Pharmaceutical metabolite identification in lettuce (Lactuca sativa) and earthworms (Eisenia fetida) using liquid chromatography coupled to high-resolution mass spectrometry and in silico spectral library.

Pharmaceuticals released into the aquatic and soil environments can be absorbed by plants and soil organisms, potentially leading to the formation of unknown metabolites that may negatively affect these organisms or contaminate the food chain. The aim of this study was to identify pharmaceutical metabolites through a triplet approach for metabolite structure prediction (software-based predictions, literature review, and known common metabolic pathways), followed by generating in silico mass spectral libraries and applying various mass spectrometry modes for untargeted LC-qTOF analysis. Therefore, Eisenia fetida and Lactuca sativa were exposed to a pharmaceutical mixture (atenolol, enrofloxacin, erythromycin, ketoprofen, sulfametoxazole, tetracycline) under hydroponic and soil conditions at environmentally relevant concentrations. Samples collected at different time points were extracted using QuEChERS and analyzed with LC-qTOF in data-dependent (DDA) and data-independent (DIA) acquisition modes, applying both positive and negative electrospray ionization. The triplet approach for metabolite structure prediction yielded a total of 3762 pharmaceutical metabolites, and an in silico mass spectral library was created based on these predicted metabolites. This approach resulted in the identification of 26 statistically significant metabolites (p < 0.05), with DDA + and DDA - outperforming DIA modes by successfully detecting 56/67 sample type:metabolite combinations. Lettuce roots had the highest metabolite count (26), followed by leaves (6) and earthworms (2). Despite the lower metabolite count, earthworms showed the highest peak intensities, closely followed by roots, with leaves displaying the lowest intensities. Common metabolic reactions observed included hydroxylation, decarboxylation, acetylation, and glucosidation, with ketoprofen-related metabolites being the most prevalent, totaling 12 distinct metabolites. In conclusion, we developed a high-throughput workflow combining open-source software with LC-HRMS for identifying unknown metabolites across various sample types.

Chlorosis as a Developmental Program in Cyanobacteria: The Proteomic Fundament for Survival and Awakening.

Cyanobacteria that do not fix atmospheric nitrogen gas survive prolonged periods of nitrogen starvation in a chlorotic, dormant state where cell growth and metabolism are arrested. Upon nutrient availability, these dormant cells return to vegetative growth within 2-3 days. This resuscitation process is highly orchestrated and relies on the stepwise reinstallation and activation of essential cellular structures and functions. We have been investigating the transition to chlorosis and the return to vegetative growth as a simple model of a cellular developmental process and a fundamental survival strategy in biology. In the present study, we used quantitative proteomics and phosphoproteomics to describe the proteomic landscape of a dormant cyanobacterium and its dynamics during the transition to vegetative growth. We identified intriguing alterations in the set of ribosomal proteins, in RuBisCO components, in the abundance of central regulators and predicted metabolic enzymes. We found O-phosphorylation as an abundant protein modification in the chlorotic state, specifically of metabolic enzymes and proteins involved in photosynthesis. Nondegraded phycobiliproteins were hyperphosphorylated in the chlorotic state. We provide evidence that hyperphosphorylation of the terminal rod linker CpcD increases the lifespan of phycobiliproteins during chlorosis.

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging.

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age.

The cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria.

Structural and functional characterisation of the cyanobacterial PetC3 Rieske protein family.

The cyanobacterium Synechocystis PCC 6803 possesses three Rieske isoforms: PetC1, PetC2 and PetC3. While PetC1 and PetC2 have been identified as alternative subunits of the cytochrome b6f complex (b6f), PetC3 was localized exclusively within the plasma membrane. The spatial separation of PetC3 from the photosynthetic and respiratory protein complexes raises doubt in its involvement in bioenergetic electron transfer. Here we report a detailed structural and functional characterization of the cyanobacterial PetC3 protein family indicating that PetC3 is not a component of the b6f and the photosynthetic electron transport as implied by gene annotation. Instead PetC3 has a distinct function in cell envelope homeostasis. Especially proteomic analysis shows that deletion of petC3 in Synechocystis PCC 6803 primarily affects cell envelope proteins including many nutrient transport systems. Therefore, the observed downregulation in the photosynthetic electron transport - mainly caused by photosystem 2 inactivation - might constitute a stress adaptation. Comprehensive in silico sequence analyses revealed that PetC3 proteins are periplasmic lipoproteins tethered to the plasma membrane with a subclass consisting of soluble periplasmic proteins, i.e. their N-terminal domain is inconsistent with their integration into the b6f. For the first time, the structure of PetC3 was determined by X-ray crystallography at an atomic resolution revealing significant high similarities to non-b6f Rieske subunits in contrast to PetC1. These results suggest that PetC3 affects processes in the periplasmic compartment that only indirectly influence photosynthetic electron transport. For this reason, we suggest to rename "Photosynthetic electron transport Chain 3" (PetC3) proteins as "periplasmic Rieske proteins" (Prp).

