Assessment of occupational exposure to stainless steel welding fumes - A human biomonitoring study.

The aim of the study was to determine the concentration of hexavalent and trivalent chromium, nickel, manganese, and iron in welding fumes (WFs) and to evaluate the significant association between the concentration of metals in the biological material of welders. The studies were conducted in welders (n = 67) and controls (n = 52). Stainless steel WFs were continuously collected in the workers' breathing zone during a shift. The serum and urine concentrations of Cr and Ni were determined by ICP-MS. The content of Mn in the whole blood was determined using ET-AAS. The content of Cr in the erythrocytes was determined using ICP-MS. The Cr concentration in the welders' urine positively correlated with a work environment concentration of Cr (R = 0.59, p < 0.0001), Cr(VI) (R = 0.58, p < 0.0001), and Cr(III) (R = 0.64, p < 0.0001) in the inhalable fraction. The Ni concentration in the welders' urine positively correlated with the Ni concentration in the inhalable and respirable fraction (R = 0.34, p < 0.005 and R = 0.44, p < 0.002). The correlation between the Mn concentration in the work environment air and the Mn concentration in the welders' whole blood (R = 0.46, p < 0.0001) was observed.

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.

Proton-coupled electron transfer at the Q(o) site: what type of mechanism can account for the high activation barrier?

In Rhodobacter sphaeroides, transfer of the first electron in quinol oxidation by the bc(1) complex shows kinetic features (a slow rate (approx. 1.5 x 10(3)/s), high activation energy (approx. 65 kJ/mol) and reorganization energy, lambda (2.5 V)) that are unexpected from Marcus theory and the distances shown by the structures. Reduction of the oxidized iron-sulfur protein occurs after formation of the enzyme-substrate complex, and involves a H-transfer in which the electron transfer occurs through the approx. 7 A of a bridging histidine forming a H-bond with quinol and a ligand to 2Fe-2S. The anomalous kinetic features can be explained by a mechanism in which the electron transfer is constrained by coupled transfer of the proton. We discuss this in the context of mutant strains with modified E(m,7) and pK for the iron-sulfur protein, and Marcus theory for proton-coupled electron transfer. We suggest that transfer of the second proton and electron involve movement of semiquinone in the Q(o) site, and rotation of the Glu of the conserved -PEWY- sequence. Mutational studies show a key role for the domain proximal to heme b(L). The effects of mutation at Tyr-302 (Tyr-279 in bovine sequence) point to a possible linkage between conformational changes in the proximal domain, and changes leading to closure of the iron-sulfur protein access channel at the distal domain.

Specific mutagenesis of the rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex.

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome c(2) oxidoreductase (bc(1) complex) of Rhodobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equivalent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B. , and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that mutations at this site can lead to modifications in the redox potential of the ISP. To study the effect of similar modifications on the thermodynamic behavior and kinetics of partial reactions of the bc(1) complex upon flash activation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assembled and able to support photosynthetic growth in all mutants. Three substitutions (Leu, Phe, Trp) led to alteration of the midpoint potential (E(m)) of the ISP and a slowing in rate of quinol oxidation, suggesting that electron transfer from quinol to the oxidized ISP controls the overall rate and that this step includes the high activation barrier. The Trp mutation led to an increase of approximately 1 pH unit in the pK value of the oxidized ISP. The pH dependence of the rate of quinol oxidation in this mutant was also shifted up by approximately 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation of the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEBS Lett. 440, 409-413].

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.

Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides.

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.

Mechanism of ubiquinol oxidation by the bc(1) complex: role of the iron sulfur protein and its mobility.

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c(1) in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c(1) and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion.

Mechanism of ubiquinol oxidation by the bc(1) complex: different domains of the quinol binding pocket and their role in the mechanism and binding of inhibitors.

