Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells.

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5'-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5'-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s-1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).

The pathogenic m.8993 T > G mutation in mitochondrial ATP6 gene prevents proton release from the subunit c-ring rotor of ATP synthase.

The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here, we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionary conservation.

Conserved role of ATP synthase in mammalian cilia.

We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis elegans. Herein, we discovered that the ciliary localization of ATP synthase is evolutionarily conserved in mammals. We showed that the ATP synthase subunit F1ß is colocalized with the cilia marker acetylated α-tubulin in both mammalian renal epithelial cells (MDCK) and normal mouse cholangiocytes (NMCs). Treatment with ATP synthase inhibitor oligomycin impaired ciliogenesis in MDCK cells, and F1ß was co-immunoprecipitated with PKD2 in mammalian cells. Our study provides evidence for the evolutionarily conserved localization of ATP synthase in cilia from worm to mammals. Defects in ATP synthase can lead to ciliary dysfunction, which may be a potential mechanism of polycystic kidney disease.

Multiomics Reveals Ectopic ATP Synthase Blockade Induces Cancer Cell Death via a lncRNA-mediated Phospho-signaling Network.

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus UTEX 2973.

Cyanobacteria are emerging as attractive organisms for sustainable bioproduction. We previously described Synechococcus elongatus UTEX 2973 as the fastest growing cyanobacterium known. Synechococcus 2973 exhibits high light tolerance and an increased photosynthetic rate and produces biomass at three times the rate of its close relative, the model strain Synechococcus elongatus 7942. The two strains differ at 55 genetic loci, and some of these loci must contain the genetic determinants of rapid photoautotrophic growth and improved photosynthetic rate. Using CRISPR/Cpf1, we performed a comprehensive mutational analysis of Synechococcus 2973 and identified three specific genes, atpA, ppnK, and rpaA, with SNPs that confer rapid growth. The fast-growth-associated allele of each gene was then used to replace the wild-type alleles in Synechococcus 7942. Upon incorporation, each allele successively increased the growth rate of Synechococcus 7942; remarkably, inclusion of all three alleles drastically reduced the doubling time from 6.8 to 2.3 hours. Further analysis revealed that our engineering effort doubled the photosynthetic productivity of Synechococcus 7942. We also determined that the fast-growth-associated allele of atpA yielded an ATP synthase with higher specific activity, while that of ppnK encoded a NAD+ kinase with significantly improved kinetics. The rpaA SNPs cause broad changes in the transcriptional profile, as this gene is the master output regulator of the circadian clock. This pioneering study has revealed the molecular basis for rapid growth, demonstrating that limited genetic changes can dramatically improve the growth rate of a microbe by as much as threefold.

The influence of l-carnitine on the expression of miRNAs in asthenospermia spermatozoa and the network regulation of the associated molecules.

l-carnitine is a natural compound that is indispensable for energy metabolism in mammals. The efficiency and safety of l-carnitine in improving sperm activity, enhancing epididymal function and treating male infertility has been widely acknowledged by clinicians. CircRNAs can regulate gene expression at the transcriptional or post-transcriptional level by serving as a molecular sponge of miRNAs with miRNA response elements. However, the detailed mechanism linking miRNA, circRNA and asthenospermia remains unclear. The present study demonstrated that hsa-miR-27b-3p, hsa-miR-151a-5p and hsa-miR-206 play an important role in the effects of l-carnitine treatment of the spermatozoa in asthenospermia patients. Furthermore, the target mRNAs of hsa-miR-206 were analysed by GO and KEGG. The results show that the target mRNAs of hsa-miR-206 may change the activity of ATP synthase and participate in the cAMP signalling pathway and the calcium signalling pathway, which may play an important role in sperm motility.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.

Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins.

Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation.

PhoU2 but Not PhoU1 as an Important Regulator of Biofilm Formation and Tolerance to Multiple Stresses by Participating in Various Fundamental Metabolic Processes in Staphylococcus epidermidis.

