Biomedical subjects

F A Wollman

Publications and source records attributed to F A Wollman.

At least 19 recordsLinked to original sources

PubMed2001

State transitions reveal the dynamics and flexibility of the photosynthetic apparatus.

The chloroplast-based photosynthetic apparatus of plants and algae associates various redox cofactors and pigments with approximately 70 polypeptides to form five major transmembrane protein complexes. Among these are two photosystems that have distinct light absorption properties but work in series to produce reducing equivalents aimed at the fixation of atmospheric carbon. A short term chromatic adaptation known as 'State transitions' was discovered thirty years ago that allows photosynthetic organisms to adapt to changes in light quality and intensity which would otherwise compromise the efficiency of photosynthetic energy conversion. A two-decade research effort has finally unraveled the major aspects of the molecular mechanism responsible for State transitions, and their physiological significance has been revisited. This review describes how a-still elusive-regulatory kinase senses the physiological state of the photosynthetic cell and triggers an extensive supramolecular reorganization of the photosynthetic membranes. The resulting picture of the photosynthetic apparatus is that of a highly flexible energy convertor that adapts to the ever-changing intracellular demand for ATP and/or reducing power.

Cytochrome b Group↗

PubMed2001

Possible role for molecular chaperones in assembly and repair of photosystem II.

Genes of the HSP70 chaperone family are induced by light. In Chlamydomonas reinhardtii, the induction of HSP70 (70 kDa heat shock protein) chaperones by light results in a partial protection of photosystem II against damage by photoinhibitory conditions. Underexpression of a chloroplast-localized HSP70 protein caused an increased sensitivity of photosystem II to light. Overexpression of this protein had a protective effect. Fluorescence measurements and studies of the turnover of photosystem II core components suggest that this HSP70 might function in both the protection and the regeneration of photosystem II. This concept is supported by fractionation studies in which the plastid HSP70 was found associated with chloroplast membranes. Because the light-induced elevation of HSP70 levels provides protection for photosystem II, we examined whether the chloroplast is involved in this regulation and found that mutants defective in plastid-localized chlorophyll synthesis, i.e. the insertion of Mg(2+) into protoporphyrin IX are impaired in the induction of HSP70 by light. Exogenous addition of Mg-protoporphyrin in the dark induced the genes. The combined results support a model in which chlorophyll precursors are essential in the signalling from chloroplast to nucleus that regulates the chaperone genes.

Animals↗

PubMed2001

Assembly-controlled regulation of chloroplast gene translation.

Studies of the biogenesis of the photosynthetic protein complexes in the unicellular green alga Chlamydomonas reinhardtii have pointed to the importance of the concerted expression of nuclear and chloroplast genomes. The accumulation of chloroplast- and nuclear-encoded subunits is concerted, most often as a result of the rapid proteolytic disposal of unassembled subunits, but the rate of synthesis of some chloroplast-encoded subunits from photosynthetic protein complexes, designed as CES proteins (Controlled by Epistasy of Synthesis), is regulated by the availability of their assembly partners from the same complex. Cytochrome f, a major subunit of the cytochrome b(6)f complex is a model protein for the study of the CES process. In the absence of subunit IV, another subunit of the cytochrome b(6)f complex, its synthesis is decreased by 90%. This results from a negative autoregulation of cytochrome f translation initiation, mediated by a regulatory motif carried by the C-terminal domain of the unassembled protein [Choquet, Stern, Wostrikoff, Kuras, Girard-Bascou and Wollman (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4380-4385]. Using site-directed mutagenesis, we have characterized this regulatory motif. We discuss the possible implications regarding the mechanism of the CES process for cytochrome f expression. We have studied the possible generalization of this mechanism to other CES proteins.

5' Untranslated Regions↗

PubMed2001

TCA1, a single nuclear-encoded translational activator specific for petA mRNA in Chlamydomonas reinhardtii chloroplast.

