In vitro synthesis of the cyanelle proteins of Cyanophora paradoxa by isolated cyanelles and cyanelle RNA

Cyanelles isolated from the alga Cyanophora paradoxa Korschikoff synthesized cyanelle proteins in vitro. This synthesis was stimulated by light and totally inhibited by chloramphenicol. Cycloheximide had only a small inhibitory effect. Electrophoretic separation of the labelled soluble cyanelle proteins yielded at least 20 discrete polypeptides. The RNA isolated from the cyanelles and the whole cells was successfully translated in a rabbit reticulocyte-lysate system.Abbreviations poly(A)^-RNA, poly(A)⁺RNA nonadenylated, polyadenylated RNA; - SDS sodium dodecyl sulfate     

Effect of precursor protein phosphorylation on import into isolated chloroplasts from Chlamydomonas.

In higher plants, chloroplast-destined precursor proteins are thought to be phosphorylated. Mediated by a specific 14-3-3 protein, these phosphorylated proteins bind to the chloroplast surface and are subsequently imported into the chloroplast. We demonstrate that also in the green alga Chlamydomonas reinhardtii the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase becomes phosphorylated by a plant protein kinase and that the phosphorylation site is located in the transit peptide. The phosphorylation status of the precursor protein regulates its import into chloroplasts especially at an early step during this process. The possible physiological function is discussed.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

A class-I intron in a cyanelle tRNA gene from Cyanophora paradoxa: phylogenetic relationship between cyanelles and plant chloroplasts

Cyanelles are photosynthetic organelles which are considered as intermediates between cyanobacteria and chloroplasts, and which have been found in unicellular eukaryotes such as Cyanophora paradoxa. The nucleotide sequence of a 667-bp region of the cyanelle genome from Cyanophora paradoxa containing genes coding for tRNA(UUCGlu) and tRNA(UAALeu) has been determined. The gene coding for tRNA(UAALeu) is split by a 232-bp intron which has a secondary structure typical for class-I structured introns and which is closely related to the intron located in the corresponding gene from liverwort and higher plant chloroplasts. It appears therefore that these tRNA(UAALeu) genes are all derived from one common ancestral gene which already contained a class-I intron.     

Unfolding and refolding of a purified precursor protein during import into isolated mitochondria.

A purified mitochondrial precursor protein unfolds to a protease-sensitive conformation at the surface of isolated mitochondria before being imported into the organelles. This unfolding is stimulated by a potential across the mitochondrial inner membrane, but does not require ATP. In contrast, import of the surface-bound unfolded precursor requires ATP, but no potential; it is accompanied by a refolding inside the mitochondria.     

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of β-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope’s invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a β-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.     

The nucleotide sequence of five ribosomal protein genes from the cyanelles ofCyanophora paradoxa: Implications concerning the phylogenetic relationship between cyanelles and chloroplasts

Summary The nucleotide sequences of the ribosomal protein genesrps18, rps19, rpl2, rpl33, and partial sequence ofrpl22 from cyanelles, the photosynthetic organelles of the protistCyanophora paradoxa, have been determined. These genes form two clusters oriented in opposite and divergent directions. One cluster contains therpl33 andrps18 genes; the other contains therpl2, rps19, andrpl22 genes, in that order. Phylogenetic trees were constructed from both the DNA sequences and the deduced protein sequences of cyanelles,Euglena gracilis and land plant chloroplasts, andEscherichia coli, using parsimony or maximum likelihood methods. In addition, a phylogenetic tree was built from a distance matrix comparing the number of nucleotide substitutions per site. The phylogeny inferred from all these methods suggests that cyanelles fall within the chloroplast line of evolution and that the evolutionary distances between cyanelles and land plant chloroplasts are shorter than betweenE. gracilis chloroplasts and land plant chloroplasts.     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

Ferredoxin and ribosomal protein S10 are encoded on the cyanelle genome of Cyanophora paradoxa

The petF and rsp10 genes of the cyanellar genome of the taxonomically ambiguous flagellate Cyanophora paradoxa have been cloned, mapped, and sequenced. In higher plants these genes are not encoded in the chloroplast DNA, but are encoded in the nucleus. The C. paradoxa petF gene predicts a protein of 99 amino acids (aa) which is more similar to type-I ferredoxins of diverse cyanobacteria than to those of green algae, dinoflagellates, and higher plants. The rsp10 gene (rspJ) predicts a protein of 105 aa which is about 50% identical and 71% homologous to the proteins of Escherichia coli and Mycoplasma capricolum. The results are discussed within the context of the endosymbiotic origins of chloroplasts from cyanobacteria.     