PhoU, a conserved protein that has been proposed to coordinate phosphate import, is a negative regulator of drug tolerance in most bacteria. In Staphylococcus epidermidis, the role of PhoU in biofilm formation and drug tolerance has not yet been investigated. Two PhoU homologs in the genome of S. epidermidis have been identified by the presence of the conserved motif E(D)XXXD of PhoU. We separately constructed ΔphoU1 and ΔphoU2 mutants of S. epidermidis strain 1457. The ΔphoU2 mutant displayed growth retardation, a weakened biofilm formation capacity, a higher sensitivity to H2O2, and reduced tolerance to multiple antibiotics. However, deletion of phoU1 had no effect on those. We compared the transcriptome profiles of the ΔphoU2 and ΔphoU1 mutants with that of the parent strain. In the ΔphoU2 mutant, expression of genes related to inorganic phosphate uptake was significantly upregulated (pst operon) and the levels of intracellular inorganic polyphosphate (polyP) were increased. In the ΔphoU2 mutant, expression of enzymes in the pentose phosphate pathway (PPP) was downregulated and less NADP (NADPH) was detected, consistent with the high sensitivity to H2O2 and the growth retardation of the ΔphoU2 mutant. The upregulated expression of ATP synthase was consistent with the high intracellular ATP content in the ΔphoU2 mutant, which may have been related to the lower drug tolerance of the ΔphoU2 mutant. This study demonstrates that PhoU2, but not PhoU1, in S. epidermidis regulates bacterial growth, biofilm formation, oxidative stress, and drug tolerance in association with alterations to inorganic phosphate metabolism, the pentose phosphate pathway, galactose metabolism, the tricarboxylic acid (TCA) or citric cycle, glycolysis and gluconeogenesis, and respiratory reactions.IMPORTANCE PhoU is widely conserved throughout the bacterial kingdom and plays an important role in response to stress and metabolic maintenance. In our study, two PhoU homologs were found in S. epidermidis The function of phoU2, but not phoU1, in S. epidermidis is related to growth, drug tolerance, the oxidative stress response, polyP levels, and ATP accumulation. In addition, phoU2 regulates biofilm formation. Hence, phoU2 is a regulator of both drug tolerance and biofilm formation, which are two bacterial properties that present major challenges to the clinical treatment of infections. Analysis of differential gene expression revealed that phoU2 is involved in fundamental metabolic processes, such as the PPP pathway. These findings indicate that phoU2 is a crucial regulator in S. epidermidis.

The stimulating role of subunit F in ATPase activity inside the A1-complex of the Methanosarcina mazei Gö1 A1AO ATP synthase.

A1AO ATP synthases couple ion-transport of the AO sector and ATP synthesis/hydrolysis of the A3B3-headpiece via their stalk subunits D and F. Here, we produced and purified stable A3B3D- and A3B3DF-complexes of the Methanosarcina mazei Gö1 A-ATP synthase as confirmed by electron microscopy. Enzymatic studies with these complexes showed that the M. mazei Gö1 A-ATP synthase subunit F is an ATPase activating subunit. The maximum ATP hydrolysis rates (Vmax) of A3B3D and A3B3DF were determined by substrate-dependent ATP hydrolysis experiments resulting in a Vmax of 7.9 s(-1) and 30.4 s(-1), respectively, while the KM is the same for both. Deletions of the N- or C-termini of subunit F abolished the effect of ATP hydrolysis activation. We generated subunit F mutant proteins with single amino acid substitutions and demonstrated that the subunit F residues S84 and R88 are important in stimulating ATP hydrolysis. Hybrid formation of the A3B3D-complex with subunit F of the related eukaryotic V-ATPase of Saccharomyces cerevisiae or subunit ε of the F-ATP synthase from Mycobacterium tuberculosis showed that subunit F of the archaea and eukaryotic enzymes are important in ATP hydrolysis.

Effects of Sulfate Reduction on Trichloroethene Dechlorination by Dehalococcoides-Containing Microbial Communities.

