Nuclear genome codes for chloroplast ribosomal proteins in Acetabularia. I. Isolation and characterization of chloroplast ribosomal particles

A method is described for the isolation of chloroplast ribosomes from Acetabularia cells in yields sufficient for the characterization of these particles. Ribosomal particles sedimenting with 70S, 56S, 44S, and 30S have been obtained. The monoribosome sediments with 70S and dissociates into a larger 44S and a smaller 30S subunit. The sedimentation behaviour of the particles as well as the equilibrium between monoribosomes and their subunits is not influenced by the centrifugation step as could be revealed by formaldehyde fixation.     

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 kinetic complexity of Acetabularia chloroplast DNA.

The kinetic complexity of Acetabularia cliftonii chloroplast DNA is 1.52 +/- 0.26 . 10(9) daltons, compared to 0.2 .10(9) daltons for Chlamydomonas chloroplast DNA. There is an average of three genomes per chloroplast. The unusually large size of the Acetabularia genome may reflect the ancient evolutionary history of this organism.     

Evidence that nuclear genes code for several chloroplast ribosomal proteins

Electrophoresis of the proteins of the 50S chloroplast ribosome subunit reveals that there are at least two detectable differencesbetween Nicotiana tabacum and N. glauca. Nuclear genes contain the information for these two polypeptides since they are transmitted to F₁ interspecific hybrids independently of the maternal parent.     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.     

The chloroplast ribosomal intron of Chlamydomonas reinhardii codes for a polypeptide related to mitochondrial maturases.

The sequences of the 888bp chloroplast ribosomal intron and of the flanking 23S rRNA gene regions of Chlamydomonasreinhardii have been established. The intron can be folded with a secondary structure which is typical of group I introns of fungal mitochondrial genes. It contains a 489bp open reading frame encoding a potential polypeptide that is related to mitochondrial maturases.     

Sites of synthesis of chloroplast ribosomal proteins in Chlamydomonas

Cells of Chlamydomonas reinhardtii were pulse-labeled in vivo in the presence of inhibitors of cytoplasmic (anisomycin) or chloroplast (lincomycin) protein synthesis to ascertain the sites of synthesis of chloroplast ribosomal proteins. Fluorographs of the labeled proteins, resolved on two-dimensional (2-D) charge/SDS and one-dimensional (1-D) SDS-urea gradient gels, demonstrated that five to six of the large subunit proteins are products of chloroplast protein synthesis while 26 to 27 of the large subunit proteins are synthesized on cytoplasmic ribosomes. Similarly, 14 of 31 small subunit proteins are products of chloroplast protein synthesis, while the remainder are synthesized in the cytoplasm. The 20 ribosomal proteins shown to be made in the chloroplast of Chlamydomonas more than double the number of proteins known to be synthesized in the chloroplast of this alga.     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.     

Chloroplast ribosomal protein L32 is encoded in the chloroplast genome

The 50 S subunit of chloroplast ribosomes was prepared from tobacco leaves. The proteins were fractionated and the N-terminal amino acid sequence of a 14 kDa protein was determined. This sequence matches the N-terminal sequence deduced from ORF55 located between ndhF and trnL on the small single-copy region of tobacco chloroplast DNA. The deduced protein shows homology to E. coli and B. stearothermophilus L32 proteins, and it has been named as CL32 and ORF55 as rpl32. The tobacco chloroplast genome therefore contains 21 different ribosomal protein genes.     

Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.

Six ribosomal proteins are specific to higher plant chloroplast ribosomes [Subramanian, A.R. (1993) Trends Biochem. Sci.18, 177-180]. Three of them have been fully characterized [Yamaguchi, K., von Knoblauch, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465; Yamaguchi, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28466-28482]. The remaining three plastid-specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N-terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs). PSRP-3 exists in two forms (alpha/beta, N-terminus free and blocked by post-translational modification), whereas PSRP-2 and PSRP-4 appear, from MS data, to be unmodified. PSRP-2 contains two RNA-binding domains which occur in mRNA processing/stabilizing proteins (e.g. U1A snRNP, poly(A)-binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis. PSRP-3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga. The ycf65 gene is absent from the chloroplast genomes of higher plants. Therefore, we suggest that Psrp-3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle-to-nucleus gene transfer in chloroplast evolution. PSRP-4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis. We would therefore suggest that PSRP-4 is an example of gene capture (via horizontal gene transfer) during chloro-ribosome emergence. Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database. All six PSRPs are nucleus-encoded. The cytosolic precursors of PSRP-2, PSRP-3, and PSRP-4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively. Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.     

Chloroplast membranes of the green alga Acetabularia mediterranea. II. Topography of the chloroplast membrane

The localization of the chlorophyll-protein complexes inside the thylakoid membrane of Acetabularia mediterranea was determined by fractionating the chloroplast membrane with EDTA and Triton X-100, by using pronase treatment, and by labeling the surface-exposed proteins with 125I. The effects of the various treatments were established by electrophoresis of the solubilized membrane fractions and electron microscopy. After EDTA and pronase treatment, the membrane structure was still intact. Only the two chlorophyll-protein complexes of 67,000 and 152,000 daltons and an additional polypeptides were found in the membrane before the EDTA and pronase treatment. The 125,000 dalton complex seems to be buried inside the lipid layer. The 23,000 dalton subunit of the 67,000 dalton complex is largely exposed to the surface of the EDTA-insoluble membrane and only the chlorophyll-binding subunit of 21,500 daltons is buried inside the lipid layer.     