Functional characterization of the small regulatory subunit PetP from the cytochrome b6f complex in Thermosynechococcus elongatus.

The cyanobacterial cytochrome b(6)f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b(6)f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700(+) reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b(6)f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b(6)f complexes with different functions that might be correlated with supercomplex formation.

The proteome and lipidome of Synechocystis sp. PCC 6803 cells grown under light-activated heterotrophic conditions.

Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.

A simple method for decomposition of peracetic acid in a microalgal cultivation system.

A cost-efficient process devoid of several washing steps was developed, which is related to direct cultivation following the decomposition of the sterilizer. Peracetic acid (PAA) is known to be an efficient antimicrobial agent due to its high oxidizing potential. Sterilization by 2 mM PAA demands at least 1 h incubation time for an effective disinfection. Direct degradation of PAA was demonstrated by utilizing components in conventional algal medium. Consequently, ferric ion and pH buffer (HEPES) showed a synergetic effect for the decomposition of PAA within 6 h. On the contrary, NaNO3, one of the main components in algal media, inhibits the decomposition of PAA. The improved growth of Chlorella vulgaris and Synechocystis PCC6803 was observed in the prepared BG11 by decomposition of PAA. This process involving sterilization and decomposition of PAA should help cost-efficient management of photobioreactors in a large scale for the production of value-added products and biofuels from microalgal biomass.

The bacterial proteogenomic pipeline.

BACKGROUND: Proteogenomics combines the cutting-edge methods from genomics and proteomics. While it has become cheap to sequence whole genomes, the correct annotation of protein coding regions in the genome is still tedious and error prone. Mass spectrometry on the other hand relies on good characterizations of proteins derived from the genome, but can also be used to help improving the annotation of genomes or find species specific peptides. Additionally, proteomics is widely used to find evidence for differential expression of proteins under different conditions, e.g. growth conditions for bacteria. The concept of proteogenomics is not altogether new, in-house scripts are used by different labs and some special tools for eukaryotic and human analyses are available. RESULTS: The Bacterial Proteogenomic Pipeline, which is completely written in Java, alleviates the conducting of proteogenomic analyses of bacteria. From a given genome sequence, a naïve six frame translation is performed and, if desired, a decoy database generated. This database is used to identify MS/MS spectra by common peptide identification algorithms. After combination of the search results and optional flagging for different experimental conditions, the results can be browsed and further inspected. In particular, for each peptide the number of identifications for each condition and the positions in the corresponding protein sequences are shown. Intermediate and final results can be exported into GFF3 format for visualization in common genome browsers. CONCLUSIONS: To facilitate proteogenomics analyses the Bacterial Proteogenomic Pipeline is a set of comprehensive tools running on common desktop computers, written in Java and thus platform independent. The pipeline allows integrating peptide identifications from various algorithms and emphasizes the visualization of spectral counts from different experimental conditions.

The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains.

The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids.

Assessing earthworm exposure to a multi-pharmaceutical mixture in soil: unveiling insights through LC-MS and MALDI-MS analyses, and impact of biochar on pharmaceutical bioavailability.

In the European circular economy, agricultural practices introduce pharmaceutical (PhAC) residues into the terrestrial environment, posing a potential risk to earthworms. This study aimed to assess earthworm bioaccumulation factors (BAFs), the ecotoxicological effects of PhACs, the impact of biochar on PhAC bioavailability to earthworms, and their persistence in soil and investigate earthworm uptake mechanisms along with the spatial distribution of PhACs. Therefore, earthworms were exposed to contaminated soil for 21 days. The results revealed that BAFs ranged from 0.0216 to 0.329, with no significant ecotoxicological effects on earthworm weight or mortality (p > 0.05). Biochar significantly influenced the uptake of 14 PhACs on the first day (p < 0.05), with diminishing effects over time, and affected significantly the soil-degradation kinetics of 16 PhACs. Moreover, MALDI-MS analysis revealed that PhAC uptake occurs through both the dermal and oral pathways, as pharmaceuticals were distributed throughout the entire earthworm tissue without specific localization. In conclusion, this study suggests ineffective PhAC accumulation in earthworms, highlights the influence of biochar on PhAC degradation rates in soil, and suggests that uptake can occur through both earthworm skin and oral ingestion.

Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress.

Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.

Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

N-formylkynurenine as a marker of high light stress in photosynthesis.

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganese-calcium active site. Light-dependent damage and repair occur in PSII under conditions of high light stress. The core reaction center complex is composed of the D1, D2, CP43, and CP47 intrinsic polypeptides. In this study, a new chromophore formed from the oxidative post-translational modification of tryptophan is identified in the CP43 subunit. Tandem mass spectrometry peptide sequencing is consistent with the oxidation of the CP43 tryptophan side chain, Trp-365, to produce N-formylkynurenine (NFK). Characterization with ultraviolet visible absorption and ultraviolet resonance Raman spectroscopy supports this assignment. An optical assay suggests that the yield of NFK increases 2-fold (2.2 ± 0.5) under high light illumination. A concomitant 2.4 ± 0.5-fold decrease is observed in the steady-state rate of oxygen evolution under the high light conditions. NFK is the product formed from reaction of tryptophan with singlet oxygen, which can be produced under high light stress in PSII. Reactive oxygen species reactions lead to oxidative damage of the reaction center, D1 protein turnover, and inhibition of electron transfer. Our results are consistent with a role for the CP43 NFK modification in photoinhibition.