Structures of mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from several animal sources have provided a basis for understanding the functional mechanism at the molecular level. Using structures of the chicken complex with and without inhibitors, we analyze the effects of mutation on quinol oxidation at the Q(o) site of the complex. We suggest a mechanism for the reaction that incorporates two features revealed by the structures, a movement of the iron sulfur protein between two separate reaction domains on cytochrome c(1) and cytochrome b and a bifurcated volume for the Q(o) site. The volume identified by inhibitor binding as the Q(o) site has two domains in which inhibitors of different classes bind differentially; a domain proximal to heme b(L), where myxothiazole and beta-methoxyacrylate- (MOA-) type inhibitors bind (class II), and a distal domain close to the iron sulfur protein docking interface, where stigmatellin and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiaole (UHDBT) bind (class I). Displacement of one class of inhibitor by another is accounted for by the overlap of their volumes, since the exit tunnel to the lipid phase forces the hydrophobic "tails" to occupy common space. We conclude that the site can contain only one "tailed" occupant, either an inhibitor or a quinol or one of their reaction products. The differential sensitivity of strains with mutations in the different domains is explained by the proximity of the affected residues to the binding domains of the inhibitors. New insights into mechanism are provided by analysis of mutations that affect changes in the electron paramagnetic resonance (EPR) spectrum of the iron sulfur protein, associated with its interactions with the Q(o)-site occupant. The structures show that all interactions with the iron sulfur protein must occur at the distal position. These include interactions between quinone, or class I inhibitors, and the reduced iron sulfur protein and formation of a reaction complex between quinol and oxidized iron sulfur protein. The step with high activation energy is after formation of the reaction complex, likely in formation of the semiquinone and subsequent dissociation of the complex into products. We suggest that further progress of the reaction requires a movement of semiquinone to the proximal position, thus mapping the bifurcated reaction to the bifurcated volume. We suggest that such a movement, together with a change in conformation of the site, would remove any semiquinone formed from further interaction with the oxidized [2Fe-2S] center and also from reaction with O(2) to form superoxide anion. We also identify two separate reaction paths for exit of the two protons released in quinol oxidation.

Steps toward constructing a cytochrome b6 f complex in the purple bacterium Rhodobacter sphaeroides: an example of the structural plasticity of a membrane cytochrome.

We have modified the cytochrome b subunit of the cytochrome bc1 complex from the purple bacterium Rhodobacter sphaeroides to introduce two distinctive features of cytochrome b6 f complexes. In the first one, we have split cyt b into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f complexes. In the second, an extra residue was added between His198 and Phe199, thus extending the span between the histidine ligands for the two b-hemes in helix D. The properties of the mutant strains were determined using thermodynamic and kinetic analysis. The two mutant enzymes were assembled and functioned so as to allow the photosynthetic growth of the mutant strains. For the split enzyme, we show that two independently translated fragments of cyt b are inserted in the membrane. Our results indicate a decrease in the stability of the semiquinone formed at the quinone reduction (Qi) site in this mutant. This property, characteristic for b6 f complexes, indicates the functional importance of the connecting span between helices D and E. The presence of the inserted threonine in helix D modified the spectrum and redox potential of the bL-heme, shifting the potential difference between the two b-hemes from 140 mV in the wild-type to 55 mV in the mutant strain. This change in the driving force of electron transfer through the membrane was reflected in an inability of the mutant strain to accumulate a large transmembrane electrical potential on successive flashes.

Inversions in the Chlamydomonas chloroplast genome suppress a petD 5' untranslated region deletion by creating functional chimeric mRNAs.

FUD6 is a non-photosynthetic Chlamydomonas mutant that lacks the cytochrome b6/f complex, due to a 236 bp deletion that removes the promoter and part of the 5' untranslated region (UTR) of the chloroplast petD gene, which encodes subunit IV of the complex. Two photosynthetic revertants of FUD6 that synthesized wild-type levels of subunit IV were found to contain related inversions of the chloroplast genome that resulted from recombination between small inverted repeats. These inversions created a functional chimeric petD gene that includes the promoter and part of the 5' UTR of the newly identified ycf9-psbM transciption unit, fused to the petD 5' UTR upstream of the FUD6 deletion. Accumulation of the ycf9-psbM dicistronic transcript was disrupted in the revertants, but monocistronic psbM mRNA accumulated normally. The FUD6 revertants demonstrate the ability of the Chlamydomonas chloroplast genome to undergo a large inversion without a deleterious effect on chloroplast function, reminiscent of events that have led to the evolutionary divergence of chloroplast genomes.

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.

Genetic analysis of chloroplast c-type cytochrome assembly in Chlamydomonas reinhardtii: One chloroplast locus and at least four nuclear loci are required for heme attachment.

Chloroplasts contain up to two c-type cytochromes, membrane-anchored cytochrome f and soluble cytochrome c6. To elucidate the post-translational events required for their assembly, acetate-requiring mutants of Chlamydomonas reinhardtii that have combined deficiencies in both plastid-encoded cytochrome f and nucleus-encoded cytochrome c6 have been identified and analyzed. For strains ct34 and ct59, where the phenotype displays uniparental inheritance, the mutations were localized to the chloroplast ccsA gene, which was shown previously to be required for heme attachment to chloroplast apocytochromes. The mutations in another eight strains were localized to the nuclear genome. Complementation tests of these strains plus three previously identified strains of the same phenotype (ac206, F18, and F2D8) indicate that the 11 ccs strains define four nuclear loci, CCS1-CCS4. We conclude that the products of the CCS1-CCS4 loci are not required for translocation or processing of the preproteins but, like CcsA, they are required for the heme attachment step during assembly of both holocytochrome f and holocytochrome c6. The ccsA gene is transcribed in each of the nuclear mutants, but its protein product is absent in ccs1 mutants, and it appears to be degradation susceptible in ccs3 and ccs4 strains. We suggest that Ccsl may be associated with CcsA in a multisubunit "holocytochrome c assembly complex," and we hypothesize that the products of the other CCS loci may correspond to other subunits.

