Supramolecular associations between atypical oxidative phosphorylation complexes of Euglena gracilis.

In vivo associations of respiratory complexes forming higher supramolecular structures are generally accepted nowadays. Supercomplexes (SC) built by complexes I, III and IV and the so-called respirasome (I/III2/IV) have been described in mitochondria from several model organisms (yeasts, mammals and green plants), but information is scarce in other lineages. Here we studied the supramolecular associations between the complexes I, III, IV and V from the secondary photosynthetic flagellate Euglena gracilis with an approach that involves the extraction with several mild detergents followed by native electrophoresis. Despite the presence of atypical subunit composition and additional structural domains described in Euglena complexes I, IV and V, canonical associations into III2/IV, III2/IV2 SCs and I/III2/IV respirasome were observed together with two oligomeric forms of the ATP synthase (V2 and V4). Among them, III2/IV SC could be observed by electron microscopy. The respirasome was further purified by two-step liquid chromatography and showed in-vitro oxygen consumption independent of the addition of external cytochrome c.

The mechanism of cyclic electron flow.

Apart from the canonical light-driven linear electron flow (LEF) from water to CO2, numerous regulatory and alternative electron transfer pathways exist in chloroplasts. One of them is the cyclic electron flow around Photosystem I (CEF), contributing to photoprotection of both Photosystem I and II (PSI, PSII) and supplying extra ATP to fix atmospheric carbon. Nonetheless, CEF remains an enigma in the field of functional photosynthesis as we lack understanding of its pathway. Here, we address the discrepancies between functional and genetic/biochemical data in the literature and formulate novel hypotheses about the pathway and regulation of CEF based on recent structural and kinetic information.

Maximal cyclic electron flow rate is independent of PGRL1 in Chlamydomonas.

Cyclic electron flow (CEF) is defined as a return of the reductants from the acceptor side of Photosystem I (PSI) to the pool of its donors via the cytochrome b6f. It is described to be complementary to the linear electron flow and essential for photosynthesis. However, despite many efforts aimed to characterize CEF, its pathway and its regulation modes remain equivocal, and its physiological significance is still not clear. Here we use novel spectroscopic to measure the rate of CEF at the onset of light in the green alga Chlamydomonas reinhardtii. The initial redox state of the photosynthetic chain or the oxygen concentration do not modify the initial maximal rate of CEF (60 electrons per second per PSI) but rather strongly influence its duration. Neither the maximal rate nor the duration of CEF are different in the pgrl1 mutant compared to the wild type, disqualifying PGRL1 as the ferredoxin-plastoquinone oxidoreductase involved in the CEF mechanism.

Host-pathogen biotic interactions shaped vitamin K metabolism in Archaeplastida.

Menaquinone (vitamin K2) shuttles electrons between membrane-bound respiratory complexes under microaerophilic conditions. In photosynthetic eukaryotes and cyanobacteria, phylloquinone (vitamin K1) participates in photosystem I function. Here we elucidate the evolutionary history of vitamin K metabolism in algae and plants. We show that Chlamydiales intracellular pathogens made major genetic contributions to the synthesis of the naphthoyl ring core and the isoprenoid side-chain of these quinones. Production of the core in extremophilic red algae is under control of a menaquinone (Men) gene cluster consisting of 7 genes that putatively originated via lateral gene transfer (LGT) from a chlamydial donor to the plastid genome. In other green and red algae, functionally related nuclear genes also originated via LGT from a non-cyanobacterial, albeit unidentified source. In addition, we show that 3-4 of the 9 required steps for synthesis of the isoprenoid side chains are under control of genes of chlamydial origin. These results are discussed in the light of the hypoxic response experienced by the cyanobacterial endosymbiont when it gained access to the eukaryotic cytosol.

The atypical subunit composition of respiratory complexes I and IV is associated with original extra structural domains in Euglena gracilis.

In mitochondrial oxidative phosphorylation, electron transfer from NADH or succinate to oxygen by a series of large protein complexes in the inner mitochondrial membrane (complexes I-IV) is coupled to the generation of an electrochemical proton gradient, the energy of which is utilized by complex V to generate ATP. In Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes, these respiratory complexes totalize more than 40 Euglenozoa-specific subunits along with about 50 classical subunits described in other eukaryotes. In the present study the Euglena proton-pumping complexes I, III, and IV were purified from isolated mitochondria by a two-steps liquid chromatography approach. Their atypical subunit composition was further resolved and confirmed using a three-steps PAGE analysis coupled to mass spectrometry identification of peptides. The purified complexes were also observed by electron microscopy followed by single-particle analysis. Even if the overall structures of the three oxidases are similar to the structure of canonical enzymes (e.g. from mammals), additional atypical domains were observed in complexes I and IV: an extra domain located at the tip of the peripheral arm of complex I and a "helmet-like" domain on the top of the cytochrome c binding region in complex IV.

