Covalent immobilization of proteins onto (Maleic anhydride-alt-methyl vinyl ether) copolymers: enhanced immobilization of recombinant proteins

The amazing diversity of extant photosynthetic eukaryotes is largely a result of the presence of formerly free-living photosynthesizing organisms that have been sequestered by eukaryotic hosts and established as plastids in a process known as endosymbiosis. The evolutionary history of these endosymbiotic events was traditionally investigated by studying ultrastructural features and pigment characteristics but in recent years has been approached using molecular sequence data and gene trees. Two important developments, more detailed studies of members of the Cyanobacteria (from which plastids ultimately derive) and the availability of complete plastid genome sequences from a wide variety of plant and algal lineages, have allowed a more accurate reconstruction of plastid evolution.     

The role of lipids in plastid protein transport

The elaborate compartmentalization of plant cells requires multiple mechanisms of protein targeting and trafficking. In addition to the organelles found in all eukaryotes, the plant cell contains a semi-autonomous organelle, the plastid. The plastid is not only the most active site of protein transport in the cell, but with its three membranes and three aqueous compartments, it also represents the most topologically complex organelle in the cell. The chloroplast contains both a protein import system in the envelope and multiple protein export systems in the thylakoid. Although significant advances have identified several proteinaceous components of the protein import and export apparatuses, the lipids found within plastid membranes are also emerging as important players in the targeting, insertion, and assembly of proteins in plastid membranes. The apparent affinity of chloroplast transit peptides for chloroplast lipids and the tendency for unsaturated MGDG to adopt a hexagonal II phase organization are discussed as possible mechanisms for initiating the binding and/or translocation of precursors to plastid membranes. Other important roles for lipids in plastid biogenesis are addressed, including the spontaneous insertion of proteins into the outer envelope and thylakoid, the role of cubic lipid structures in targeting and assembly of proteins to the prolamellar body, and the repair process of D1 after photoinhibition. The current progress in the identification of the genes and their associated mutations in galactolipid biosynthesis is discussed. Finally, the potential role of plastid-derived tubules in facilitating macromolecular transport between plastids and other cellular organelles is discussed.     

Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.     

In vitro import of pre-ferredoxin-NADP+-oxidoreductase from Cyanophora paradoxa into cyanelles and into pea chloroplasts

Using a novel cyanelle isolation procedure we showed that pre-ferredoxin-NADP+-oxidoreductase (pre-FNR) from C. paradoxa is translocated in vitro across the peptidoglycan-containing cyanelle envelope. Efficient import was also observed in a heterologous system with pea chloroplasts as the recipient organelles. These results support the conclusion derived from comparative analysis of plastid genome organization, that all plastids originate from a common semi-autonomous endosymbiotic ancestor.     

The growing importance of the plastid-like DNAs of the Apicomplexa

Organisms in the phylum Apicomplexa possess, in addition to their mitochondrial genome, an extrachromosomal DNA that possesses significant similarities with the extrachromosomal genomes of plastids. To date, the majority of data on these plastid-like DNAs have been obtained from the human malarial organism, Plasmodium falciparum. In common with plastid DNAs, the plastid-like DNA of P. falciparum possesses genes for DNA-dependent RNA polymerase subunits beta and beta 1 and for organellar-like large- and small-subunits ribosomal RNAs. Both the polymerase subunit and ribosomal RNA gene sequences share a number of features with those from plastid DNAs. In addition, the ribosomal RNA genes are organised in an inverted repeat arrangement, reminiscent of plastid DNAs. Additional molecular features shared between the 2 genomes are discussed. Plastid-like DNAs have also been identified in other Plasmodium species as well as Toxoplasma gondii, Eimeria tenella, Babesia bovis and a number of Sarcocystis species. A cryptic organelle often observed in apicomplexans has been proposed as the organelle that harbours the plastid-like DNAs, but conclusive evidence for this has not yet been obtained. Although approximately 1/2 of the plastid-like DNA of P. falciparum has been sequenced to date, no function has yet been ascribed to this DNA or its putative organelle. Phylogenetic inferences based on sequence data from this DNA have indicated an evolutionary origin from photosynthetic organisms, but the true provenance of the plastid-like DNAs remains to be determined. Because of the specific nature of the plastid-like DNAs, they may prove useful as effective targets for chemotherapeutics.     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56-69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O. ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P < 0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.     