In order to elucidate interactions between sulfate reduction and dechlorination, we systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. Sulfate (up to 5 mM) did not inhibit the growth or metabolism of pure cultures of the dechlorinator Dehalococcoides mccartyi 195, the sulfate reducer Desulfovibrio vulgaris Hildenborough, or the syntroph Syntrophomonas wolfei In contrast, sulfide at 5 mM exhibited inhibitory effects on growth of the sulfate reducer and the syntroph, as well as on both dechlorination and growth rates of D. mccartyi Transcriptomic analysis of D. mccartyi 195 revealed that genes encoding ATP synthase, biosynthesis, and Hym hydrogenase were downregulated during sulfide inhibition, whereas genes encoding metal-containing enzymes involved in energy metabolism were upregulated even though the activity of those enzymes (hydrogenases) was inhibited. When the electron acceptor (trichloroethene) was limiting and an electron donor (lactate) was provided in excess to cocultures and enrichments, high sulfate concentrations (5 mM) inhibited reductive dechlorination due to the toxicity of generated sulfide. The initial cell ratio of sulfate reducers to D. mccartyi (1:3, 1:1, or 3:1) did not affect the dechlorination performance in the presence of sulfate (2 and 5 mM). In contrast, under electron donor limitation, dechlorination was not affected by sulfate amendments due to low sulfide production, demonstrating that D. mccartyi can function effectively in anaerobic microbial communities containing moderate sulfate concentrations (5 mM), likely due to its ability to outcompete other hydrogen-consuming bacteria and archaea.IMPORTANCE Sulfate is common in subsurface environments and has been reported as a cocontaminant with chlorinated solvents at various concentrations. Inconsistent results for the effects of sulfate inhibition on the performance of dechlorination enrichment cultures have been reported in the literature. These inconsistent findings make it difficult to understand potential mechanisms of sulfate inhibition and complicate the interpretation of bioremediation field data. In order to elucidate interactions between sulfate reduction and reductive dechlorination, this study systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. This study provides a more fundamental understanding of the competition mechanisms between reductive dechlorination by Dehalococcoides mccartyi and sulfate reduction during the bioremediation process. It also provides insights on the significance of sulfate concentrations on reductive dechlorination under electron donor/acceptor-limiting conditions during in situ bioremediation applications. For example, at a trichloroethene-contaminated site with a high sulfate concentration, proper slow-releasing electron donors can be selected to generate an electron donor-limiting environment that favors reductive dechlorination and minimizes the sulfide inhibition effect.

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans.

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.

Regulation of the biogenesis of OXPHOS complexes in cell transition from replicating to quiescent state: involvement of PKA and effect of hydroxytyrosol.

A study is presented on the expression of mitochondrial oxidative phosphorylation complexes in exponentially growing and serum-starved, quiescent human fibroblast cultures. The functional levels of respiratory complexes I and III and complex V (adenosine triphosphate (ATP) synthase) were found to be severely depressed in serum-starved fibroblasts. The depression of oxidative phosphorylation system (OXPHOS) complexes was associated with reduced levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and the down-stream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factors (TFAM). In serum-starved fibroblasts decrease of the catalytic activity of AMP cyclic dependent protein kinase (PKA) and phosphorylation of cAMP response element-binding protein (CREB), the transcription coactivator of the PGC-1α gene, was found. Hydroxytyrosol prevented the decline in the expression of the PGC-1α transcription cascade of OXPHOS complexes in serum-starved fibroblast cultures. The positive effect of HT was associated with activation of PKA and CREB phosphorylation. These results show involvement of PKA, CREB and PGC-1α in the regulation of OXPHOS in cell transition from the replicating to the quiescent state.

FoF1-ATP synthase of Streptomyces fradiae ATCC 19609: structural, biochemical, and functional characterization.

The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FoF1-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the ß- and b-subunits of the FoF1-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FoF1-ATP synthase, i.e. α, ß, γ, δ, ε, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, ß-, α- and ε-subunits. The ß-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FoF1-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FoF1-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FoF1-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs.

Engineering rotor ring stoichiometries in the ATP synthase.

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c(11) ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c(<11), c(12), c(13), c(14), and c(>14) rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c(12) instead of c(11), is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.

