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PubMed1998

In vivo analysis of the effect of dicyclohexylcarbodiimide on electron and proton transfers in cytochrome bf complex of Chlorella sorokiniana.

The effect of N,N'-dicyclohexylcarbodiimide (DCCD) on electron and proton transfers within the cytochrome (cyt) bf complex has been analyzed in living cells of the green algae Chlorella sorokiniana under anaerobic conditions. DCCD induces a partial decoupling of the protomotive Q-cycle, in agreement with the conclusions of Wang and Beattie (1991) Arch. Biochem. Biophys. 291, 363-370. In the presence of 20 microM DCCD, we observe the development of a lag phase in the kinetics of the slow electrogenic phase associated with electron and proton transfers within the cyt bf complex. In the same conditions, the initial rate of cyt b and cyt f reduction is decreased by about 30%. We propose that in the absence of DCCD, a transmembrane movement of proton is coupled to the oxidation of plastoquinol at site Qo. In the presence of 20 microM DCCD, this redox-coupled proton pump is inhibited, and the kinetics of phase b and cyt b reduction become close to that predicted on the basis of a pure Q-cycle process. In agreement with this hypothesis, we observe that upon a weak-flash excitation, two charges are translocated through the membrane in addition to the charge translocated at the level of photosystem I. Part of this large electrogenic phase could be associated with the translocation of a proton from the stroma to the lumen. A tentative mechanism is discussed that remains in the frame of the Q-cycle but accounts for an additional proton-pumping process or for the partial decoupling observed in the presence of DCCD, as well.

Anaerobiosis↗
PubMed1998

In vivo evidence for 5'-->3' exoribonuclease degradation of an unstable chloroplast mRNA.

The acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5' untranslated region (UTR). However, when this 5' UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5' end of the petD 5' UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5' UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5'-->3' exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5' UTR protects the mRNA from 5'-->3' degradation in wild-type cells.

Alleles↗
PubMed1990

Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and cytochrome b6/f complex. In vitro control of kinase activity.

We have previously reported that the cytochrome b6/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome b6/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome b6/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome b6/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome b6/f. Phosphorylation of LHCII by fractions containing both cytochrome b6/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of beta-D-octyl glucoside but not when 3-[(cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate was used as the solubilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo-5-t-butyl-p-benzoquinone) known to inhibit cytochrome b6/f activity. However, kinase activity was inhibited by these analogues in all preparations including those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome b6/f while the deactivation may be related to a distinct quinone binding site of the enzyme molecule.

Ammonium Sulfate↗
PubMed1989

On the question of interheme electron transfer in the chloroplast cytochrome b6 in situ.

The redox properties, the site of action of the inhibitor NQNO, and the question of interheme transfer in the chloroplast cytochrome b6 have been examined with regard to the role of the b6-f complex in quinol oxidation and H+ translocation. (i) The two hemes of the cytochrome ba and bp, have similar (delta Em less than or equal to 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (Em7 = -40 mV) but are pH dependent below this range with an estimated pK = 6.7. (ii) Only half of cytochrome b6, the stromal-side heme, ba, was reducible by NADPH and ferredoxin. (iii) The 2-3-fold increase (to 0.60 +/- 0.09 heme/600 Chl) in the amplitude of flash-induced cytochrome reduction caused by NQNO was not affected when heme ba was initially reduced, implying that NQNO affects flash reduction at the site of heme bp. (iv) Multiple light flashes did not increase the amplitude of b6 reduction in the presence or absence of NQNO or show binary oscillations. Together with localization of a site of action of NQNO near heme bp, these data provide no evidence for efficient electron transfer from heme bp to heme ba as specified by the Q cycle model. (v) NQNO interaction with heme bp does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t1/2 approximately 30 ms) when heme ba was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme ba indicates that the oxidation of ba and bp may be cooperative.

Antimycin A↗
PubMed1991

Specific ribonuclease activities in spinach chloroplasts promote mRNA maturation and degradation.