Immunological similarities between specific chloroplast ribosomal proteins from Chlamydomonas reinhardtii and ribosomal proteins from Escherichia coli

Polyclonal antibodies were elicited against seven of the 33 different proteins of the large subunit of the chloroplast ribosome from Chlamydomonas reinhardtii. Three of these proteins are synthesized in the chloroplast and four are made in the cytoplasm and imported. In western blots, six of the seven antisera are monospecific for their respective large subunit ribosomal proteins, and none of these antisera cross-reacted with any chloroplast small subunit proteins from C. reinhardtii. Antisera to the three chloroplast-synthesized ribosomal proteins cross-reacted with specific Escherichia coli large subunit proteins of comparable charge and molecular weight. Only one of the four antisera to the chloroplast ribosomal proteins synthesized in the cytoplasm cross-reacted with an E. coli large subunit protein. None of the antisera cross-reacted with any E. coli small subunit proteins. On the assumption of a procaryotic, endosymbiotic origin for the chloroplast, those chloroplast ribosomal proteins still synthesized within the organelle appear to have retained more antigenic sites in common with E. coli ribosomal proteins than have those which are now the products of cytoplasmic protein synthesis. Antisera to this cytoplasmically synthesized group of chloroplast ribosomal proteins did not recognize any antigenic sites among C. reinhardtii cytoplasmic ribosomal proteins, suggesting that the genes for the cytoplasmically synthesized chloroplast ribosomal proteins either are not derived from the cytoplasmic ribosomal protein genes or have evolved to a point where no antigenic similarities remain.      

Nucleotide sequence and linkage map position of the genes for ribosomal proteins L14 and S8 in the maize chloroplast genome

The nucleotide sequence of a 1287-base-pair segment of the maize (Zea mays) chloroplast DNA, encoding chloroplast ribosomal proteins L14, S8 and the C-terminal part of L16, has been determined using the dideoxy-chain-termination method. These data from a monocot plant are compared to the corresponding data from a dicot and a lower plant and from two bacteria. The deduced amino acid sequence of maize chloroplast L14 shows 80%, 81%, 51% and 52% and that of S8 shows 75%, 58%, 39% and 38% sequence identity, respectively, to the corresponding sequences of Nicotiana tabacum, Marchantia polymorpha, Bacillus stearothermophilus and Escherichia coli. The starting map coordinates of rpL14 and rpS8 in the physical map of the maize chloroplast DNA [Larrinua, I. M., Muskavitch, K. M. T., Gubbins, E. J. and Bogorad, L. (1983) Plant Mol. Biol. 2, 129-140] are 31.330 and 31.841. The gene order is rpL16-spacer-rpL14-spacer-rpS8. Shine-Dalgarno sequences (GGA and AGGAGG) and computer-derived stem-loop structures of dyad symmetry are present in the spacers and the 3' downstream region of rpS8, respectively, but a chloroplast promoter-like sequence could not be detected suggesting that the latter might be located further upstream in this ribosomal protein gene cluster in maize chloroplast DNA.     

Comparison of ribosomal proteins of chloroplast from spinach and of E. coli

Summary A comparison of ribosomal proteins from Escherichia coli and from chloroplasts of Spinach was made using two separate methods: electrophoretic migration and immunochemical cross-reaction between blotted E. coli ribosomal proteins and chloroplast ribosomal subunits antisera. It is shown that L2 from E. coli (E-12) and L4 from chloroplasts (CS-L4) comigrated and that E-L4 immunologically cross-reacted with the isolated CS-L4 antibody. Co-migration was observed for three additional couples of 50S ribosomal proteins. It is also shown that at least one 30S E. coli ribosomal protein immuno-cross reacted with a 30S chloroplast antiserum and that three couples of 30S ribosomal proteins comigrated.     

Pea chloroplast ribosomal proteins: Characterization and site of synthesis

Summary The characterization of pea chloroplast ribosomal proteins has been carried out. The 30 S and 50 S subunits contain 24 and 32 ribosomal proteins respectively. These numbers are very similar to the numbers previously found for spinach chloroplast (Mache et al. 1980).Chloroplast-synthesized ribosomal proteins have been determined using the light driven system of protein synthesis of Ellis and Hartley (1981). It is concluded that at least 6 ribosomal proteins of the 30 S and 5 proteins of the 50 S subunits are made in chloroplasts. The possibility that 4 additional ribosomal proteins (3 of the 30 S and 1 of the 50 S subunit) are chloroplastmade, is discussed.     

Nuclear DNA codes for Nicotiana ferredoxin.

Ferredoxin was purified from 10 species of Nicotiana and spinach leaves. Fingerprints showed all to contain five major tryptic peptides. Some of the spinach peptides were different in RF and mobility from the Nicotiana peptides, but none of the Nicotiana ferredoxins had peptides which could distinguish one species of ferredoxin from another. Electrofocusing S-carbaminomethylcysteinyl ferredoxins showed spinach ferredoxin to have a more acidic and N. glutinosa ferredoxin a slightly more acidic isoelectric point than the other 9 Nicotiana species which were alike. Electro-focusing ferredoxin from the hybrid N. glutinosa female times N. glauca male resolved two bands or isozymes of ferredoxin, one corresponding to N. glutinosa, the other to N. glauca, the code for the latter having come from the DNA in the N. glauca pollen used to form the hybrid plant. N. glutinosa ferredoxin does not contain methionine and is different from N. tabacum and N. glauca ferredoxins which contain methionine. The N. glutinosa female times N. glauca male ferredoxin contained one-half the methionine found in N. glauca ferredoxin, thus confirming that some of the genetic information for ferredoxin in the hybrid was originally contained in the nuclear DNA of N. glauca.