Transketolase from Cyanophora paradoxa: In Vitro Import into Cyanelles and Pea Chloroplasts and a Complex History of a Gene Often, But Not Always, Transferred in the Context of Secondary Endosymbiosis

ABSTRACT. The glaucocystophyte Cyanophora paradoxa is an obligatorily photoautotrophic biflagellated protist containing cyanelles, peculiar plastids surrounded by a peptidoglycan layer between their inner and outer envelope membranes. Although the 136-kb cyanelle genome surpasses higher plant chloroplast genomes in coding capacity by about 50 protein genes, these primitive plastids still have to import >2,000 polypeptides across their unique organelle wall. One such protein is transketolase, an essential enzyme of the Calvin cycle. We report the sequence of the pre-transketolase cDNA from C. paradoxa and in vitro import experiments of precursor polypeptides into cyanelles and into pea chloroplasts. The transit sequence clearly indicates the localization of the gene product to cyanelles and is more similar to the transit sequences of the plant homologues than to transit sequences of other cyanelle precursor polypeptides with the exception of a cyanelle consensus sequence at the N-terminus. The mature sequence reveals conservation of the thiamine pyrophosphate binding site. A neighbor-net planar graph suggests that Cyanophora , higher plants, and the photosynthetic protist Euglena gracilis acquired their nuclear-encoded transketolase genes via endosymbiotic gene transfer from the cyanobacterial ancestor of plastids; in the case of Euglena probably entailing two transfers, once from the plastid in the green algal lineage and once again in the secondary endosymbiosis underlying the origin of Euglena's plastids. By contrast, transketolase genes in some eukaryotes with secondary plastids of red algal origin, such as Thalassiosira pseudonana , have retained the pre-existing transketolase gene germane to their secondary host.     

Homologous protein import machineries in chloroplasts and cyanelles

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa.     

Protein translocation into and within cyanelles (review)

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria in morphology, pigmentation and, especially, in the presence of a peptidoglycan wall situated between the inner and outer envelope membranes. However, it is now clear that cyanelles in fact are primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high plastid gene content of the Porphyra purpurea rhodoplast and the peptidoglycan wall of glaucocystophyte cyanelles. This means that the import apparatus of all primary plastids should be homologous. Indeed, heterologous in vitro import can now be shown in both directions, provided a phenylalanine residue essential for cyanelle import is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved explaining the efficient heterologous import of native precursors from C. paradoxa. With respect to conservative sorting in cyanelles, both the Sec and Tat pathways could be demonstrated. Another cyanobacterial feature, the dual location of the Sec translocase in thylakoid and inner envelope membranes, is also unique to cyanelles. For the first time, protease protection of internalized lumenal proteins could be shown for cyanobacteria-like, phycobilisome-bearing thylakoid membranes after import into isolated cyanelles.     

An ORF323 with homology to crtE, specifying prephytoene pyrophosphate dehydrogenase, is encoded by cyanelle DNA in the eukaryotic alga Cyanophora paradoxa.

Carotenoids are essential constituents of the light-harvesting and light-protective systems of photosynthetic organisms. The biochemistry of carotenoid biosynthesis in eukaryotes is known, whereas evidence for the genes specifying this biosynthetic pathway is scant. We report here the nucleotide sequence and expression of a gene likely encoding crtE (prephytoene pyrophosphate dehydrogenase). The reaction product of this enzyme is phytoene, a C40 carotenoid precursor common to all organisms. The gene is found in the cyanelle (plastid) DNA of an eukaryotic alga, Cyanophora paradoxa. The expression into protein of cyanelle crtE has been demonstrated in vitro. The identity and similarity scores of CrtE from cyanelles with the corresponding protein from the photosynthetic bacterium Rhodobacter capsulatus are 28.6 and 68.5%, respectively.     

The cyanelle genome of Cyanophora paradoxa, unlike the chloroplast genome, codes for the ribosomal L3 protein.

We describe a 1132 bp sequence of the cyanelle genome of Cyanophora paradoxa containing the rpl3 gene. This gene, which is not chloroplast encoded in plants, is the first of a long cyanelle ribosomal operon whose organization resembles that of the S10 operon of E. coli. We have shown that the rpl3 gene is transcribed in cyanelles as a 7500 nucleotide precursor and that the 5'-end of the mRNA starts approximately 90 nucleotides upstream from the initiation codon. However, no typical procaryotic promoter could be found for this gene. We have detected, using anti E. coli L3 antibodies, the cyanelle L3 protein in cyanelle extracts and in E. coli cells transformed with the cyanelle rpl3 gene.     