Mutations of cytochrome b6 in Chlamydomonas reinhardtii disclose the functional significance for a proline to leucine conversion by petB editing in maize and tobacco.

We have introduced a proline codon in place of a leucine codon at position 204 of the petB gene of Chlamydomonas reinhardtii. This gene modification mimics the presence of proline codons at the same position in the petB genes of maize and tobacco, which are subsequently edited to leucine codons at the RNA level. Following transformation, we observed no editing at this position in C. reinhardtii, independent of the type of proline codon we have used: the CCA codon, edited in maize, or a CCT codon. Strains carrying the introduced mutation were non phototrophic and displayed a block in photosynthetic electron transfer, consistent with a lack of cytochrome b6f activity. Thus the presence of a proline residue at position 204 in cytochrome b6 is detrimental to photosynthesis. We show that the mutant phenotype arose from a defective assembly of cytochrome b6f complexes and not from altered electron transfer properties in the assembled protein complex. Biochemical comparison of the proline-containing transformants with a cytochrome b6 mutant deficient in heme-attachment indicates that their primary defect is at the level of assembly of apocytochrome b6 with the bh heme, thereby preventing assembly of the whole cytochrome b6f complex.

Molecular genetic identification of a pathway for heme binding to cytochrome b6.

Heme binding to cytochrome b6 is resistant, in part, to denaturing conditions that typically destroy the noncovalent interactions between the b hemes and their apoproteins, suggesting that one of two b hemes of holocytochrome b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b6, and to identify mutants that are blocked at one step of this pathway. Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines that coordinate the bh or bl hemes to the apoprotein were created. These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b6, which allowed us to specifically identify cytochrome b6 with altered bh or bl ligation. In gabaculine-treated (i.e. heme-depleted) wild type and site-directed mutant strains, we established that (i) the single immunoreactive band, observed in strains carrying the bl site-directed mutations, corresponds to apocytochrome b6 and (ii) the additional band present in strains carrying bh site-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b6. Five nuclear mutants (ccb strains) that are defective in holocytochrome b6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochrome b6 assembly, because the ccb strains can synthesize other b cytochromes and all c-type cytochromes. The ccb strains, which define four nuclear loci (CCB1, CCB2, CCB3, and CCB4), provide the first evidence that a b-type cytochrome requires trans-acting factors for its heme association.

Maturation of pre-apocytochrome f in vivo. A site-directed mutagenesis study in Chlamydomonas reinhardtii.

The biosynthesis of cytochrome f is a multistep process which requires processing of the precursor protein and covalent ligation of a c-heme upon membrane insertion of the protein. The crystal structure of a soluble form of cytochrome f has revealed that one axial ligand of the c-heme is provided by the alpha-amino group of Tyr1 generated upon cleavage of the signal sequence from the precursor protein (Martinez S. E., Huang D., Szczepaniak A., Cramer W.A., and Smith J. L. (1994) Structure 2, 95-105). We therefore investigated, by site-directed mutagenesis, the possible interplay between protein processing and heme attachment to cytochrome f in Chlamydomonas reinhardtii. These modifications were performed by chloroplast transformation using a petA gene encoding the full-length precursor protein and also a truncated version lacking the C-terminal membrane anchor. We first substituted the two cysteinyl residues responsible for covalent ligation of the c-heme, by a valine and a leucine, and showed that heme binding is not a prerequisite for cytochrome f processing. In another series of experiments, we replaced the consensus cleavage site for the thylakoid processing peptidase, AQA, by an LQL sequence. The resulting transformants were nonphototrophic and displayed delayed processing of the precursor form of cytochrome f, but nonetheless both the precursor and processed forms showed heme binding and assembled in cytochrome b6f complexes. Thus, pre-apocytochrome f adopts a suitable conformation for the cysteinyl residues to be substrates of the heme lyase and pre-holocytochrome f folds in an assembly-competent conformation. In the last series of experiments, we compared the rates of synthesis and degradation of the various forms of cytochrome f in the four types of transformants under study: (i) the C terminus membrane anchor apparently down-regulates the rate of synthesis of cytochrome f and (ii) degradation of misfolded forms of cytochrome f occurs by a proteolytic system intimately associated with the thylakoid membranes.