We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5' untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of cytochrome b(6)f complex petA mRNA. The high number of TCA1 alleles, together with the absence of genetic evidence for other nuclear loci controlling translation of the chloroplast petA gene, strongly suggests that TCA1 is the only trans-acting factor. We studied the assembly-dependent regulation of cytochrome f translation--known as the CES process--in TCA1-mutated contexts. In the presence of a leaky tca1 allele, we observed that the regulation of cytochrome f translation was now exerted within the limits of the restricted translational activation conferred by the altered version of TCA1 as predicted if TCA1 was the ternary effector involved in the CES process.

5' Untranslated Regions↗

PubMed2001

The light sensitivity of ATP synthase mutants of Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii mutants defective in the chloroplast ATP synthase are highly sensitive to light. The ac46 mutant is affected in the MDH1 gene, required for production or stability of the monocistronic atpH mRNA encoding CF(O)-III. In this and other ATP synthase mutants, we show that short-term exposure to moderate light intensities-a few minutes-induces an inhibition of electron transfer after the primary quinone acceptor of photosystem II (PSII), whereas longer exposure-several hours-leads to a progressive loss of PSII cores. An extensive swelling of thylakoids accompanies the initial inhibition of electron flow. Thylakoids deflate as PSII cores are lost. The slow process of PSII degradation involves the participation of ClpP, a chloroplast-encoded peptidase that is part of a major stromal protease Clp. In the light of the above findings, we discuss the photosensitivity of ATP synthase mutants with respect to the regular photoinhibition process that affects photosynthetic competent strains at much higher light intensities.

Adenosine Triphosphatases↗

PubMed2001

The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing.

In eubacteria and mitochondria, Hsp70 chaperone activity is controlled by the nucleotide exchange factor GrpE. We have identified the chloroplastic GrpE homolog of Chlamydomonas, CGE1, as an approximately 26-kD protein coimmunoprecipitating with the stromal HSP70B protein. When expressed in Escherichia coli, CGE1 can functionally replace GrpE and interacts physically with DnaK. CGE1 is encoded by a single-copy gene that is induced strongly by heat shock and slightly by light. Alternative splicing generates two isoforms that differ only by two residues in the N-terminal part. The larger form is synthesized preferentially during heat shock, whereas the smaller one dominates at lower temperatures. Fractions of both HSP70B and CGE1 associate with chloroplast membranes in an ATP-sensitive manner. By colorless native PAGE and pulse labeling, CGE1 monomers were found to assemble rapidly into dimers and tetramers. In addition, CGE1 was found to form ATP-sensitive complexes with HSP70B of approximately 230 and approximately 120 kD, the latter increasing dramatically after heat shock.

Alternative Splicing↗

PubMed2001

The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture.

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Amino Acid Sequence↗

PubMed2000

Contrasted effects of inhibitors of cytochrome b6f complex on state transitions in Chlamydomonas reinhardtii: the role of Qo site occupancy in LHCII kinase activation.

We have investigated the relationship between the occupancy of the Q(o) site in the cytochrome b(6)f complex and the activation of the LHCII protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII proteins, while subunit V of the cytochrome b(6)f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b(6)f that relates the activation of the LHCII kinase to the occupancy of the Q(o) site and the movement of the Rieske protein.

Animals↗

PubMed2000

A new subunit of cytochrome b6f complex undergoes reversible phosphorylation upon state transition.