Plastids are widespread and ancient in parasites of the phylum Apicomplexa

Current evidence supports the presence of a non-photosynthetic chloroplast-like organelle in several apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii. This apicomplexan organelle, referred to here as the "plastid", may have been acquired through a primary or secondary endosymbiosis of a photosynthetic organism. Alternatively, apicomplexan plastids may have been acquired through several independent endosymbiotic events, as appears to be the case for the acquisition of chloroplasts by dinoflagellates. The likelihood of multiple origins of an apicomplexan plastid is enhanced by the close evolutionary relatedness of apicomplexan and dinoflagellate taxa. In this study, we have tested the hypothesis that apicomplexan plastids are derived from a single ancient ancestor. Two lines of evidence supporting this hypothesis are presented. First, this study supports the widespread presence of plastid DNA in apicomplexan species. Second, the topologies of the phylogenetic trees derived from plastid and nuclear-encoded rRNA gene sequences suggest the co-evolution of the DNAs localised in these two compartments. Taken together, these data support a single ancient lineage for the plastids of parasites in the phylum Apicomplexa.     

The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants

The photosynthetic gene rbcL has been lost or dramatically altered in some lineages of nonphotosynthetic parasitic plants, but the dynamics of these events following loss of photosynthesis and whether rbcL has sustained functionally significant changes in photosynthetic parasitic plants are unknown. To assess the changes to rbcL associated with the loss of functional constraints for photosynthesis, nucleotide sequences from nonparasitic and parasitic plants of Scrophulariales were used for phylogeny reconstruction and character analysis. Plants in this group display a broad range of parasitic abilities, from photosynthetic ("hemiparasites") to nonphotosynthetic ("holoparasites"). With the exception of Conopholis (Orobanchaceae), the rbcL locus is present in all parasitic plants of Scrophulariales examined. Several holoparasitic genera included in this study, including Boschniakia, Epifagus, Orobanche, and Hyobanche, have rbcL pseudogenes. However, the holoparasites Alectra orobanchoides, Harveya capensis, Harveya purpurea, Lathraea clandestina, Orobanche corymbosa, O. fasciculata, and Striga gesnerioides have intact open reading frames (ORFs) for the rbcL gene. Phylogenetic hypotheses based on rbcL are largely in agreement with those based on sequences of the nonphotosynthetic genes rps2 and matK and show a single origin of parasitism, and loss of photosynthesis and pseudogene formation have been independently derived several times in Scrophulariales. The mutations in rbcL in nonparasitic and hemiparasitic plants would result in largely conservative amino acid substitutions, supporting the hypothesis that functional proteins can experience only a limited range of changes, even in minimally photosynthetic plants. In contrast, ORFs in some holoparasites had many previously unobserved missense substitutions at functionally important amino acid residues, suggesting that rbcL genes in these plants have evolved under relaxed or altered functional constraints.     

Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1 Mb region from map positions 64% to 92% of the genome

The contiguous sequence of 1,003,450 bp spanning map positions 64% to 92% of the genome of Synechocystis sp. strain PCC6803 has been deduced. Computer analysis of the sequence predicts that this region contains at least 818 potential ORFs, in which 255 (31%) were either genes that had already been identified or their homologues, 84 (10%) were homologues to registered hypothetical genes, and 149 (18%) showed weak similarities to reported genes. The remaining 330 ORFs showed no apparent similarity to any reported genes or carried no significant protein motifs. The potential ORFs as a whole occupied 86% of the sequenced region, implying compact arrangement of genes in the genome. As to the structural RNA genes, one rRNA operon consisting of 5,028 bp and at least 11 species of tRNA genes were identified. It is noteworthy that 10 out of the 11 tRNA species showed significant sequence similarities to tRNAs reported in plant chloroplasts. As other notable unique sequences, three classes of IS-like elements each with characteristics typical of IS elements were identified, and a typical unit of WD(Trp-Asp)-repeats which have only been detected in the regulatory proteins of eukaryotes was identified within the large 5,079-bp ORF located at map position 69%.     