The peculiar NPQ regulation in the stramenopile Phaeomonas sp. challenges the xanthophyll cycle dogma.

In changing light conditions, photosynthetic organisms develop different strategies to maintain a fine balance between light harvesting, photochemistry, and photoprotection. One of the most widespread photoprotective mechanisms consists in the dissipation of excess light energy in the form of heat in the photosystem II antenna, which participates to the Non Photochemical Quenching (NPQ) of chlorophyll fluorescence. It is tightly related to the reversible epoxidation of xanthophyll pigments, catalyzed by the two enzymes, the violaxanthin deepoxidase and the zeaxanthin epoxidase. In Phaeomonas sp. (Pinguiophyte, Stramenopiles), we show that the regulation of the heat dissipation process is different from that of the green lineage: the NPQ is strictly proportional to the amount of the xanthophyll pigment zeaxanthin and the xanthophyll cycle enzymes are differently regulated. The violaxanthin deepoxidase is already active in the dark, because of a low luminal pH, and the zeaxanthin epoxidase shows a maximal activity under moderate light conditions, being almost inactive in the dark and under high light. This light-dependency mirrors the one of NPQ: Phaeomonas sp. displays a large NPQ in the dark as well as under high light, which recovers under moderate light. Our results pinpoint zeaxanthin epoxidase activity as the prime regulator of NPQ in Phaeomonas sp. and therefore challenge the deepoxidase-regulated xanthophyll cycle dogma.

Impact of a mutation in the mitochondrial LSU rRNA gene from Chlamydomonas reinhardtii on the activity and the assembly of respiratory-chain complexes.

Two substitutions A1090G and A1098C (together called the m mutation) located in the conserved GTPase domain of the mitochondrial LSU rRNA gene were recently shown to weakly compensate for the phenotypical effect of a -1T frameshift mutation in the mitochondrial cox1 gene of C. reinhardtii. In order to analyze the impact of the m mutation on the mitochondrial translational machinery, a strain carrying the m mutation but wild-type for the cox1 gene was isolated. We found that the growth and the respiratory rate of the m mutant were affected and that the activities of complexes I, III, and IV, all containing mitochondria-encoded subunits, were lowered. In contrast the activities of complex II and of the alternative oxidase, both encoded exclusively by the nuclear genome, were not modified. The steady-state levels of complex I enzyme and of several components of the respiratory complexes I, III, and IV were also reduced in the mutant. We moreover showed that m did not suppress other frameshift or UGA stop mutations which affect mitochondrial genes.

Structure of the telomeric ends of mt DNA, transcriptional analysis and complex I assembly in the dum24 mitochondrial mutant of Chlamydomonas reinhardtii.

The dum24 mutant of Chlamydomonas reinhardtii contains four types of altered mitochondrial linear genomes: two types of deleted monomers and two types of dimers resulting from fusions between some monomers via their deleted ends. All molecules lack at least cob, nd4 and the 3' end of nd5, three adjacent genes located in the left part of the genome. We present evidence showing that in dum24, as in other deletion mutants, the deletions extend to the left telomeric end, and propose that the only replicative forms in the mutants are the dimeric DNA molecules that possess intact telomeric structures at both ends. Two abnormally large transcripts produced from chimeric genes are detected in dum24, which throws some light on the location of potential promoter sequences and processing signals in the mitochondrial genome. Using BN-PAGE analysis and immunological methods to detect complex I, we further show that dum24 mitochondria do not possess the normal multimeric complex I (850 kDa), but produce a smaller, partially assembled, complex (650 kDa), demonstrating a role for ND4 and/or ND5 subunits(s) in complex I assembly.

Mutations inactivating mitochondrial genes in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is now becoming a useful model for the study of mitochondrial genetics in a photosynthetic organism. The small (15.8 kb) mitochondrial genome C. reinhardtii has been sequenced completely and all the genes have been identified. Several mutants inactivated in mitochondrial genes encoding components of the respiratory complexes I, III and IV have been characterized at the molecular level. Assembly of complex I in several mutant strains and mapping of mitochondrial mutations by recombinational analysis are also described.

Mutants of Chlamydomonas reinhardtii deficient in mitochondrial complex I: characterization of two mutations affecting the nd1 coding sequence.

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) comprises more than 30 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components of complex I are coded for by mitochondrial genes. Three mutants deprived of complex I activity and displaying slow growth in the dark were isolated after mutagenic treatment with acriflavine. A genetical analysis demonstrated that two mutations (dum20 and dum25) affect the mitochondrial genome whereas the third mutation (dn26) is of nuclear origin. Recombinational analyses showed that dum20 and dum25 are closely linked on the genetic map of the mitochondrial genome and could affect the nd1 gene. A sequencing analysis confirmed this conclusion: dum20 is a deletion of one T at codon 243 of nd1; dum25 corresponds to a 6-bp deletion that eliminates two amino acids located in a very conserved hydrophilic segment of the protein.