A 15.2-kDa polypeptide, encoded by the nuclear gene PETO, was identified as a novel cytochrome b(6)f subunit in Chlamydomonas reinhardtii. The PETO gene product is a bona fide subunit, subunit V, of the cytochrome b(6)f complex, because (i) it copurifies with the other cytochrome b(6)f subunits in the early stages of the purification procedure, (ii) it is deficient in cytochrome b(6)f mutants accumulating little of the complex, and (iii) it colocalizes with cytochrome f, which migrates between stacked and unstacked membrane regions upon state transition. Sequence analysis and biochemical characterization of subunit V shows that it has a one transmembrane alpha-helix topology with two large hydrophilic domains extending on the stromal and lumenal side of the thylakoid membranes, with a lumenal location of the N terminus. Subunit V is reversibly phosphorylated upon state transition, a unique feature that, together with its topological organization, points to the possible role of subunit V in signal transduction during redox-controlled short term and long term adaptation of the photosynthetic apparatus in eukaryotes.

Amino Acid Sequence↗

PubMed2000

Identification of the initiation codon for the atpB gene in Chlamydomonas chloroplasts excludes translation of a precursor form of the beta subunit of the ATP synthase.

The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.

5' Untranslated Regions↗

PubMed2000

Post-transcriptional defects in tobacco chloroplast mutants lacking the cytochrome b6/f complex.

A variety of post-transcriptional mechanisms govern the synthesis and assembly of photosynthetic protein complexes in chloroplasts. To test whether such mechanisms are conserved between photosynthetic algae and vascular plants, we have interrupted the chloroplast petA, petB and petD genes of tobacco, which encode three subunits of the cytochrome b6/f complex, and compared our results to those previously obtained with Chlamydomonas reinhardtii. As expected, the mutants exhibited high chlorophyll fluorescence, consistent with the loss of a functional cytochrome b6/f complex. Unlike the corresponding mutants of Chlamydomonas, however, cytochrome f was barely detectable in the DeltapetB or DeltapetD mutants. The amounts of petB- and petD-containing mRNAs were reduced in the mutants compared to wild-type plants, but the remaining mRNA was normally associated with polysomes. In contrast, there was a decrease in polysome association of the polycistronic petA mRNA in the DeltapetB and DeltapetD mutants, suggesting that the synthesis of cytochrome f may be decreased in the absence of cytochrome b6 or SUIV. These results are discussed in light of the translational autoregulation model that has been proposed for cytochrome b6/f complex assembly in Chlamydomonas.

Chloroplasts↗

PubMed2000

Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex.

In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels. It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease.

Adenosine Triphosphatases↗

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

Functional characterization of Chlamydomonas mutants defective in cytochrome f maturation.

We have altered the N terminus of cytochrome f by site-directed mutagenesis of the chloroplast petA gene in Chlamydomonas reinhardtii. We have replaced the tyrosine residue, Tyr(32), located immediately downstream of the processing site Ala(29)-Gln(30)-Ala(31) by a proline. Tyr(32) is the N terminus of the mature protein and serves as the sixth axial ligand to the heme iron. This mutant, F32P, accumulated different forms of holocytochrome f and assembled them into the cytochrome b(6)f complex. The strain was able to grow phototrophically. Our results therefore contradict a previous report (Zhou, J., Fernandez-Velasco, J. G., and Malkin, R. (1996) J. Biol. Chem. 271, 1-8) that a mutation, considered to be identical to the mutation described here, prevented cytochrome b(6)f assembly. A comparative functional characterization of F32P with F29L-31L, a site-directed processing mutant in which we had replaced the processing site by a Leu(29)-Gln(30)-Leu(31) sequence (2), revealed that both mutants accumulate high spin cytochrome f, with an unusual orientation of the heme and low spin cytochrome f with an alpha-band peak at 552 nm. Both hemes have significantly lower redox potentials than wild type cytochrome f. We attribute the high spin form to uncleaved pre-holocytochrome f and the low spin form to misprocessed forms of cytochrome f that were cleaved at a position different from the regular Ala(29)-Gln-Ala(31) motif. In contrast to F29L-31L, F32P displayed a small population of functional cytochrome f, presumably cleaved at Ala(29), with characteristics close to those of wild type cytochrome f. The latter form would account for cytochrome b(6)f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P.

Amino Acid Sequence↗

PubMed1999

The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.