Mitochondrial gene rearrangement in the sea cucumber genus Cucumaria

A novel mitochondrial tRNA gene arrangement is described for two species of sea cucumber. The mitochondrial tRNA gene cluster common to sea stars, sea urchins, and the sea cucumber Parastichopus californicus has been significantly modified in the genus Cucumaria as a result of dispersal of the tRNA genes into two separate areas of the genome. The tRNA genes in the novel clusters are interspersed with short unassigned sequences (UASs). Alignment of the two separated novel clusters indicates that the rearrangement was most likely the result of a tandem duplication of approximately 7 kb, encompassing the putative control region, the tRNA cluster, NADH dehydrogenase subunits 1 and 2, the large ribosomal RNA (lrRNA), cytochrome oxidase subunit I, and tRNAArg. Subsequently, deletion of the duplicated lrRNA and protein-coding genes occurred. In addition, the degeneration of one of each of the duplicated tRNA gene pairs has resulted in the interspersed UAS segments observed in each cluster. In contrast, the second copy of the putative control region has been maintained with a very high degree of sequence conservation, suggesting either some functional constraint or concerted evolution for the duplicated element. Analysis of gene organization in other sea cucumber species may provide (1) important insights into the mechanism of mitochondrial gene rearrangements and (2) an informative character set for deep-level phylogenetic analysis of this echinoderm class.     

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.     

Evolution of a mitochondrial tRNA PHE gene in A. thaliana: import of cytosolic tRNA PHE into mitochondria

Previously we have described a putative tRNATyr in Arabidopsis thaliana mitochondria, the sequence of which is different from that of other plant mitochondrial tRNATyr genes. We show here that this tRNATyr gene sequence is present in several copies in the mitochondrial genome of A. thaliana. One copy of these tRNATyr gene sequences, termed here tRNATyr-1, could encode a functional tRNA. Expression analysis has shown that the tRNATyr-1 gene is cotranscribed with the downstream tRNAGlu gene, and that the corresponding mature-sized tRNA is present in mitochondria. We also show that the native tRNATyr gene, similar to the mitochondrial tRNATyr genes found in plants, is present in the A. thaliana mitochondrial genome and expressed. The tRNATyr-1 gene has been previously suggested to be derived from a tRNAPhe gene sequence. We show here that, as a consequence, there is no tRNAPhe gene in the mitochondrial genome of A. thaliana and that a cytosolic tRNAPhe is imported in A. thaliana mitochondria.     

Is E37, a major polypeptide of the inner membrane from plastid envelope, an S-adenosyl methionine-dependent methyltransferase?

Using antibodies raised against E37, one of the major polypeptides of the inner membrane from the chloroplast envelope, it has been demonstrated that a single immunologically related polypeptide was present in total protein extracts from various higher plants (monocots and dicots), in photosynthetic and non-photosynthetic tissues from young spinach plantlets, as well as in the cytoplasmic membrane from the cyanobacteria Synechococcus. This ubiquitous distribution of E37 strongly suggests that this protein plays an envelope-specific function common to all types of plastids. Comparison of tobacco and spinach E37 amino acid sequences deduced from the corresponding cDNA demonstrates that consensus motifs for S-adenosyl methionine-dependent methyltransferases are located in both sequences. This hypothesis was confirmed using a biochemical approach. It was demonstrated that E37, together with two minor spinach chloroplast envelope polypeptides of 32 and 39 kDa, can be specifically photolabeled with [3H]-S-adenosyl methionine upon UV-irradiation. Identification of E37 as a photolabeled polypeptide was established by immunoprecipitation. Furthermore, photolabeling of the three envelope polypeptides was specifically inhibited by very low concentration of S-adenosyl homocysteine, thus providing evidence for the presence within these proteins of S-adenosyl methionine- and S-adenosyl homocysteine-binding sites that were closely associated. Taken as a whole these results strongly suggest that E37 is an ubiquitous plastid envelope protein that probably has an S-adenosyl methionine-dependent methyltransferase activity. The 32 and 39 kDa envelope polypeptides probably have a similar methyltransferase activity.     