ATP synthase: an identified target gene of bantam in paired female Schistosoma japonicum.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. An increasing number of schistosome miRNAs have been identified and are expected possibly involved in differentiation, development, and metabolism. However, limited information is available concerning the target genes of schistosome miRNAs. In the present study, the key target genes of bantam, an abundant miRNA found in paired female Schistosoma japonicum, were predicted by bioinformatics analysis and Solexa technology. Luciferase reporter assay and bantam mimic assay were applied in combination to further verify the targets of bantam. Results showed that ATP synthase (CAX76793.1), one of the three selected predicted targets, was confirmed as the target of bantam; bantam mimic assay results also showed that the two other predicted targets, namely, ataxia telangiectasia mutated (ATM)-related (XP_002571630.1), and ribosomal protein L30 (CAX72575.1), were not confirmed as targets. This research proposed the design and significance of reasonable biological experiments that could be performed to identify miRNA target genes in schistosomes.

A pangenomic analysis of the Nannochloropsis organellar genomes reveals novel genetic variations in key metabolic genes.

BACKGROUND: Microalgae in the genus Nannochloropsis are photosynthetic marine Eustigmatophytes of significant interest to the bioenergy and aquaculture sectors due to their ability to efficiently accumulate biomass and lipids for utilization in renewable transportation fuels, aquaculture feed, and other useful bioproducts. To better understand the genetic complement that drives the metabolic processes of these organisms, we present the assembly and comparative pangenomic analysis of the chloroplast and mitochondrial genomes from Nannochloropsis salina CCMP1776. RESULTS: The chloroplast and mitochondrial genomes of N. salina are 98.4% and 97% identical to their counterparts in Nannochloropsis gaditana. Comparison of the Nannochloropsis pangenome to other algae within and outside of the same phyla revealed regions of significant genetic divergence in key genes that encode proteins needed for regulation of branched chain amino synthesis (acetohydroxyacid synthase), carbon fixation (RuBisCO activase), energy conservation (ATP synthase), protein synthesis and homeostasis (Clp protease, ribosome). CONCLUSIONS: Many organellar gene modifications in Nannochloropsis are unique and deviate from conserved orthologs found across the tree of life. Implementation of secondary and tertiary structure prediction was crucial to functionally characterize many proteins and therefore should be implemented in automated annotation pipelines. The exceptional similarity of the N. salina and N. gaditana organellar genomes suggests that N. gaditana be reclassified as a strain of N. salina.

Split photosystem protein, linear-mapping topology, and growth of structural complexity in the plastid genome of Chromera velia.

The canonical photosynthetic plastid genomes consist of a single circular-mapping chromosome that encodes a highly conserved protein core, involved in photosynthesis and ATP generation. Here, we demonstrate that the plastid genome of the photosynthetic relative of apicomplexans, Chromera velia, departs from this view in several unique ways. Core photosynthesis proteins PsaA and AtpB have been broken into two fragments, which we show are independently transcribed, oligoU-tailed, translated, and assembled into functional photosystem I and ATP synthase complexes. Genome-wide transcription profiles support expression of many other highly modified proteins, including several that contain extensions amounting to hundreds of amino acids in length. Canonical gene clusters and operons have been fragmented and reshuffled into novel putative transcriptional units. Massive genomic coverage by paired-end reads, coupled with pulsed-field gel electrophoresis and polymerase chain reaction, consistently indicate that the C. velia plastid genome is linear-mapping, a unique state among all plastids. Abundant intragenomic duplication probably mediated by recombination can explain protein splits, extensions, and genome linearization and is perhaps the key driving force behind the many features that defy the conventional ways of plastid genome architecture and function.

The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae.

Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50%). Cells were lysed by sonication, which was optimised to obtain 54-94% cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu(2+) as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and ß-tubulins and ATP synthase (ß subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae.

A c subunit with four transmembrane helices and one ion (Na+)-binding site in an archaeal ATP synthase: implications for c ring function and structure.

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na(+)-DCCD competition experiments revealed only one binding site for DCCD and Na(+), indicating that the mature c subunit of this A(1)A(O) ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na(+)-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na(+)-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na(+)-specific under in vivo conditions, comparable with the Na(+)-dependent V(1)V(O) ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na(+)-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A(1)A(O) ATP synthases.