high photosynthetic activity under adverse growth conditions.

The EMBO Journal, 1995

The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membran... more The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cwlS-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles a func- tional PSI complex and is capable of photoautotrophic growth. However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex. These results contrast with those obtained previously with a cyano- bacterial psaF-, psaJ-double mutant where no phenotype was apparent.

The EMBO Journal, 1994

Stability of the chloroplast psbD mRNA encoding the D2 protein of the photosystem II reaction cen... more Stability of the chloroplast psbD mRNA encoding the D2 protein of the photosystem II reaction center is drastically decreased in the nuclear photosynthetic mutant nac2-26 of Chlamydomonas reinhardtii. Using biolistic transformation and genetic crosses we have introduced chimeric genes consisting of thepsbD leader fused to a reporter gene into the chloroplast in both wild-type and mutant nuclear backgrounds. The chimeric message is destabilized in the latter, but not in the former case, indicating that the 74 nt psbD leader includes one of the target sites for psbD RNA degradation in the absence of wild-type NAC2 function. Increased instability of the psbD leader in mutant versus wild-type chloroplast lysates is also demonstrated in vitro and the primary cleavage sites have been mapped. The instability of the psbD RNA in the mutant correlates with the loss of binding of a 47 kDa protein to the psbD leader RNA, suggesting that this factor acts as message stabilizer in wild-type.

The EMBO Journal, 1986

Communicated by J.-D.Rochaix Dl and D2, two chloroplast proteins with apparent mol. wt of 32 000-... more Communicated by J.-D.Rochaix Dl and D2, two chloroplast proteins with apparent mol. wt of 32 000-34 000, play an important role in the photo- synthetic reactions mediated by the membrane-bound protein complex of photosystem II (PSII). We have isolated and characterized an uniparental, non-photosynthetic mutant of Chlamydomonas reinhardtii and show that the mutation is in the chloroplast gene psbD, coding for D2. A 46 bp direct DNA duplication in the coding region of the mutant gene causes a frame-shift which results in a psbD transcript coding for 186 amino acid residues instead of the normal 352. The trun- cated D2 peptide is never seen, even after pulse-labeling, suggesting that the mutant protein is very unstable. In addition, little or no Dl protein is detected in this mutant although the gene and normal levels of mRNA for Dl are present in mutant cells. All other core PSI1 proteins are synthesized and inserted into the membrane fraction, but never accumulate. These results suggest that D2 contributes not only to the stabilization of the PSII complex in the membrane, but also may play a specific role in the regulation of the Dl protein, either at the translational or post-translational level.

Molecular and Cellular Biology, 1994

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previous... more 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 Plant cell, Jan 30, 2014

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in... more Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some...

THE PLANT CELL ONLINE, 2001

The Ycf3 protein is essential for the accumulation of the photosystem I (PSI) complex and acts at... more 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.

The Plant Cell, 2009

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in ph... more Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstruc...

The Plant Cell, 1997

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, tha... more 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.

The Plant Cell, 1989

Plants and green algae can develop resistance to herbicides that block photosynthesis by competin... more Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSll electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSll electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, Qe of PSII, provide a strong basis of support for structural and functional models of the PSll reaction center.

The Journal of Cell Biology, 1998

Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the ... more Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the 5′ untranslated region of the chloroplast psbC mRNA of the green alga Chlamydomonas reinhardtii. These analyses revealed that RNA-binding proteins of 30–32, 46, 47, 60, and 80 kD are associated with chloroplast membranes. The buoyant density and the acyl lipid composition of these membranes are compatible with their origin being the inner chloroplast envelope membrane. However, unlike previously characterized inner envelope membranes, these membranes are associated with thylakoids. One of the membrane-associated RNA-binding proteins appears to be RB47, which has been reported to be a specific activator of psbA mRNA translation. These results suggest that translation of chloroplast mRNAs encoding thylakoid proteins occurs at either a subfraction of the chloroplast inner envelope membrane or a previously uncharacterized intra-chloroplast compartment, which is physically associated with thy...

The Journal of Cell Biology, 2002

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are inv... more 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.

The EMBO Journal, 2001

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving trans-splicing of... more 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 ®rst 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 ®rst psaA exon transcript. These partners, in association with other factors, form a chloroplast RNP particle that is required for the splicing of the ®rst intron of psaA and which may be the counterpart of eukaryotic snRNPs involved in nuclear splicing.

The EMBO Journal, 1999

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving two steps of tran... more In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving two steps of transsplicing 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.

Proceedings of the National Academy of Sciences, 2000

Genetic analysis has revealed that the accumulation of several chloroplast mRNAs of the green alg... more 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.

Proceedings of the National Academy of Sciences, 2010

The ability of plants to adapt to changing light conditions depends on a protein kinase network i... more The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana , called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core pr...