The Cu(II)-repressible plastidic cytochrome c. Cloning and sequence of a complementary DNA for the pre-apoprotein

We have cloned a complementary DNA for pre-apocytochrome c-552 from Chlamydomonas reinhardtii. The deduced sequence of the mature protein shows high homology to those of cytochromes c-553 from cyanobacteria. Its homology to mitochondrial cytochrome c or bacterial photosynthetic cytochrome c2 is lower and appears to be concentrated in sequences around amino acids involved in the interaction with heme. With respect to primary sequence, the "transit sequence" for cytochrome c-552 appears to show no homology to other transit sequences for nuclear encoded chloroplast proteins. However, based on analogy to transit sequences for other proteins (Daldal, F., Cheng, S., Applebaum, J., Davidson, E., and Prince, R. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2012-2016; Goldschmidt-Clermont, M., and Rahire, M. (1986) J. Mol. Biol. 191, 421-432; Smeekens, S., de Groot, M., van Binsbergen, J., and Weisbeek, P. (1986) Cell 46, 365-375) the transit sequence of cytochrome c-552 can be divided into envelope-traversing and thylakoid-traversing domains. Cytochrome c-552 appears to encoded by a single nuclear gene in C. reinhardtii. The gene is expressed exclusively in Cu(II)-deficient cells.     

The biosynthesis of membrane and soluble plastidic c-type cytochromes of Chlamydomonas reinhardtii is dependent on multiple common gene products.

Cytochrome c6 functions in the thylakoid lumen to catalyze electron transfer from reduced cytochrome f of the cytochrome b6f complex to P700+ of photosystem I. The biogenesis of mature cyt c6 from cytosolically translated pre-apocytochrome c6 involves numerous post-translational modifications including the proteolytic removal of a transit sequence and the covalent attachment of heme to two cysteinyl thiols on the apoprotein. Here, we report on the characterization of a previously unrecognized class of non-allelic mutants of Chlamydomonas reinhardtii that are blocked at the conversion of apocyt c6 to holocyt c6. The mutants are acetate requiring since they are also deficient in cyt f, cyt b and the Rieske FeS protein. Pulse-chase studies indicate that heme attachment is not required for the two-step processing of pre-apocytochrome c6 to apocyt c6, but is required for the stability of the mature protein. This is in contrast to the biosynthesis of mitochondrial cyt c1 where heme attachment is required for the second processing step. We propose that the assembly of both holocytochrome c6 and the cytochrome b6f complex are dependent on common gene products, possibly those involved in heme delivery or metabolism. This is the first suggestion that multiple loci are involved in the biosynthesis of both plastidic c-type cytochromes.     

A carboxysomal carbon-concentrating mechanism in the cyanelles of the 'coelacanth' of the algal world, Cyanophora paradoxa?

Cyanelles are the peculiar plastids of glaucocystophyte algae that retained a peptidoglycan wall from the ancestral cyanobacterial endosymbiont. All cyanobacteria and most algae possess an inorganic carbon-concentrating mechanism (CCM) that involves a microcompartment--carboxysomes in prokaryotes and pyrenoids in eukaryotes--harboring the bulk of cellular (plastidic) Rubisco. In the case of the living fossil, Cyanophora paradoxa, the existence of a CCM was a matter of debate. Microarray data revealing 142 CO(2)-responsive genes (induced or repressed through a shift from high to low CO(2) conditions), gas exchange measurements and measurements of photosynthetic affinity provided strong support for a CCM. We favor a recent hypothesis that glaucocystophyte cyanelles as the closest cousins to cyanobacteria among plastids contain 'eukaryotic carboxysomes': bicarbonate enrichment within cyanelles should be considerably higher than in chloroplasts with their pyrenoid-based CCM. Thus, the stress-bearing function of the peptidoglycan layer, the other unique heritage, would be indispensable. An isolation method for cyanelle 'carboxysomes' was developed and the protein components other than Rubisco analyzed by MS. Rubisco activase was identified and corroborated by western blotting. The well-established cyanelle in vitro import system allows to use them as 'honorary cyanobacteria': assembly processes of supramolecular structures as phycobilisomes and carboxysomes thus can be studied after import of nucleus-encoded precursor proteins and subsequent fractionation. Even minor components can easily be tracked and a surprisingly dynamic view is obtained. Labeled pre-activase was imported into isolated cyanelles and 30% of the mature protein was found to be incorporated into the carboxysome fraction. A final decision between carboxysome or pyrenoid must await the identification of cyanelle carbonic anhydrase and, especially, the demonstration of shell proteins.     