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

Background  

Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems.     

Homologous and heterologous protein import into mitochondria isolated from the green alga Chlamydomonas reinhardtii

We have established a homologous system for studying mitochondrial protein import in Chlamydomonas reinhardtii, using C. reinhardtii precursor proteins and mitochondria isolated from C. reinhardtii. The precursors of the F₁ ATP synthase subunit and the Rieske FeS protein were imported into mitochondria with high efficiency, while the F₁ subunit precursor was imported with much lower efficiency. The import of heterologous precursor proteins from higher plants was also less efficient. The precursor of the C. reinhardtii PsaF chloroplast protein was converted into a protease-protected form upon incubation with mitochondria. In vitro processing studies revealed that in contrast to the situation in higher plants, the processing of the precursors was catalysed by a soluble, matrix-located peptidase.     

Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria.

In order to study the role of protein unfolding during post-translational protein import into mitochondria, we destabilized the structure of a mitochondrial precursor protein by site-directed mutagenesis. The precursor consisted of the first 16 residues of the yeast cytochrome oxidase subunit IV precursor fused to mouse dihydrofolate reductase. Labilization of the folded precursor structure was monitored by increased susceptibility to protease and diminished ability of methotrexate to block import of the precursor into isolated yeast mitochondria. On comparing the original precursor with two mutant forms that were destabilized to different degrees, increased labilization correlated with an increased rate and efficiency of import into mitochondria. This supports the view that the precursor must unfold in order to enter the mitochondria.     

Properties of the Photosynthetic System and DNA of Cyanophora paradoxa Cyanelles

The cyanelle from the photosynthetic biflagellate protist Cyanophora paradoxa has been studied in terms of its photosynthetic properties. Structurally, the cyanelle resembles unicellular cyanobacteria. The cyanelle is readily released from the host cell by means of the French press. The isolated cyanelle shows typical photosystem I and photosystem II activities as well as phenazine methosulfate-mediated photophosphorylation. The kinetic parameters K_m and V_max were determined for CO₂ fixation in the cyanelle and cells of C. paradoxa and compared to a cyanobacterium. The determined values were not much different, although the cyanobacterium had a significantly greater rate of CO₂ fixation, and the cyanelle was least active in this regard. Photosystem I chlorophyll-protein complex is readily isolated from the thylakoid membrane. In all these respects, the photosynthetic apparatus of the cyanelle resembles that of cyanobacteria. No nitrogen fixation activity was observed. Attempts to regenerate the isolated cyanelle were not successful, but in some cases, an unidentified cyanobacterium grew up in standing cultures of C. paradoxa cyanelles. Buoyant density data indicate that the strain of C. paradoxa we have investigated differs from that employed by others, since our strain shows a value of 1.716 grams per cubic centimeter and others report values of 1.695 and 1.691.     

New insights into the import mechanism of the ferredoxin precursor into chloroplasts.

We have investigated the import pathway of the nuclear-encoded chloroplast protein ferredoxin. By using purified precursor protein and washed intact chloroplasts in a defined in vitro uptake system, we show that preferredoxin is fully import-competent by itself. In addition, we show also that the in vitro, in a wheat germ lysate, synthesized preferredoxin is not stably associated with another protein. Import is dependent only on ATP and does not require the presence of cytosolic proteins. Translocation could be largely stimulated by the thiol reducing agent dithiothreitol (DTT). To determine whether DTT acts on the precursor or on the chloroplast, we modified the 5 cysteines in the precursor by a reaction with iodoacetamide, thereby preventing the formation of disulfide bridges in the precursor. The import of this modified precursor was still stimulated by the addition of DTT, indicating that DTT had a stimulating effect on the chloroplast import machinery. In the case of the modified precursor, the import must have taken place without iron-sulfur cluster attachment in the stroma. The modified precursor could be imported with a similar efficiency as the parent precursor showing that import takes place independently from cofactor assembly.     

Isolation of the plastid FtsZ gene from Cyanophora paradoxa (Glaucocystophyceae, Glaucocystophyta)

Plastids of glaucocystophytes are termed cyanelles and retain primitive features, such as a peptidoglycan wall. We isolated a full‐length prokaryotic plastid division gene, FtsZ, from the glaucocystophyte alga Cyanophora paradoxa Korshikov (CpFtsZ‐cy). CpftsZ‐cy has a chloroplast‐targeting signal at the N‐teminus. Immunofluorescence microscopy showed that CpFtsZ‐cy forms a ring‐like structure at the division plane of cyanelles.