Ariane Atteia

Pyruvate formate-lyase and a novel route of eukaryotic ATP synthesis in Chlamydomonas mitochondria

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.

A Proteomic Survey of Chlamydomonas reinhardtii Mitochondria Sheds New Light on the Metabolic Plasticity of the Organelle and on the Nature of the α-Proteobacterial Mitochondrial Ancestor

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.

Identification of Novel Mitochondrial Protein Components of Chlamydomonas reinhardtii. A Proteomic Approach

Pure mitochondria of the photosynthetic algaChlamydomonas reinhardtii were analyzed using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The major oxidative phosphorylation complexes were resolved: F1F0-ATP synthase, NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase. The oligomeric states of these complexes were determined. The F1F0-ATP synthase runs exclusively as a dimer, in contrast to the C. reinhardtii chloroplast enzyme, which is present as a monomer and subcomplexes. The sequence of a 60-kD protein, associated with the mitochondrial ATP synthase and with no known counterpart in any other organism, is reported. This protein may be related to the strong dimeric character of the algal F1F0-ATP synthase. The oxidative phosphorylation complexes resolved by BN-PAGE were separated into their subunits by second dimension sodium dodecyl sulfate-PAGE. A number of polypeptides were identified mainly on the basis of their N-terminal sequence. Core I and II subunits of complex III were characterized, and their proteolytic activities were predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits in cytochrome c oxidase was demonstrated. Other mitochondrial proteins like the chaperone HSP60, the alternative oxidase, the aconitase, and the ADP/ATP carrier were identified. BN-PAGE was also used to approach the analysis of the major chloroplast protein complexes of C. reinhardtii.

Anaerobic energy metabolism in unicellular photosynthetic eukaryotes.

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.

Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii

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.

New Insights into the Unique Structure of the F0F1-ATP Synthase from the Chlamydomonad Algae Polytomella sp. and Chlamydomonas reinhardtii

In this study, we investigate the structure of the mitochondrial F0F1-ATP synthase of the colorless alga Polytomella sp. with respect to the enzyme of its green close relative Chlamydomonas reinhardtii. It is demonstrated that several unique features of the ATP synthase in C. reinhardtii are also present in Polytomella sp. The α- and β-subunits of the ATP synthase from both algae are highly unusual in that they exhibit extensions at their N- and C-terminal ends, respectively. Several subunits of the Polytomella ATP synthase in the range of 9 to 66 kD have homologs in the green alga but do not have known equivalents as yet in mitochondrial ATP synthases of mammals, plants, or fungi. The largest of these so-called ASA (ATP Synthase-Associated) subunits, ASA1, is shown to be an extrinsic protein. Short heat treatment of isolated Polytomella mitochondria unexpectedly dissociated the otherwise highly stable ATP synthase dimer of 1,600 kD into subcomplexes of 800 and 400 kD, assigned as the ATP synthase monomer and F1-ATPase, respectively. Whereas no ASA subunits were found in the F1-ATPase, all but two were present in the monomer. ASA6 (12 kD) and ASA9 (9 kD), predicted to be membrane bound, were not detected in the monomer and are thus proposed to be involved in the formation or stabilization of the enzyme. A hypothetical configuration of the Chlamydomonad dimeric ATP synthase portraying its unique features is provided to spur further research on this topic.

Characterization of the α and β-subunits of the F0F1-ATPase from the alga Polytomella spp., a colorless relative of Chlamydomonas reinhardtii

The isolation and partial characterization of the oligomycin-sensitive F0F1-ATP synthase/ATPase from the colorless alga Polytomella spp. is described. Purification was performed by solubilization with dodecyl-β-d-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F1 sector of mitochondrial ATPases. The α-subunit, which migrates on SDS-polyacrylamide gels with an apparent molecular mass of 55 kDa, was identified by the N-terminal sequencing of 47 residues. This subunit exhibited a short extension at its N-terminus highly similar to the one described for the unicellular alga Chlamydomonas reinhardtii (Nurani, G. and Franzen L.-G. (1996) Plant Mol. Biol. 31, 1105–1116). In whole mitochondria, the α-subunit was susceptible to limited proteolytic digestion induced by heat. An endogenous protease removed the first 22 residues of the mature α-subunit. Subunit β was also identified by N-terminal sequencing of 31 residues. This subunit of 63 kDa exhibited a higher apparent molecular mass than α, as judged by its mobility on denaturing polyacrylamide gel electrophoresis. This β-subunit is 7–8 kDa larger than the β-subunits of other mitochondrial ATPases. It is suggested that the β-subunit from Polytomella spp. may have a C-terminal extension similar to that described for the green alga C. reinhardtii (Franzen, L.-G. and Falk, G. (1992) Plant Mol. Biol. 19, 771–780). In addition, it was found that the C-terminal extension of the β-subunit of C. reinhardtii showed homology with the endogenous ATPase inhibitors from various sources and with the ϵ-subunit from the F0F1-ATP synthase from Escherichia coli, which is considered to be a functional homolog of the inhibitor proteins. The data reported here provide the first biochemical evidence for a close relationship between the colorless alga Polytomella spp. and its photosynthetic counterpart C. reinhardtii. It is also suggested that the C-terminal extensions of the β-subunits of the ATP synthases from these algae, may play a regulatory role in these enzymes.

Genomic, Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.

An extremely radioresistant green eukaryote for radionuclide bio-decontamination in the nuclear industry

Nuclear activities generate radioactive elements which require processes for their decontamination. Although biological remediation has proved to be 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 well-known prokaryote Deinococcus radiodurans. The 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, 1000–450 000 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.

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp.

ABSTRACT Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.