Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved

Summary Antibodies to individual chloroplast ribosomal (r-)proteins ofChlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness ofChlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast,Escherichia coli, and the cyanobacteriumAnabaena 7120. In addition,³⁵S-labeled chloroplast r-proteins from large and small subunits ofC. reinhardtii were coelectrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts,E. coli, andAnabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In constrast, when³⁵S-labeled chloroplast r-proteins from large and small subunits of a closely related speciesC. smithii were coelectrophoresed with unlabeledC. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility.Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins.Anabaena r-proteins showed the greatest immunological similarity toC. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins inC. reinhardtii showed much higher levels of cross-reactivity with r-proteins fromAnabaena, spinach, andE. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins ofC. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). FourE. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two otherE. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity.     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

Studies of ribosomal proteins of yeast species and their hybrids gel electrophoresis and immunochemical cross-reactions

Summary The cytoplasmic ribosomal proteins (r-proteins) of seventeen yeast species of the genera Saccharomyces and Kluyveromyces were analyzed by one-dimensional gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The electrophoretic patterns of cytoplasmic r-proteins from different species display extensive differences in both the 40S and the 60S subunit. Relatedness of species suggested by r-protein patterns correlates well with that based on DNA/DNA homology (Bicknell and Douglas 1970). Immunochemical cross-reactions and antibiotic susceptibility tests were also used to compare different species.Analyses of r-proteins from two different interspecific hybrids showed that their ribosomes were hybrid, containing r-proteins from both parents. These findings are discussed in relation to the evolution of yeast species and the regulation of expression of r-proteins in cucaryotes.     

The cytoplasmic ribosomes of Chlamydomonas reinhardtii: characterization of antibiotic sensitivity and cycloheximide-resistant mutants

Summary In vitro protein synthesis was used to characterize the antibiotic sensitivity of cytoplasmic ribosomes from wild-type and antibiotic-resistant strains of Chlamydomonas reinhardtii. Cytoplasmic ribosomes from two cycloheximide-resistant mutants, act-1 and act-2, were resistant to the antibiotic in vitro. The alteration effected by the act-1 mutation, which was dominant in diploids, was localized to the large subunit of the cytoplasmic ribosomes, but no ribosomal protein alterations were detected using two-dimensional gel electrophoresis. The act-2 mutation, which was semidominant in diploids, was frequently associated with a charge alteration in the large subunit ribosomal protein (r-protein) cyL38 that segregated independently from the antibiotic-resistant phenotype in crosses.     

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostreptonresistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.     

The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome

Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.     

Ribosome specificity for the formation of guanosine polyphosphates

Ribosomes obtained from $\text{Bacillus brevis}$ (ATCC 8185) were slightly active in synthesizing guanosine polyphosphates, which activity was greatly stimulated by addition of $\text{Escherichia coli}$ stringent factor. $\text{Chlamydomonas reinhardtii}$ chloroplast ribosomes did not produce guanosine polyphosphates on incubation but responded with abundant synthesis to addition of the stringent factor from $\text{E. coli}$. In contrast, cytoplasmic ribosomes from the same organism did not respond. Interchange experiments between either subunit from chloroplasts with the $\text{E. coli}$ counterparts showed good activity. When the small subunit of cytoplasmic $\text{Chlamydomonas}$ ribosomes was combined with the large subunit of $\text{E. coli}$ or of chloroplasts, a small but definite response was obtained.     

Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii

Summary Mutations at seven recombinationally distinct chloroplast loci confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii. Assays of polynucleotide-directed amino acid incorporation by ribosomes reconstituted from mutant and wild type subunits demonstrate that streptomycin, neamine/kanamycin and spectinomycin resistance mutations specifically affect the small ribosomal subunit, whereas mutations to erythromycin resistance affect the large subunit. Although in each case the subunit site of antibiotic resistance is the same as that observed in analogous mutations in Escherichia coli, the number of loci conferring resistance to a given antibiotic differs in the two organisms. We have previously shown that streptomycin resistance mutations in Chlamydomonas map at five discrete loci (one nuclear and four chloroplast), and that mutations to neamine/kanamycin and spectinomycin resistance appear to define a single chloroplast locus. Results presented here confirm our previous report that all chloroplast erythromycin resistance mutations isolated to date fall into two recombinationally distinct loci, and indicate that mutants at one of these loci may be further divided on the basis of their level of cross resistance to other macrolide antibiotics.     

Ribosomal proteins

Summary A comparison of the protein patterns of the 70S and 80S ribosomes from various plants, E. coli and yeast by disc-gel electrophoresis has shown the following relations: 1. There is a greater similarity between chloroplast ribosomes from various plants than between chloroplast and cytoplasmic ribosomes obtained from the same plant. 2. The protein patterns of the cytoplasmic ribosomes from bean, spinach and tobacco are more similar to each other than when compared to that of wheat germ. 3. At least one band is common to cytoplasmic ribosomes from all plants tested. 4. Only very few bands with identical mobilities are observed between chloroplast and E. coli ribosomes and between cytoplasmic plant and yeast ribosomes.     

Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes

Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one- and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.     

High heterogeneity within the ribosomal proteins of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.Patrick Giavalisco, Daniel Wilson are contributed equally to this work.     

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.      

Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins

Antibodies raised against Drosophila melanogaster ribosomal proteins (r-proteins) were used to examine possible structural relationships between eukaryotic and prokaryotic r-proteins. The antisera were raised against either groups of r-proteins or individually purified r-proteins. Two antisera showed a cross-reaction with total Escherichia coli r-proteins in Ouchterlony double immunodiffusion assays: an antiserum against the D. melanogaster small subunit protein S14 (anti-S14) and an antiserum against a group of D. melanogaster r-proteins (anti-TP80). The specificity of the antisera and the identity of the homologous E. coli r-proteins were characterized by using immunooverlay and immunoblot assays. These assays indicated that anti-S14 was highly specific for protein S14 and anti-TP80 was a multispecific serum that recognized several of the D. melanogaster ribosomal proteins. The E. coli protein homologous to D. melanogaster protein S14 was identified as E. coli protein S6. By adsorption of the anti-TP80 serum, we determined that D. melanogaster protein 7/8 is homologous to the acidic E. coli protein L7/L12. D. melanogaster acidic protein 13 was also shown to be immunologically related to D. melanogaster protein 7/8.This research was supported by National Institutes of Health Grant GM23410 awarded to WYC. LMS was the recipient of a predoctoral fellowship from Molecular, Cellular, and Developmental Biology Training Grant PHS T32 CM07227. We are very grateful to Dr. Anthony Mahowald for providing us with embryos.     

Cross-genomic analysis of the translational systems of various organisms

We have characterized the genes encoding ribosomal proteins (r-proteins) as well as other translation-related factors of 15 eubacteria and four archaebacteria, and the genes for the mitochondrial r-proteins of Saccharomyces cerevisiae by using the complete genomic nucleotide sequence data of these organisms. In eubacteria, including two species of Mycoplasma, the operon structure of the r-protein genes is well conserved, while their relative orientation and chromosomal location are quite divergent. The operon structure of the r-protein genes in archaebacteria, on the other hand, is quite different from eubacteria and also among themselves. In addition, many archaebacterial r-proteins show similarity to rat cytoplasmic r-proteins. Nonetheless, characteristic features of several genes encoding proteins of functional importance are well conserved throughout the bacterial species including archaebacteria, as well as in S. cerevisiae. We searched for the genes encoding mitochondrial r-proteins in yeast by combining informatics and genetic experiments. Furthermore, we characterized some of the r-proteins genes by exchanging portions between Escherichia coli and S. cerevisiae and performed functional analysis of some of the genes from different evolutionary points of view. Our work may be extended towards phylogenetic analysis of organisms producing secondary metabolites of various sorts. Journal of Industrial Microbiology & Biotechnology (2001) 27, 163–169. Received 21 September 1999/ Accepted in revised form 22 September 2000     

Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins

Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.     

E. coli ribosomal proteins are cross reactive with antibody prepared against Chlamydomonas reinhardi chloroplast ribosomal subunit

Summary Antisera prepared against purified Chlamydomonas reinhardi small chloroplast ribosomal subunit, judged homogenous by sucrose gradient velocity sedimentation and RNA gel electrophoresis was immunologically cross reactive with E. coli ribosomal proteins. The results of three different experimental approaches, namely Ouchterlony double diffusion, sucrose gradient velocity sedimentation and two dimensional crossed immunoelectrophoresis indicate that both E. coli ribosomal subunits and the chloroplast large ribosomal subunit contain proteins which show antigenic similarity to the chloroplast small ribosomal subunit proteins. However, cytoplasmic ribosomal subunits did not contain proteins which were cross reactive with immune antisera.     

Establishment of Arabidopsis thaliana ribosomal protein RPL23A-1 as a functional homologue of Saccharomyces cerevisiae ribosomal protein L25

Arabidopsis thaliana ribosomal protein (r-protein) RPL23A-1 shows 54% amino acid sequence identity to the Saccharomyces cerevisiae equivalent r-protein, L25. AtRPL23A-1 also shows high amino acid sequence identity to members of the L23/L25 r-protein family in other species. R-protein L25 in S. cerevisiae has been identified as a primary rRNA-binding protein that directly binds to a specific site on yeast 26S rRNA. It is translocated to the nucleolus where it binds to 26S rRNA during early large ribosome subunit assembly; this binding is thought to play an important role in ribosome assembly. The S. cerevisiae mutant strain YCR61 expresses L25 when grown on galactose, but not glucose, medium. Transformation of YCR61 with a shuttle vector containing the AtRPL23A-1 cDNA allowed transformed colonies to grow in and on glucose selection medium. R-protein AtRPL23A-1 can complement the L25 mutation, demonstrating the functional equivalence of the two r-proteins and introducing AtRPL23A-1 as the first plant member of the L23/L25 r-protein family.     

Ribosome profiles and riboproteomes of healthy and Potato virus A- and Agrobacterium-infected Nicotiana benthamiana plants

Nicotiana benthamiana is an important model plant for plant–microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG-tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA- or Agrobacterium-infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r-proteins). The N. benthamiana riboproteome revealed approximately 6600 r-protein hits representing 424 distinct r-proteins that were members of 71 of the expected 81 r-protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r-protein composition. In PVA-infected plants, the number of identified r-protein paralogues was lower than in Agrobacterium-infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA-infected plants co-purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease-VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen-infected N. benthamiana will be useful for studies on the specific use of r-protein paralogues to control translation in infected plants.     

rRNA Maturation in Yeast Cells Depleted of Large Ribosomal Subunit Proteins

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.