Protein transport into "complex" diatom plastids utilizes two different targeting signals

The plastids found in diatoms and other chromophytic algae are completely enclosed by four membranes in contrast to chloroplasts of higher plants, which are surrounded by only two membranes. The bipartite targeting sequence of diatom nuclear-encoded plastid proteins contains an endoplasmic reticulum signal sequence and, based on sequence comparison, a transit peptide-like domain similar to that which targets proteins into the plastids of higher plants. By performing heterologous import experiments using the precursor of the gamma subunit of the chloroplast ATPase from the diatom Odontella sinensis we were able to show that protein import into diatom plastids is at least a two-step event. We demonstrate that the first step involves co-translational transport through endoplasmic reticulum membranes and that there is an additional targeting step which is similar to the import of precursor proteins into chloroplasts of higher plants and green algae indicating that the transit peptide-like domain of the diatom precursor is functionally equivalent to the respective targeting signal of higher plants. Our results suggest that the transit peptide depending targeting mechanism in plastids has apparently remained relatively unchanged over the course of evolution, with only the peptidase cleavage site significantly modified.     

The yellow variegated mutant of Arabidopsis is plastid autonomous and delayed in chloroplast biogenesis

The yellow variegated mutant of Arabidopsis thaliana is characterized by bright-yellow true leaves that turn green- and white-sectored as leaf development proceeds. Variegation is due to the action of a nuclear recessive gene. Whereas cells in the green sectors contain morphologically normal chloroplasts, cells in the yellow and white sectors are heteroplastidic and contain plastids with rudimentary lamellar structures, as well as some normal-appearing chloroplasts. This indicates that plastids in yellow variegated are affected differently by the nuclear mutation (the mutant is "plastid autonomous"). Genetic analyses have revealed that yellow variegated is an allele of the var2 locus, and that defective plastids are not maternally inherited. The traits of plastid autonomy and lack of maternal inheritance of the plastid defect set var2 apart from other nuclear gene-induced variegations and define a novel class of variegation mutant. The primary lesion in var2 probably does not involve a blockage in the pathways of pigment biosynthesis. Under high temperatures or low light conditions, plant growth is retarded and mutant plants are nearly all-green. Considered together, our data suggest that var2 is delayed in chloroplast biogenesis. We suggest that the stochastic pattern of variegation in the mutant may be due to an interplay of factors that regulate var2 gene expression and factors that mediate rates of cell and plastid division. Plastids with a critical threshold of the partially functional var2 protein are green, while plastids containing less than the threshold of var2 activity are white.     

Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: the existence of genes possibly involved in chloroplast division

The complete nucleotide sequence of the chloroplast genome (150,613 bp) from the unicellular green alga Chlorella vulgaris C-27 has been determined. The genome contains no large inverted repeat and has one copy of rRNA gene cluster consisting of 16S, 23S, and 5S rRNA genes. It contains 31 tRNA genes, of which the tRNALeu(GAG) gene has not been found in land plant chloroplast DNAs analyzed so far. Sixty-nine protein genes and eight ORFs conserved with those found in land plant chloroplasts have also been found. The most striking is the existence of two adjacent genes homologous to bacterial genes involved in cell division, minD and minE, which are arranged in the same order in Escherichia coli. This finding suggests that the mechanism of chloroplast division is similar to bacterial division. Other than minD and minE homologues, genes encoding ribosomal proteins L5, L12, L19, and S9 (rpl5, rpl12, rpl19, and rps9); a chlorophyll biosynthesis Mg chelating subunit (chlI); and elongation factor EF-Tu (tufA), which have not been reported from land plant chloroplast DNAs, are present in this genome. However, many of the new chloroplast genes recently found in red and brown algae have not been found in C. vulgaris. Furthermore, this algal species possesses two long ORFs related to ycf1 and ycf2 that are exclusively found in land plants. These observations suggest that C. vulgaris is closer to land plants than to red and brown algae.