We have used an in vitro system to characterize ribonuclease activities present in spinach chloroplasts. We show that 3' end maturation of petD mRNA, which encodes subunit IV of the cytochrome b6/f complex, is affected by a 33-kDa protein that binds to a hairpin structure at the 3' end of the mature mRNA. Binding of the 33-kDa protein to the petD hairpin structure decreases the efficiency of 3' end maturation, probably by impeding the progress of the processive 3'-5' exonuclease activity involved in chloroplast mRNA processing. A two-base mutation in the stem of the petD hairpin structure creates a novel recognition site for a ribonuclease which competes with the normal processing exonuclease activity. This mutation results in a very low 3' end processing efficiency for mutant petD transcripts, and instead generates a second processing product that lacks a complete hairpin structure. An endonuclease activity which is biochemically distinct from the previously characterized exonuclease activities has also been identified. This endonuclease activity is EDTA-insensitive, and cleaves petD RNA both at the termination codon and at the mature RNA 3' end. Cleavage of petD mRNA at the termination codon leads to rapid degradation of upstream RNA. The possible roles of these ribonuclease activities in chloroplast mRNA decay in vivo are discussed.

Base Sequence↗
PubMed1997

The spinach chloroplast endoribonuclease CSP41 cleaves the 3'-untranslated region of petD mRNA primarily within its terminal stem-loop structure.

3'-Untranslated region stem-loop structures are major determinants of chloroplast mRNA stability. The 3' stem-loop region of spinach petD precursor mRNA (pre-mRNA), a chloroplast gene encoding subunit IV of the cytochrome b6.f complex, forms a stable RNA-protein complex in vitro with chloroplast stem-loop binding proteins (CSPs) of 55, 41, and 29 kDa. We have previously purified CSP41 and cloned the corresponding cDNA. In vitro studies demonstrated that CSP41 is a bifunctional protein that displays both endoribonuclease and RNA-binding activities. In this work, the RNase activity of CSP41 is further characterized using the bacterially expressed protein. Our data show that CSP41 cleaves both single-stranded and double-stranded RNAs but not DNA. However, it exhibits a preference for stem-loop-containing RNAs. When the 3'-untranslated region of petD pre-mRNA is provided as a substrate, CSP41 specifically cleaves it within the stem-loop region, implying that CSP41 has an important role in the control of petD mRNA stability. Our data also show that the sequence-specific RNA-binding activity of CSP41 affects the rate, but not the specificity, of its RNase activity, suggesting that CSP41 is probably involved in other events of chloroplast RNA metabolism in addition to RNA degradation. By analyzing C-terminal deletions of CSP41, the RNase domain was located between amino acid residues 73 and 191.

Amino Acid Sequence↗
PubMed2004

Molecular control of a bimodal distribution of quinone-analogue inhibitor binding sites in the cytochrome b(6)f complex.

The 3.0-3.1A X-ray structures of the cytochrome b(6)f complex from Mastigocladus laminosus and Chlamydomonas reinhardtii obtained in the presence of the p-side quinone-analogue inhibitor tridecyl-stigmatellin (TDS) are very similar. A difference occurs in the p-side binding position of TDS. In C.reinhardtii, TDS binds in the ring-in mode, as previously found for stigmatellin in X-ray structures of the cytochrome bc(1) complex. In this mode, the H-bonding chromone ring moiety of the TDS bound in the Q(p) niche is proximal to the ISP [2Fe-2S] cluster, and its 13 carbon tail extends through a portal to the large inter-monomer quinone-exchange cavity. However, in M.laminosus, TDS binds in an oppositely oriented ring-out mode, with the tail inserted toward the Q(p) niche through the portal and the ring caught in the quinone-exchange cavity that is 20A away from the [2Fe-2S] cluster. Site-directed mutagenesis of residues that might determine TDS binding was performed with the related transformable cyanobacterium Synechococcus sp. PCC 7002. The following changes in the sensitivity of electron transport activity to TDS and stigmatellin were observed: (a) little effect of mutation L193A in cytochrome b(6), which is proximal to the chromone of the ring-out TDS; (b) almost complete loss of sensitivity by mutation L111A in the ISP cluster binding region, which is close to the chromone of the ring-in TDS; (c) a ten and 60-fold increase associated with the mutation L81F in subunit IV. It was inferred that only the ring-in binding mode, in which the ring interacts with residues near the ISP, is inhibitory, and that residue 81 of subunit IV, which resides at the immediate entrance to the Q(p) niche, controls the relative binding affinity of inhibitor at the two different binding sites.

Amino Acid Sequence↗
PubMed1999

Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation.

Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elements II and III, located in the center and near the 3' end of the 5' untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

Algal Proteins↗
PubMed2000

The role of chloroplast electron transport and metabolites in modulating Rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase.

Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP(+) ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO(2) assimilation rates decreased with decreasing ribulose 1, 5-bisphosphate concentrations. In anti-b/f plants, CO(2) assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway.

