Catherine de Vitry

Molecular Genetic Identification of a Pathway for Heme Binding to Cytochrome b 6

Heme binding to cytochromeb 6 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 b 6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b 6, 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 b 6, which allowed us to specifically identify cytochrome b 6 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 b 6 and (ii) the additional band present in strains carrying bhsite-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b 6. Five nuclear mutants (ccb strains) that are defective in holocytochromeb 6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochromeb 6 assembly, because the ccbstrains can synthesize other b cytochromes and allc-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.

A specific c-type cytochrome maturation system is required for oxygenic photosynthesis

Oxygenic photosynthesis is an important bioenergetic process that maintains the Earths atmosphere and allows carbon fixation. A critical enzyme in this process, the cytochrome b6f complex, differs from other protein complexes of the same family by an unusual covalently attached cofactor chemically defined as a c′ heme. We have identified a set of pioneer proteins that carry the biogenesis of this c′ heme and started their characterization. They are encoded by the genomes of all organisms performing oxygenic photosynthesis, whatever their phylogenetic distances. These proteins are thus among the few that distinguish photosynthetic cells evolving oxygen from other types of living cells.

Thylakoid targeting of Tat passenger proteins shows no ΔpH dependence in vivo

The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane ΔpH. In this paper, we assess the ΔpH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the ΔpH in vivo. Using experimental conditions in which the trans‐thylakoid ΔpH was almost zero, we found no evidence for a ΔpH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a ΔpH under physiological conditions, but becomes ΔpH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors.

Function of the polypeptides of the photosystem II reaction center in Chlamydomonas reinhardtii. Localization of the primary reactants

Abstract The distribution of the primary quinone and of the pheophytin acceptors has been studied in PS II particles isolated from Chlamydomonas reinhardtii , with respect to the distribution of the apoproteins of the two chlorophyll-protein complexes associated with the PS II core. We show that photoreduction of the primary quinone requires the presence of the 50 and 47 kDa polypeptides. On the contrary, charge separation between P-680 and the pheophytin acceptor molecules can occur within the chlorophyll-protein complex of which the 50 kDa polypeptide is the apoprotein. Functional analysis of the PS II fractions shows that an active PS II center contains one photoreducible quinone and one photoreducible pheophytin per 45 chlorophyll molecules. Stoichiometric analysis of the PS II fractions shows that a PS II reaction center contains 45 chlorophyll molecules associated with most likely one copy of the 50 kDa and the 47 kDa polypeptides.

Effect of inhibitors, redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Abstract Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of 14 C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q B , ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o -Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o -phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q A to Q B ) occurs via displacement of UQ from the Q B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q B to Q − B but is practically unaffected by reduction of Q A to Q − A in the absence of Q B . UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q B site.

The 4-kDa nuclear-encoded PetM polypeptide of the chloroplast cytochrome b6f complex. Nucleic acid and protein sequences, targeting signals, transmembrane topology.

The 4-kDa subunit of cytochrome bf complex encoded by the nuclear PetM gene in Chlamydomonas reinhardtii has been characterized. 38 of the 39 residues of the mature protein have been established by Edman degradation, a cDNA clone encoding the complete precursor has been isolated and sequenced, and a 0.6-kb transcript detected. The deduced amino acid sequence of the precursor includes an N-terminal transit peptide of 60 amino acids with stromal targeting features. Examination of the sequence suggests that PetM spans the membrane as a single transmembrane α-helix, which is supported by its non-extractability following dissociating treatments. When PetM and PetG, another small subunit of the bf complex, are folded into α-helices, an array of identical residues becomes apparent. Proteolysis data, charge distribution, and homology with PetG are consistent with a lumenal localization of the N terminus of PetM.

Analysis of the Nucleus-Encoded and Chloroplast-Targeted Rieske Protein by Classic and Site-Directed Mutagenesis of Chlamydomonas

Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron–sulfur protein 2Fe–2S subunit of the chloroplast cytochrome b6f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b6f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe–2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.

Thylakoid FtsH Protease Contributes to Photosystem II and Cytochrome b6f Remodeling in Chlamydomonas reinhardtii under Stress Conditions

FtsH is a ubiquitous protease that mediates the degradation of membrane proteins. The chloroplast FtsH is known for its involvement in repairing photosystem II damaged by light. This work reports the characterization of an ftsh mutant in Chlamydomonas and demonstrates the wider role of FtsH in thylakoid membrane protein maintenance under light as well as nutrient stresses. FtsH is the major thylakoid membrane protease found in organisms performing oxygenic photosynthesis. Here, we show that FtsH from Chlamydomonas reinhardtii forms heterooligomers comprising two subunits, FtsH1 and FtsH2. We characterized this protease using FtsH mutants that we identified through a genetic suppressor approach that restored phototrophic growth of mutants originally defective for cytochrome b6f accumulation. We thus extended the spectrum of FtsH substrates in the thylakoid membranes beyond photosystem II, showing the susceptibility of cytochrome b6f complexes (and proteins involved in the ci heme binding pathway to cytochrome b6) to FtsH. We then show how FtsH is involved in the response of C. reinhardtii to macronutrient stress. Upon phosphorus starvation, photosynthesis inactivation results from an FtsH-sensitive photoinhibition process. In contrast, we identified an FtsH-dependent loss of photosystem II and cytochrome b6f complexes in darkness upon sulfur deprivation. The D1 fragmentation pattern observed in the latter condition was similar to that observed in photoinhibitory conditions, which points to a similar degradation pathway in these two widely different environmental conditions. Our experiments thus provide extensive evidence that FtsH plays a major role in the quality control of thylakoid membrane proteins and in the response of C. reinhardtii to light and macronutrient stress.

A novel pathway of cytochrome c biogenesis is involved in the assembly of the cytochrome b6f complex in arabidopsis chloroplasts.

We recently characterized a novel heme biogenesis pathway required for heme ci′ covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme ci′ binding.

Photosynthetic growth despite a broken Q-cycle

Central in respiration or photosynthesis, the cytochrome bc1 and b6f complexes are regarded as functionally similar quinol oxidoreductases. They both catalyse a redox loop, the Q-cycle, which couples electron and proton transfer. This loop involves a bifurcated electron transfer step considered as being mechanistically mandatory, making the Q-cycle indispensable for growth. Attempts to falsify this paradigm in the case of cytochrome bc1 have failed. The rapid proteolytic degradation of b6f complexes bearing mutations aimed at hindering the Q-cycle has precluded so far the experimental assessment of this model in the photosynthetic chain. Here we combine mutations in Chlamydomonas that inactivate the redox loop but preserve high accumulation levels of b6f complexes. The oxidoreductase activity of these crippled complexes is sufficient to sustain photosynthetic growth, which demonstrates that the Q-cycle is dispensable for oxygenic photosynthesis.