Conservative sorting in a primitive plastid. The cyanelle of Cyanophora paradoxa

Higher plant chloroplasts possess at least four different pathways for protein translocation across and protein integration into the thylakoid membranes. It is of interest with respect to plastid evolution, which pathways have been retained as a relic from the cyanobacterial ancestor ('conservative sorting'), which ones have been kept but modified, and which ones were developed at the organelle stage, i.e. are eukaryotic achievements as (largely) the Toc and Tic translocons for envelope import of cytosolic precursor proteins. In the absence of data on cyanobacterial protein translocation, the cyanelles of the glaucocystophyte alga Cyanophora paradoxa for which in vitro systems for protein import and intraorganellar sorting were elaborated can serve as a model: the cyanelles are surrounded by a peptidoglycan wall, their thylakoids are covered with phycobilisomes and the composition of their oxygen-evolving complex is another feature shared with cyanobacteria. We demonstrate the operation of the Sec and Tat pathways in cyanelles and show for the first time in vitro protein import across cyanobacteria-like thylakoid membranes and protease protection of the mature protein.     

Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope.

The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Schmid, G. Allmaier, B. Pfanzagl, W. Löffelhardt, C. Quintela, M. A. de Pedro, and W. Stanek, Biol. Mass Spectrom. 22:524-536, 1993). In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized. An average glycan chain length of 51 disaccharide units was determined by the transfer of [U-14C]galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan. The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing.     

What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein?

Whereas it is widely accepted that the transit peptide of the precursor for the light-harvesting chlorophyll a/b protein (preLHCP) is responsible for targeting this polypeptide to chloroplasts, the signals which govern its intraorganellar targeting appears to be transit peptide-mediated for plastocyanin (Smeekins, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375) and several other nuclear-encoded, thylakoid luminal proteins. To determine whether a similar mechanism operates for LHCP (an integral thylakoid protein), we have used oligonucleotide-directed mutagenesis to delete the proposed transit sequence from a petunia precursor of this polypeptide. Intact preLHCP and the deletion mutant product have been expressed in vitro, and their abilities to integrate into purified thylakoids have been compared. We have found that both polypeptides insert into thylakoids correctly, provided the latter are supplemented with a membrane-free stromal extract and Mg.ATP. Our results clearly demonstrate that whereas the transit peptide is required for transport into chloroplasts, thylakoid integration of preLHCP is determined by mature portions of the polypeptide. In addition, we note that transit peptide removal has little effect on the apparent solubility of the in vitro translation products.     

Amino acid sequences of cytochrome c-554(548) and cytochrome c'' from a halophilic denitrifying bacterium of the genus Paracoccus.

The amino acid sequences of the cytochromes c-554(548) and c' from the moderately halophilic bacterium Paracoccus sp., I.A.M. 203 (= A.T.C.C. 12084, N.C.I.B. 8669) have been determined. Cytochrome c-554(548) consists of a single polypeptide chain of 83 residues, and dimerizes strongly. The most similar protein of known sequence is the N-terminal half of the dihaem cytochrome c4, and other related proteins include the cytochrome c-554(547) of Thiobacillus neapolitanus and the cytochrome c-553 of Desulfovibrio vulgaris. Cytochrome c', which has a single polypeptide chain of 132 residues, is similar in sequence to cytochromes c' from phototrophic and denitrifying bacteria, but only shows about 36% sequence identity to the most similar protein of known sequence. Both of the Paracoccus proteins have a considerable excess of acidic amino acid side chains over basic ones, and a higher proportion of their basic amino acids is arginine than is usual in cytochromes c. Both these characteristics seem to be adaptations to increase the stability of the proteins in an environment of high ionic strength. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication 50140 (24 pp.) at the British Library (Lending Division), Boston Spa, Yorkshire LS23 7BQ, U.K. from which copies are available on prepayment.     

Analyses of ribosomal RNA sequences from glaucocystophyte cyanelles provide new insights into the evolutionary relationships of plastids