Adenosine Monophosphate↗
PubMed1994

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.

Animals↗
PubMed1994

The petD gene is transcribed by functionally redundant promoters in Chlamydomonas reinhardtii chloroplasts.

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

Animals↗
PubMed1997

Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins.

This work establishes the potential of Bio-Beads as a simple alternative to conventional dialysis for removing detergent and for obtaining 2D crystals of integral membrane proteins useful for structure analysis by electron crystallography. Kinetic and equilibrium aspects of removal of different detergents by adsorption onto hydrophobic Bio-Beads SM2 have been systematically investigated and extended to 2D crystallization of different prototypic membrane proteins, including: (a) Ca2+ ATPase from sarcoplasmic reticulum; (b) melibiose permease from Escherichia coli; (c) cytochrome b6f from Chlamydomonas reinhardtii. Different crystals could be produced from all protein preparations, with optical diffraction down to 20-25 A in negative stain.

Animals↗
PubMed1994

RNA editing of tobacco petB mRNAs occurs both in chloroplasts and non-photosynthetic proplastids.

We found an RNA editing site in the protein coding region of tobacco (Nicotiana tabacum) petB transcripts. This editing (CCA to CUA) leads to an amino acid conversion from proline to leucine. It is observed not only in chloroplasts isolated from tobacco leaves but also in non-photosynthetic proplastids isolated from a tobacco cell culture. Also unspliced pre-mRNA shows complete editing. The editing site is the same as that recently observed in the maize petB transcripts which restores the codon for a highly conserved leucine residue, suggesting that RNA editing at this site is critical for the protein (cytochrome b6) function.

Base Sequence↗
PubMed1998

Rapid diversification of marine picophytoplankton with dissimilar light-harvesting structures inferred from sequences of Prochlorococcus and Synechococcus (Cyanobacteria).

Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade. Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support. This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae. Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus. Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages. Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes. Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions.

Animals↗
PubMed2000

Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum.

Plastid DNA was purified from the dinoflagellate Amphidinium operculatum. The genes atpB, petD, psaA, psbA and psbB have been shown to reside on single-gene minicircles of a uniform size of 2.3-2.4 kb. The psaA and psbB genes lack conventional initiation codons in the expected positions, and may use GTA for translation initiation. There are marked biases in codon preference. The predicted PsbA protein lacks the C-terminal extension which is present in all other photosynthetic organisms except Euglena gracilis, and there are other anomalies elsewhere in the predicted amino acid sequences. The non-coding regions of the minicircles contain a "core" region which includes a number of stretches that are highly conserved across all minicircles and modular regions that are conserved within subsets of the minicircles.

Amino Acid Sequence↗
PubMed2006

Regulatory sequences of orthologous petD chloroplast mRNAs are highly specific among Chlamydomonas species.

The 5' untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5' UTR. To further analyze these elements, we compared 5' UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5' UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5' UTRs was tested in C. reinhardtii chloroplasts using beta-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.

5' Untranslated Regions↗
PubMed1984

A redox study of the electron transport pathway responsible for generation of the slow electrochromic phase in chloroplasts.

The amplitude of the slow phase of the electrochromic bandshift and the dark redox state of cytochrome b6, as well as its flash-induced turnover, have been measured as a function of ambient redox potential between +200 and -200 mV. Formation of a quinol-like donor with an Em,7 = +100 +/- 10 mV is required for generation of the slow phase. 80-100% of the amplitude of this signal with a t 1/2 = 3-4 ms is observed at -200 mV where cytochrome b6 was almost fully reduced (Em,7 of dark and flash-induced photoreduction was -30 mV and -75 mV, respectively). The change in the photoreduction of cytochrome b6 above 0 mV had an Em,7 of +50 mV, about 50 mV more negative than the midpoint at this pH for the onset of the slow electrochromic change. At potentials below -140 mV the amplitude of b6 photoreduction becomes small or negligible. The nature of the cytochrome b6 photoresponse is changed at potentials below -140 mV from a net photoreduction with a t1/2 = approximately less than 1 ms to a photooxidation with a t1/2 = 15-20 ms that is substantially slower than the electrochromic band-shift with a t1/2 = 3-4 ms. It is concluded that the slow electrochromic phase probably does not arise from a mechanism involving a turnover of cytochrome b6. From consideration of the possible flash-induced electron-transfer steps and alternative mechanisms for generation of the slow phase, it is suggested that it may arise from a redox-linked H+ pump involving the high potential iron-sulfur protein.

Chloroplasts↗