Unique properties vs. common themes: the atypical cyanobacterium Gloeobacter violaceus PCC 7421 is capable of state transitions and blue-light-induced fluorescence quenching.

The atypical unicellular cyanobacterium Gloeobacter violaceus PCC 7421, which diverged very early during the evolution of cyanobacteria, can be regarded as a key organism for understanding many structural, functional, regulatory and evolutionary aspects of oxygenic photosynthesis. In the present work, the performance of two basic photosynthetic adaptation/protection mechanisms, common to all other oxygenic photoautrophs, had been challenged in this ancient cyanobacterium which lacks thylakoid membranes: state transitions and non-photochemical fluorescence quenching. Both low temperature fluorescence spectra and room temperature fluorescence transients show that G. violaceus is capable of performing state transitions similar to evolutionarily more recent cyanobacteria, being in state 2 in darkness and in state 1 upon illumination by weak blue or far-red light. Compared with state 2, variable fluorescence yield in state 1 is strongly enhanced (almost 80%), while the functional absorption cross-section of PSII is only increased by 8%. In contrast to weak blue light, which enhances fluorescence yield via state 1 formation, strong blue light reversibly quenches Chl fluorescence in G. violaceus. This strongly suggests regulated heat dissipation which is triggered by the orange carotenoid protein whose presence was directly proven by immunoblotting and mass spectrometry in this primordial cyanobacterium. The results are discussed in the framework of cyanobacterial evolution.

Agrobacterium tumefaciens Type IV and Type VI Secretion Systems Reside in Detergent-Resistant Membranes.

Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen Agrobacterium tumefaciens. Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in A. tumefaciens.

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803.

The cytochrome b(6)f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b(6)f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane. Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.

Remodeling of photosynthetic electron transport in Synechocystis sp. PCC 6803 for future hydrogen production from water.

Photosynthetic microorganisms such as the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) can be exploited for the light-driven synthesis of valuable compounds. Thermodynamically, it is most beneficial to branch-off photosynthetic electrons at ferredoxin (Fd), which provides electrons for a variety of fundamental metabolic pathways in the cell, with the ferredoxin-NADP+ Oxido-Reductase (FNR, PetH) being the main target. In order to re-direct electrons from Fd to another consumer, the high electron transport rate between Fd and FNR has to be reduced. Based on our previous in vitro experiments, corresponding FNR-mutants at position FNR_K190 (Wiegand, K., et al.: "Rational redesign of the ferredoxin-NADP-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production". Biochim Biophys Acta, 2018) have been generated in Synechocystis cells to study their impact on the cellular metabolism and their potential for a future hydrogen-producing design cell. Out of two promising candidates, mutation FNR_K190D proved to be lethal due to oxidative stress, while FNR_K190A was successfully generated and characterized: The light induced NADPH formation is clearly impaired in this mutant and it shows also major metabolic adaptations like a higher glucose metabolism as evidenced by quantitative mass spectrometric analysis. These results indicate a high potential for the future use of photosynthetic electrons in engineered design cells - for instance for hydrogen production. They also show substantial differences of interacting proteins in an in vitro environment vs. physiological conditions in whole cells.

Metabolism controls dimerization of the chloroplast FoF1 ATP synthase in Chlamydomonas reinhardtii.

Dimers and oligomers of F-type ATP synthases have been observed previously in mitochondria of various organisms and for the CF(o)F(1) ATP synthase of chloroplasts of Chlamydomonas reinhardtii. In contrast to mitochondria, however, dimers of chloroplast ATP synthases dissociate at elevated phosphate concentration. This suggests a regulation by cell physiological processes. Stable isotope labeling of living cells and blue-native PAGE have been employed to quantitate changes in the ratio of monomeric to dimeric CF(o)F(1) ATP synthase. Chlamydomonas reinhardtii cells were cultivated photoautotrophically in the presence of (15)N and photomixotrophically at natural (14)N abundance, respectively. As compared to photoautotrophic growth, an increased assembly of ATP synthase dimers on the expense of preexisting monomers during photomixotrophic growth was observed, demonstrating a metabolic control of the dimerization process.

OXPHOS Supercomplexes: respiration and life-span control in the aging model Podospora anserina.

Recent biochemical evidence has indicated the existence of respiratory supercomplexes as well as ATP synthase oligomers in the inner mitochondrial membrane of different eukaryotes. We have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This aging model is able to respire by either the standard or the alternative pathway. In the latter, electrons are directly transferred from ubiquinol to the alternative oxidase (AOX) and thus bypass complexes III and IV. We showed that the two pathways are composed of distinct respiratory supercomplexes. These data are of significance for the understanding of both respiratory pathways as well as of life-span control and aging.