Glaucocystophyte algae (sensu Kies, Berl. Deutsch. Bot. Ges. 92, 1979) contain plastids (cyanelles) that retain the peptidoglycan wall of the putative cyanobacterial endosymbiont; this and other ultrastructural characters (e.g., unstacked thylakoids, phycobilisomes) have suggested that cyanelles are primitive plastids that may represent undeveloped associations between heterotrophic host cells (i.e., glaucocystophytes) and cyanobacteria. To test the monophyly of glaucocystophyte cyanelles and to determine their evolutionary relationship to other plastids, complete 16S ribosomal RNA sequences were determined for Cyanophora paradoxa, Glaucocystis nostochinearum, Glaucosphaera vacuolata, and Gloeochaete wittrockiana. Plastid rRNAs were analyzed with the maximum-likelihood, maximumparsimony, and neighbor joining methods. The phylogenetic analyses show that the cyanelles of C. paradoxa, G. nostochinearum, and G. wittrockiana form a distinct evolutionary lineage; these cyanelles presumably share a monophyletic origin. The rDNA sequence of G. vacuolata was positioned within the nongreen plastid lineage. This result is consistent with analyses of nuclear-encoded rRNAs that identify G. vacuolata as a rhodophyte and support its removal from the Glaucocystophyta. Results of a global search with the maximumlikelihood method suggest that cyanelles are the first divergence among all plastids; this result is consistent with a single loss of the peptidoglycan wall in plastids after the divergence of the cyanelles. User-defined tree analyses with the maximum-likelihood method indicate, however, that the position of the cyanelles is not stable within the rRNA phylogenies. Both maximumparsimony and neighbor-joining analyses showed a close evolutionary relationship between cyanelles and nongreen plastids; these phylogenetic methods were sensitive to inclusion/exclusion of the G. wittrockiana cyanelle sequence. Base compositional bias within the G. wittrockiana 16S rRNA may explain this result. Taken together the phylogenetic analyses are interpreted as supporting a near-simultaneous radiation of cyanelles and green and nongreen plastids; these organelles are all rooted within the cyanobacteria.Correspondence to: D. Bhattacharya     

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.     

Characterization of three soluble c-type cytochromes isolated from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705

Three soluble, low molecular mass cytochromes c (Mr 8000-15,000) were isolated and purified from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705. On the basis of their alpha: absorbance peaks in the reduced forms, they were named cytochromes c550, c552 and c555. Cytochrome c552 reacted very fast, c555 very slowly and c550 not at all with carbon monoxide. The complete amino acid sequence (73 residues) of cytochrome c552 was established which identifies it as a monoheme, class I cytochrome c with some remote similarity to the cytochrome c6 family.     

Ribosomal protein L35: identification in spinach chloroplasts and isolation of a cDNA clone encoding its cytoplasmic precursor

We describe the isolation of spinach chloroplast ribosomal protein L35 and characterization of a cDNA clone encoding its cytoplasmic precursor. This protein was only recently identified in ribosomes, but the sequences of four L35 genes have now been reported and confirm its presence in eubacteria, chloroplasts, and cyanelles. Using N-terminal sequence data, oligonucleotides were designed and a cDNA library was screened. The nucleotide sequence of the cDNA clones shows that the spinach L35 protein is encoded as a precursor of 159 residues, comprising a mature protein of 73 residues and a transit peptide of 86 residues. The cleavage site for forming the mature protein is deduced to be Thr-Val-Phe-Ala decreases Ala-Lys-Gly-Tyr. The L35 protein in the photosynthetic organelle of the protozoan Cyanophora paradoxa is encoded in the organelle DNA [Bryant & Stirewalt (1990) FEBS Lett. 259, 273-280]. The corresponding gene has not been found in the chloroplast DNA of a lower plant (liverwort) and two higher plants. Our results demonstrate that the L35 protein in a higher plant (spinach) is encoded in the nucleus. This finding, in light of the endosymbiont hypothesis, suggests an organelle to nucleus transfer of the L35 gene at the evolutionary beginnings of land plants.     

The amino acid sequence of cytochrome c from the blowfly Lucilia cuprina.

The amino acid sequence of cytochrome c isolated from the sheep blowfly Lucilia cuprina has been determined by comparison of the compositions of the tryptic peptides to those predicted from the published sequences of cytochromes c from other insects. Cytochrome c from L. cuprina differs at a single residue when compared to cytochrome c from the screw worm fly Haematobia irritans, a species belonging to the same order as the blowfly. This substitution, proline for alanine, has been located at position 44 in the protein chain.     

The cyanelle genome of Cyanophora paradoxa encodes ribosomal proteins not encoded by the chloroplasts genomes of higher plants

The rpl35, rpl20, rpl5, rps8, and a portion of the rpl6 genes of the cyanelle genome of Cyanophora paradoxa have been cloned, mapped and sequenced. Homologs of the rpl35, rpl5, and rpl6 genes are not found in the chloroplasts of higher plants. The rpl35 genes most likely form a dicistronic operon which is located upstream from the apcE-apcA-apcB locus of the cyanelle and which is divergently transcribed from this locus. The rpl5, rpl8, and rpl6 genes probably form a part of a larger cluster of genes encoding components of the cyanellar ribosomes. These genes are organized in a fashion similar to that observed in all procaryotes examined to date, with the exception that the rps14 gene is not found between the rpl5 and rps8 coding sequences. Hypotheses concerning the origins of cyanelles and chloroplasts are discussed.