Proceedings of the National Academy of Sciences, 1987

We have cloned a cDNA encoding a 20-kDa polypeptide, oxygen-evolving enhancer protein 2 (OEE2), i... more We have cloned a cDNA encoding a 20-kDa polypeptide, oxygen-evolving enhancer protein 2 (OEE2), in Chlamydomonas reinhardtii. This polypeptide has been implicated in photosynthetic oxygen evolution, and it is associated with the photosystem II complex, the site of oxygen evolution in all higher plants and algae. The sequence of OEE2 cDNA, the deduced amino acid sequence of the preprotein, the N-terminal protein sequence of mature OEE2 protein, and the coding regions of the single OEE2 gene are presented. The protein is synthesized with a 57-amino acid N-terminal transit peptide that directs the transport of this polypeptide across three cellular membranes. A nuclear mutant of C. reinhardtii, deficient in oxygen-evolving activity, is shown to be specifically missing OEE2 polypeptides. This mutation, which results in the complete absence of all OEE2 mRNA and protein, does not affect the accumulation of other photosystem II polypeptides or their mRNAs.

Proceedings of the National Academy of Sciences, 2011

Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled ... more Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8 , indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL...

Proceedings in Life Sciences, 1984

The EMBO Journal, 1994

We have engineered and analyzed a chloroplast mutant of Chlamydomonas reinhardtii that lacks ycf8... more We have engineered and analyzed a chloroplast mutant of Chlamydomonas reinhardtii that lacks ycf8, the chloroplast open reading frame 8, which is highly conserved in location and predicted amino acid sequence in land plants and C.reinhardtii. The ycf8 sequence was replaced with the aadA cassette which confers resistance to spectinomycin when expressed in the chloroplast. Although the mutant is able to grow phototrophically, photosystem II function and cell growth are impaired under stress conditions such as high light intensity and diminished chloroplast protein synthesis induced by spectinomycin. Use of an antibody generated against the ycf8 product has revealed that this hydrophobic polypeptide is associated with photosystem II, based on its severely reduced levels in various photosystem II- deficient mutants and on its copurification with photosystem II. This protein, therefore, appears to be (i) a novel photosystem II subunit and (ii) required for maintaining optimal photosystem II activity under adverse growth conditions. Key words: chloroplast open reading frame/photosystem 11/stress A small chloroplast ORF located downstream of, and co- transcribed with, psbB has been found in Chlamydomonas reinhardtii (Monod et al., 1992). This ORF, designated ycJ8 for 'hypothetical chloroplast open reading frame 8', is evolutionarily conserved from C. reinhardtii throughout all higher plants examined and it could encode a polypeptide between 31 and 38 amino acids. While the organization of most protein-encoding chloroplast genes in C.reinhardtii differs from that found in higher plants, a striking exception is provided by the psbB region in which the relative arrangement of psbB, ycJ8, psbN and psbH is conserved (Monod et al., 1992; Johnson and Schmidt, 1993). However, in contrast to higher plants where petB and petD are part of the psbB operon, these genes are located elsewhere on the chloroplast genome of C. reinhardtii (Biischlen et al., 1991). All homologs ofycJ8 encode a negatively charged glutamic acid as the second residue, a hydrophobic central region and several positively charged amino acids in the C-terminal region (Monod et al., 1992). This ORF, therefore, has the potential to encode a small membrane-spanning polypeptide. Furthermore, since ycJ8 is co-transcribed with one of the principal polypeptides of the PSII complex, it has been tempting to speculate that the ycJ8 product is associated with, and of functional importance to, this photosynthetic complex. Here we demonstrate that ycJ8 is expressed as part of the psbB-psbH operon and that its product is specifically associated with the PSII complex and, thus, represents a new photosystem II subunit. We also show that inactivation of ycJ8 puts the PSIH complex at a disadvantage under certain stress conditions, such as high light and reduced chloroplast protein synthesis induced by spectinomycin. This is the first identification of a small PSIH subunit that appears to be essential for maintaining high photosynthetic activity under adverse growth conditions.

The EMBO Journal, 1995

The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membran... more The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cwlS-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles a func- tional PSI complex and is capable of photoautotrophic growth. However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex. These results contrast with those obtained previously with a cyano- bacterial psaF-, psaJ-double mutant where no phenotype was apparent.

The EMBO Journal, 1994

Stability of the chloroplast psbD mRNA encoding the D2 protein of the photosystem II reaction cen... more Stability of the chloroplast psbD mRNA encoding the D2 protein of the photosystem II reaction center is drastically decreased in the nuclear photosynthetic mutant nac2-26 of Chlamydomonas reinhardtii. Using biolistic transformation and genetic crosses we have introduced chimeric genes consisting of thepsbD leader fused to a reporter gene into the chloroplast in both wild-type and mutant nuclear backgrounds. The chimeric message is destabilized in the latter, but not in the former case, indicating that the 74 nt psbD leader includes one of the target sites for psbD RNA degradation in the absence of wild-type NAC2 function. Increased instability of the psbD leader in mutant versus wild-type chloroplast lysates is also demonstrated in vitro and the primary cleavage sites have been mapped. The instability of the psbD RNA in the mutant correlates with the loss of binding of a 47 kDa protein to the psbD leader RNA, suggesting that this factor acts as message stabilizer in wild-type.