Conversion of cytochrome f to a soluble form in vivo in Chlamydomonas reinhardtii.

We introduced a stop codon in place of the ATT codon encoding Ile283 (numbered from the Met initiation codon) in the petA gene from Chlamydomonas reinhardtii. The resulting protein was expected to be truncated on its carboxy-terminus end, lacking the last 35 amino acids. This region of the polypeptide sequence encompasses a hydrophobic stretch assumed to anchor the protein in the thylakoid membrane. Once introduced in whole cells of C. reinhardtii by chloroplast transformation, the modified petA gene expressed a truncated apoprotein which was efficiently converted to a truncated holocytochrome f. This protein accumulated in the lumen of the thylakoids in a soluble form. Thus the conversion of preapocytochrome f to holocytochrome f does not require an interaction with the membrane through its C-terminus anchor. We show that the rest of the cytochrome b6f complex failed to accumulate in the transformants, most probably because of a lack of interaction between soluble cytochrome f and the other cytochrome b6f subunits. However, soluble cytochrome f was still able to donate electrons to photosystem I, which is indicative of its ability to maintain interactions with plastocyanin. The control of the rate of synthesis of cytochrome f by the neighboring subunit, suIV (Kuras & Wollman (1994) EMBO J. 13, 1019-1027), was not observed with the truncated cytochrome f. This observation suggests that either the transmembrane anchor of cytochrome f contains a target for the regulation of cytochrome f translation by suIV or there is a transient form of membrane-bound cytochrome f which is highly sensitive to proteolysis at an early post-translational stage.

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.

The assembly of cytochrome b6/f complexes: an approach using genetic transformation of the green alga Chlamydomonas reinhardtii.

As an approach to the study of the biogenesis of the cytochrome b6/f complex, we characterized the behaviour of its constitutive subunits in mutant strains of Chlamydomonas reinhardtii bearing well-defined mutations. To this end, we have constructed three deletion mutant strains, each lacking one of the major chloroplast pet genes: the delta petA, delta petB and delta petD strains were unable to synthesize cyt f, cyt b6 and subunit IV (suIV) respectively. Western blotting analysis, pulse-labelling and pulse-chase experiments allowed us to compare the cellular accumulation, the rates of synthesis and the turnover of the cyt b6/f subunits remaining in the various strains. We show that the rates of synthesis of cyt b6 and suIV are independent of the presence of the other subunits of the complex but that their stabilization in the thylakoid membranes is a concerted process, with a marked dependence of suIV stability on the presence of cyt b6. In contrast, mature cyt f was stable in the absence of either suIV or cyt b6 but its rate of synthesis was severely decreased in these conditions. We conclude that the stoichiometric accumulation of the chloroplast-encoded subunits of the cyt b6/f complex results from two regulation processes: a post-translational regulation leading to the proteolytic disposal of unassembled cyt b6 and suIV and a co-translational (or early post-translational) regulation which ensures the production of cyt f next to its site of assembly.

Extensive accumulation of an extracellular L-amino-acid oxidase during gametogenesis of Chlamydomonas reinhardtii.

In a previous study [Bulté, L. & Wollman, F.-A. (1992) Eur. J. Biochem. 204, 327-336], we identified a novel gamete-specific polypeptide of Chlamydomonas reinhardtii, M alpha. This 66-kDa polypeptide reacts with antibodies to cytochrome f and accumulates in gametes only in conditions that promote destabilisation of the cytochrome b6/f complex. Here, we show that M alpha is not a modification product of cytochrome f, but is part of protein M, a high-molecular-mass L-amino-acid oxidase located in the periplasm. It catalyzes oxidation of all L-amino acids tested, except cysteine. Using phenylalanine as a substrate, saturation of the enzymatic rate is reached at 2 microM. These characteristics suggest that protein M may operate in vivo as an efficient scavanger of ammonium from extracellular amino acids. The enzyme contains non-covalently bound FAD. It exists in two forms with essentially similar enzymatic properties, of 1.2-1.3 MDa and 0.9-1.0 MDa, respectively. The lighter form is an oligomer of M alpha, while the heavier form contains, in addition to M alpha, a second polypeptide of 135 kDa, M beta, in a molar ratio of 3-4 M alpha/M beta. Both polypeptides are glycosylated.

Activation of the catalytic core of a group I intron by a remote 3' splice junction.

Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions. Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo. Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction. Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation. A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing. A role for these interactions in mediating putative alternative splicing events is discussed.