Amino Acid Sequence↗

PubMed1999

A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition.

Dark-grown Chlamydomonas reinhardtii cultures that were illuminated at low fluence rates before exposure to high-light conditions exhibited a faster rate of recovery from photoinhibition than did dark-grown cells that were directly exposed to photoinhibitory conditions. This pretreatment has been shown to induce the expression of several nuclear heat shock protein 70 (HSP70) genes, including HSP70B, encoding a chloroplast-localized chaperone. To investigate a possible role of plastidic HSP70B in photoprotection and repair of photosystem II, which is the major target of photoinhibition, we have constructed strains overexpressing or underexpressing HSP70B. The effect of light stress on photosystem II in nuclear transformants harboring HSP70B in the sense or antisense orientation was monitored by measuring variable fluorescence, flash-induced charge separation, and relative amounts of various photosystem II polypeptides. Underexpression of HSP70B caused an increased light sensitivity of photosystem II, whereas overexpression of HSP70B had a protective effect. Furthermore, the reactivation of photosystem II after photoinhibition was enhanced in the HSP70B-overexpressing strain when compared with the wild type, both in the presence or absence of synthesis of chloroplast-encoded proteins. Therefore, HSP70B may participate in vivo both in the molecular protection of the photosystem II reaction centers during photoinhibition and in the process of photosystem II repair.

Animals↗

PubMed1998

Glu78, from the conserved PEWY sequence of subunit IV, has a key function in cytochrome b6f turnover.

We have investigated the structure to function relationship at the Qo site in cytochrome b6f complexes in vivo. To this end, we created site-directed mutants of Chlamydomonas reinhardtii, at position 78 in the sequence of subunit IV. The target glutamic acid, present in the highly conserved 77PEWY80 sequence, was changed to residues of different polarities which did not prevent the functional assembly of cytochrome b6f complexes. Spectroscopic analysis performed in anaerobic conditions in vivo revealed distinct alterations in cytochrome b6f function, depending on the nature of the substituted residue. The semiconservative E78D substitution, in which only the length of the side chain is reduced, retained the functional features of the wild-type configuration. The E78K and E78L substitutions caused a significant decrease, by factors of 3 and 5, respectively, in the rate of the concerted oxidation process at the Qo site without a change in the affinity of Qo for reduced plastoquinones. The E78Q and E78N substitutions modified the characteristics of cytochrome b6f turnover under repetitive flash illumination. They caused a large increase in the electrogenicity of the electron-transfer reactions through the mutated cytochrome b6f complex. This increase was specifically sensitive to the electrical component of the proton-motive force. Surprisingly, despite the larger number of charges translocated across the membrane per charge injected in the high potential chain, the reduction phase for cytochrome b6 became barely detectable in the mutants, unless inhibitors at the Qi site were present. We show that similar functional characteristics can be observed with the cytochrome b6f complex in the wild-type in anaerobic conditions, provided a single flash illumination regime is used. These observations suggest that cytochrome b6f turnover may involve a mechanism implying an extra proton pumping activity.

Amino Acid Sequence↗

PubMed1998

Translation of cytochrome f is autoregulated through the 5' untranslated region of petA mRNA in Chlamydomonas chloroplasts.

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b6f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5' untranslated region of petA mRNA, which encodes cytochrome f, and the C-terminal domain of the unassembled protein. This model for the regulation of cytochrome f translation explains both the decreased rate of cytochrome f synthesis in vivo in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented. When expressed from a chimeric mRNA containing the atpA 5' untranslated region, cytochrome f no longer showed an assembly-dependent regulation of translation. Conversely, the level of antibiotic resistance conferred by a chimeric petA-aadA-rbcL gene was shown to depend on the state of assembly of cytochrome b6f complexes and on the accumulation of the C-terminal domain of cytochrome f. We discuss the possible ubiquity of the CES process in organellar protein biogenesis.

Animals↗