Proteins of mammalian mitochondrial ribosomes.

The bovine mitochondrial system is being developed as a model system for studies on mammalian mitochondrial ribosomes. Information is emerging on the structural organization and RNA binding properties of proteins in these mitochondrial ribosomes. Unexpectedly, these ribosomes appear to interact directly with GTP, via a high affinity binding site on the small subunit. Despite major differences in their RNA content and physical properties, mammalian mitochondrial and cytoplasmic ribosomes contain about the same number of proteins. The proteins in each kind of ribosome have a similar size distribution, and both sets are entirely coded by nuclear genes, raising the possibility that these different ribosomes may contain the same set of proteins. Comparison of bovine mitochondrial and cytoplasmic r-proteins by co-electrophoresis in two-dimensional gels reveals that most of the cytoplasmic ribosomal proteins are more basic than the mitochondrial ribosomal proteins, and that none are co-migratory with mitochondrial ribosomal proteins, suggesting that the proteins in the two ribosomes are different. To exclude the possibility that the electrophoretic differences result only from post-translational modification of otherwise identical proteins, antibodies against several proteins from the large subunit of bovine mitochondrial ribosomes were tested against cytoplasmic ribosomes by solid phase radioimmunoassay and against cytoplasmic ribosomal proteins on Western blots. The lack of cross-reaction of these antibodies with cytoplasmic r-proteins suggests that mitochondrial ribosomal proteins have different primary structures and thus are most likely encoded by a separate set of nuclear genes.     

Mass spectrometry of intact ribosomes

The ability to maintain intact ribosomes in the mass spectrometer has enabled research into their changes in conformation and interactions. In the mass spectrometer, it is possible to induce dissociation of proteins from the intact ribosome and, in conjunction with atomic structures, to understand the factors governing their release. We have applied this knowledge to interpret the structural basis for release of proteins from ribosomes for which no high resolution structures are available, such as complexes with elongation factor G and ribosomes from yeast. We also describe how improvements in technology and understanding have widened the scope of our research and lead to dramatic improvements in quality and information available from spectra of intact ribosomes.     

A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments

Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes. During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum. An understanding of these processes will require the detailed structure of a eukaryotic ribosome. To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution. In general, we find that the active sites for protein synthesis and translocation have been highly conserved. It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove. By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center. In addition, both the small and large subunits are built around a dense tubular network. Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network. We surmise that many of these features correspond to rRNA, based on biochemical and structural data.Ribosomal function is critically dependent on the specific association of small and large subunits. Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes. In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit. Moreover, a novel bridge is formed between the platform and the base of the L1 domain. Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers. Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation.     

Properties of human mitochondrial ribosomes

Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.     

Modeling a minimal ribosome based on comparative sequence analysis

We have determined the three-dimensional organization of ribosomal RNAs and proteins essential for minimal ribosome function. Comparative sequence analysis identifies regions of the ribosome that have been evolutionarily conserved, and the spatial organization of conserved domains is determined by mapping these onto structures of the 30S and 50S subunits determined by X-ray crystallography. Several functional domains of the ribosome are conserved in their three-dimensional organization in the Archaea, Bacteria, Eucaryotic nuclear, mitochondria and chloroplast ribosomes. In contrast, other regions from both subunits have shifted their position in three-dimensional space during evolution, including the L11 binding domain and the alpha-sarcin-ricin loop (SRL). We examined conserved bridge interactions between the two ribosomal subunits, giving an indication of which contacts are more significant. The tRNA contacts that are conserved were also determined, highlighting functional interactions as the tRNA moves through the ribosome during protein synthesis. To augment these studies of a large collection of comparative structural models sampled from all major branches on the phylogenetic tree, Caenorhabditis elegans mitochondrial rRNA is considered individually because it is among the smallest rRNA sequences known. The C.elegans model supports the large collection of comparative structure models while providing insight into the evolution of mitochondrial ribosomes.     