The EMBO Journal, 1986

Communicated by J.-D.Rochaix Dl and D2, two chloroplast proteins with apparent mol. wt of 32 000-... more Communicated by J.-D.Rochaix Dl and D2, two chloroplast proteins with apparent mol. wt of 32 000-34 000, play an important role in the photo- synthetic reactions mediated by the membrane-bound protein complex of photosystem II (PSII). We have isolated and characterized an uniparental, non-photosynthetic mutant of Chlamydomonas reinhardtii and show that the mutation is in the chloroplast gene psbD, coding for D2. A 46 bp direct DNA duplication in the coding region of the mutant gene causes a frame-shift which results in a psbD transcript coding for 186 amino acid residues instead of the normal 352. The trun- cated D2 peptide is never seen, even after pulse-labeling, suggesting that the mutant protein is very unstable. In addition, little or no Dl protein is detected in this mutant although the gene and normal levels of mRNA for Dl are present in mutant cells. All other core PSI1 proteins are synthesized and inserted into the membrane fraction, but never accumulate. These results suggest that D2 contributes not only to the stabilization of the PSII complex in the membrane, but also may play a specific role in the regulation of the Dl protein, either at the translational or post-translational level.

Molecular and Cellular Biology, 1994

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previous... more 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 Plant cell, Jan 30, 2014

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in... more Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some...

THE PLANT CELL ONLINE, 2001

The Ycf3 protein is essential for the accumulation of the photosystem I (PSI) complex and acts at... more 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.

The Plant Cell, 2009

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in ph... more Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstruc...

The Plant Cell, 1997

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, tha... more 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.

The Plant Cell, 1989

Plants and green algae can develop resistance to herbicides that block photosynthesis by competin... more Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSll electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSll electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, Qe of PSII, provide a strong basis of support for structural and functional models of the PSll reaction center.

The Journal of Cell Biology, 1998

Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the ... more Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the 5′ untranslated region of the chloroplast psbC mRNA of the green alga Chlamydomonas reinhardtii. These analyses revealed that RNA-binding proteins of 30–32, 46, 47, 60, and 80 kD are associated with chloroplast membranes. The buoyant density and the acyl lipid composition of these membranes are compatible with their origin being the inner chloroplast envelope membrane. However, unlike previously characterized inner envelope membranes, these membranes are associated with thylakoids. One of the membrane-associated RNA-binding proteins appears to be RB47, which has been reported to be a specific activator of psbA mRNA translation. These results suggest that translation of chloroplast mRNAs encoding thylakoid proteins occurs at either a subfraction of the chloroplast inner envelope membrane or a previously uncharacterized intra-chloroplast compartment, which is physically associated with thy...

The Journal of Cell Biology, 2002

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are inv... more 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.

The EMBO Journal, 2001

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving trans-splicing of... more 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 ®rst 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 ®rst psaA exon transcript. These partners, in association with other factors, form a chloroplast RNP particle that is required for the splicing of the ®rst intron of psaA and which may be the counterpart of eukaryotic snRNPs involved in nuclear splicing.

The EMBO Journal, 1999

In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving two steps of tran... more In Chlamydomonas reinhardtii, the psaA mRNA is assembled by a process involving two steps of transsplicing 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.

Proceedings of the National Academy of Sciences, 2000

Genetic analysis has revealed that the accumulation of several chloroplast mRNAs of the green alg... more 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.

Proceedings of the National Academy of Sciences, 2010

The ability of plants to adapt to changing light conditions depends on a protein kinase network i... more The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana , called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core pr...

Proceedings of the National Academy of Sciences, 1987

We have cloned a cDNA encoding a 20-kDa polypeptide, oxygen-evolving enhancer protein 2 (OEE2), i... more We have cloned a cDNA encoding a 20-kDa polypeptide, oxygen-evolving enhancer protein 2 (OEE2), in Chlamydomonas reinhardtii. This polypeptide has been implicated in photosynthetic oxygen evolution, and it is associated with the photosystem II complex, the site of oxygen evolution in all higher plants and algae. The sequence of OEE2 cDNA, the deduced amino acid sequence of the preprotein, the N-terminal protein sequence of mature OEE2 protein, and the coding regions of the single OEE2 gene are presented. The protein is synthesized with a 57-amino acid N-terminal transit peptide that directs the transport of this polypeptide across three cellular membranes. A nuclear mutant of C. reinhardtii, deficient in oxygen-evolving activity, is shown to be specifically missing OEE2 polypeptides. This mutation, which results in the complete absence of all OEE2 mRNA and protein, does not affect the accumulation of other photosystem II polypeptides or their mRNAs.

Proceedings of the National Academy of Sciences, 2011

Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled ... more Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8 , indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL...