The unicellular green alga Chlamydomonas reinhardtii is a prime model for deciphering processes occurring in the intracellular compartments of the photosynthetic cell. Organelle-specific proteomic studies have started to delineate its various subproteomes, but sequence-based prediction software is necessary to assign proteins subcellular localizations at whole genome scale. Unfortunately, existing tools are oriented toward land plants and tend to mispredict the localization of nuclear-encoded algal proteins, predicting many chloroplast proteins as mitochondrion targeted. We thus developed a new tool called PredAlgo that predicts intracellular localization of those proteins to one of three intracellular compartments in green algae: the mitochondrion, the chloroplast, and the secretory pathway. At its core, a neural network, trained using carefully curated sets of C. reinhardtii proteins, divides the N-terminal sequence into overlapping 19-residue windows and scores the probability that they belong to a cleavable targeting sequence for one of the aforementioned organelles. A targeting prediction is then deduced for the protein, and a likely cleavage site is predicted based on the shape of the scoring function along the N-terminal sequence. When assessed on an independent benchmarking set of C. reinhardtii sequences, PredAlgo showed a highly improved discrimination capacity between chloroplast- and mitochondrion-localized proteins. Its predictions matched well the results of chloroplast proteomics studies. When tested on other green algae, it gave good results with Chlorophyceae and Trebouxiophyceae but tended to underpredict mitochondrial proteins in Prasinophyceae. Approximately 18% of the nuclear-encoded C. reinhardtii proteome was predicted to be targeted to the chloroplast and 15% to the mitochondrion.

Pyruvate formate-lyase (PFL) catalyzes the non-oxidative conversion of pyruvate to formate and acetyl-CoA. PFL and its activating enzyme (PFL-AE) are common among strict anaerobic and microaerophilic prokaryotes but are very rare among eukaryotes. In a proteome survey of isolated Chlamydomonas reinhardtii mitochondria, we found several PFL-specific peptides leading to the identification of cDNAs for PFL and PFL-AE, establishing the existence of a PFL system in this photosynthetic algae. Anaerobiosis and darkness led to increased PFL transcripts but had little effect on protein levels, as determined with antiserum raised against C. reinhardtii PFL. Protein blots revealed the occurrence of PFL in both chloroplast and mitochondria purified from aerobically grown cells. Mass spectrometry sequencing of C. reinhardtii mitochondrial proteins, furthermore, identified peptides for phosphotransacetylase and acetate kinase. The phosphotransacetylase-acetate kinase pathway is a common route of ATP synthesis or acetate assimilation among prokaryotes but is novel among eukaryotes. In addition to PFL and pyruvate dehydrogenase, the algae also expresses pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde/alcohol dehydrogenase. Among eukaryotes, the oxygen producer C. reinhardtii has the broadest repertoire of pyruvate-, ethanol-, and acetate-metabolizing enzymes described to date, many of which were previously viewed as specific to anaerobic eukaryotic lineages.

Living cells are able to harvest energy by coupling exergonic electron transfer between reducing and oxidising substrates to the generation of chemiosmotic potential. Whereas a wide variety of redox substrates is exploited by prokaryotes resulting in very diverse layouts of electron transfer chains, the ensemble of molecular architectures of enzymes and redox cofactors employed to construct these systems is stunningly small and uniform. An overview of prominent types of electron transfer chains and of their characteristic electrochemical parameters is presented. We propose that basic thermodynamic considerations are able to rationalise the global molecular make-up and functioning of these chemiosmotic systems. Arguments from palaeogeochemistry and molecular phylogeny are employed to discuss the evolutionary history leading from putative energy metabolisms in early life to the chemiosmotic diversity of extant organisms. Following the Occam's razor principle, we only considered for this purpose origin of life scenarios which are contiguous with extant life. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.

Anaerobic metabolic pathways allow unicellular organisms to tolerate or colonize anoxic environments. Over the past ten years, genome sequencing projects have brought a new light on the extent of anaerobic metabolism in eukaryotes. A surprising development has been that free-living unicellular algae capable of photoautotrophic lifestyle are, in terms of their enzymatic repertoire, among the best equipped eukaryotes known when it comes to anaerobic energy metabolism. Some of these algae are marine organisms, common in the oceans, others are more typically soil inhabitants. All these species are important from the ecological (O(2)/CO(2) budget), biotechnological, and evolutionary perspectives. In the unicellular algae surveyed here, mixed-acid type fermentations are widespread while anaerobic respiration, which is more typical of eukaryotic heterotrophs, appears to be rare. The presence of a core anaerobic metabolism among the algae provides insights into its evolutionary origin, which traces to the eukaryote common ancestor. The predicted fermentative enzymes often exhibit an amino acid extension at the N-terminus, suggesting that these proteins might be compartmentalized in the cell, likely in the chloroplast or the mitochondrion. The green algae Chlamydomonas reinhardtii and Chlorella NC64 have the most extended set of fermentative enzymes reported so far. Among the eukaryotes with secondary plastids, the diatom Thalassiosira pseudonana has the most pronounced anaerobic capabilities as yet. From the standpoints of genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism in C. reinhardtii remains the best characterized among photosynthetic protists. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle.

International audienceNuclear activities generate radioactive elements which require processes for their decontamination. Although biological remediation has proved efficient in industrial applications, no biotechnology solution is currently operational for highly radioactive media. Such a solution requires organisms that accumulate radionuclides while withstanding radioactivity. This paper describes the potentialities of an extremophile autotrophic eukaryote, Coccomyxa actinabiotis nov. sp., that we isolated from a nuclear facility and which withstands huge ionizing radiation doses, up to 20,000 Gy. Half the population survives 10,000 Gy, which is comparable to the hyper-radioresistant wellknown prokaryote Deinococcus radiodurans. Cell metabolic profile investigated by nuclear magnetic resonance was hardly affected by radiation doses of up to 10,000 Gy. Cellular functioning completely recovered within a few days. This outstanding microalga also strongly accumulates radionuclides, including 238U, 137Cs, 110mAg, 60Co, 54Mn, 65Zn, and 14C (decontamination above 85% in 24 h, concentration factor, 1,000-450,000 mL g-1 fresh weight). In 1 h, the microalga revealed as effective as the conventional physico-chemical ion-exchangers to purify nuclear effluents. Using this organism, an efficient real-scale radionuclide bio-decontamination process was performed in a nuclear fuel storage pool with an important reduction of waste volume compared to the usual physico-chemical process. The feasibility of new decontamination solutions for the nuclear industry and for environmental clean-up operations is demonstrated

Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.