Physiochemical properties of rat liver mitochondrial ribosomes

In the present study, the physiochemical properties of rat liver mitochondrial ribosomes were examined and compared with Escherichia coli ribosomes. The sedimentation and translational diffusion coefficients as well as the molecular weight and buoyant density of rat mitochondrial ribosomes were determined. Sedimentation coefficients were established using the time-derivative algorithm (Philo, J. S. (2000) Anal. Biochem. 279, 151-163). The sedimentation coefficients of the intact monosome, large subunit, and small subunit were 55, 39, and 28 S, respectively. Mitochondrial ribosomes had a particle composition of 75% protein and 25% RNA. The partial specific volume was 0.688 ml/g, as determined from the protein and RNA composition. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.41 g/cm(3). The molecular masses of mitochondrial and E. coli ribosomes determined by static light-scattering experiments were 3.57 +/- 0.14 MDa and 2.49 +/- 0.06 MDa, respectively. The diffusion coefficient obtained from dynamic light-scattering measurements was 1.10 +/- 0.01 x 10(-7) cm(2) s(-1) for mitochondrial ribosomes and 1.72 +/- 0.03 x 10(-7) cm(2) s(-1) for the 70 S E. coli monosome. The hydration factor determined from these hydrodynamic parameters were 4.6 g of water/g of ribosome and 1.3 g/g for mitochondrial and E. coli ribosomes, respectively. A calculated hydration factor of 3.3 g/g for mitochondrial ribosomes was also obtained utilizing a calculated molecular mass and the Svedberg equation. These measurements of solvation suggest that ribosomes are highly hydrated structures. They are also in agreement with current models depicting ribosomes as porous structures containing numerous gaps and tunnels.     

Ribosomes from a relC mutant strain of Escherichia coli show altered activity with bacterial release factor-1

Ribosomes from a relC mutant of Escherichia coli, JF505, are altered in the large subunit protein L11. This protein has abnormal mobility on gel electrophoresis. The ribosomes have a lowered specific activity for release factor-1 which is intermediate between that found for ribosomes containing normal L11 and that for L11 lacking ribosomes. JF505 ribosomes are as sensitive to inactivation of in vitro termination by thiostrepton as normal ribosomes when the antibiotic is added in dimethylsulphoxide but less sensitive when it is added in ethanol.     

Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.     

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.     

Native protein mass spectrometry: from intact oligomers to functional machineries

The development of electrospray ionization coupled to mass spectrometry has enabled the analysis of very large intact protein complexes, even when they are held together by weak non-covalent interactions. Together with equally spectacular advances in mass spectrometric instrumentation, a new field has emerged, termed native protein mass spectrometry, which focuses on the structural and functional analysis of the dynamics and interactions occurring in protein complexes. In the past two years, several important progressive steps in technologies have been reported that have led to exciting applications ranging from the detailed study of equilibria between different quaternary structures as influenced by environmental changes or binding of substrates or cofactors, to the analysis of intact nano-machineries, such as whole virus particles, proteasomes and ribosomes.     

Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics

Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug–ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.     

The structure of ribosomes as indicated by studies on tetramers from hypothermic chick embryos

Ribosome tetramers induced in chick embryos by exposure to cold, and tetramers of large subunits derived from them, have been studied by electron microscopy and sucrose-density-gradient analysis. Individual ribosomes of the normal tetramer are elongated bean-shaped structures, 220-280A by 195A (1A=10(-1)nm) with a cleft in the outer edge which divides the two-dimensional image into two unequal ends. Most of the tetramers appear to attach to the surface of the electron-microscope grid by one preferred face. The subunits of the large-subunit tetramers have a round outline and no cleft. About 25% of the subunits of these tetramers have a line running radially across the particle. The dissociation of tetramers into large-subunit tetramers and small subunits has been shown to be reversible. Mixtures of these particles from sucrose-density-gradient fractions were reassociated to give a tetramer with the same sedimentation coefficient as the original tetramer and with the same structure as viewed in the electron microscope. The results indicate that the cleft is a property of the complete ribosome, and that it marks the position of the small subunit. The reversibility of the dissociation also strengthens the view that no change in the large subunit occurs during dissociation or reassociation, i.e. that the sites of interaction between ribosomes in both types of tetramer are the same. The conclusions affect the interpretation of electron-micrograph images and an anomaly in the relationship between the two types of tetramer is discussed.     

Association of maternal and newly synthesized ribosomes with membranous noncytoskeletal structures in Xenopus laevis embryonic cells

In Xenopus laevis embryos a high concentration of both KCl and 0.5% DOC (sodium deoxycholate) is needed for maximal extraction of ribosomes and polysomes. We studied the nature of the structures that keep ribosomes and polysomes immobilized within the cytoplasm of embryonic cells at cleavage through tailbud stages, using various combinations of a low-salt buffer (20 mM KCl), a high-salt buffer (500 mM KCl), 0.5% DOC, and 0.5% Triton X-100. With a low-salt buffer and 0.5% DOC, but not Triton X-100, 80S ribosomal monomers and polysomes were liberated from the cytoplasmic rapidly sedimenting structures (RSS) to the soluble fraction. With a high-salt buffer (500 mM KCl), ribosomes were solubilized as 60S and 40S subunits together with about one-half of the total polysomes. When cells were homogenized in a low-salt buffer with added inhibitors of the cytoskeleton (cytochalasin B or colchicine), the majority of polysomes but not ribosomes were solubilized. These results provide evidence for the following conclusions. 1) Polysomes are bound to cytoskeletal structures in Xenopus embryos, but ribosomes, both maternal and newly synthesized, are associated with membranous noncytoskeletal structures. 2) The membranous structures consist of two compartments, one high-salt sensitive and the other high-salt resistant. 3) Ribosomes of the high-salt resistant group increase in amount with developmental stage and appear to be the precursor to the ribosomes of the high-salt sensitive group.     

Multiple defects in translation associated with altered ribosomal protein L4

The ribosomal proteins L4 and L22 form part of the peptide exit tunnel in the large ribosomal subunit. In Escherichia coli, alterations in either of these proteins can confer resistance to the macrolide antibiotic, erythromycin. The structures of the 30S as well as the 50S subunits from each antibiotic resistant mutant differ from wild type in distinct ways and L4 mutant ribosomes have decreased peptide bond-forming activity. Our analyses of the decoding properties of both mutants show that ribosomes carrying the altered L4 protein support increased levels of frameshifting, missense decoding and readthrough of stop codons during the elongation phase of protein synthesis and stimulate utilization of non-AUG codons and mutant initiator tRNAs at initiation. L4 mutant ribosomes are also altered in their interactions with a range of 30S-targeted antibiotics. In contrast, the L22 mutant is relatively unaffected in both decoding activities and antibiotic interactions. These results suggest that mutations in the large subunit protein L4 not only alter the structure of the 50S subunit, but upon subunit association, also affect the structure and function of the 30S subunit.     

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.     

The action mode of the ribosome-inactivating protein α-sarcin

Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.     

TRANSFER OF PROTEINS FROM THE CHLOROPLAST TO VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): A PATHWAY FOR DEGRADATION

Several chloroplast proteins were detected by immunoelectron microscopy within dense granules in cytoplasmic vacuoles in the alga Chlamydomonas reinhardtii Dangeard. Transfer from chloroplast to vacuoles of two major, pulse-labeled polypeptides, the large subunit of rubisco and the α subunit of ATPase, which are synthesized on chloroplast ribosomes, was demonstrated by the recovery of these polypeptides in vacuolar granules over a several-hour time period. The ultrastructure of cryofixed algal cells was examined to search for structures that would provide insight into the transfer of chloroplast proteins to vacuoles. Micrographs showed that the two membranes of the envelope were appressed, with no detectable intermembrane space, over most of the chloroplast surface. Protrusions of the outer membrane of the envelope were occasionally found that enclosed stroma, with particles similar in size to chloroplast ribosomes, but generally not thylakoid membranes. These observations suggest that chloroplast material, especially the stromal phase, was extruded from the chloroplast in membrane-bound structures, which then interacted with Golgi-derived vesicles for degradation of the contents by typical lysosomal activities. A protein normally targeted to vacuoles through the endomembrane system for incorporation into the cell wall was detected in Golgi structures and vacuolar granules but not the chloroplast.     

The structures of antibiotics bound to the E site region of the 50 S ribosomal subunit of Haloarcula marismortui: 13-deoxytedanolide and girodazole

Crystal structures of the 50 S ribosomal subunit from Haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-Deoxytedanolide binds to the E site of the 50 S subunit at the same location as the CCA of tRNA, and thus appears to inhibit protein synthesis by competing with deacylated tRNAs for E site binding. Girodazole binds near the E site region, but is somewhat buried and may inhibit tRNA binding by interfering with conformational changes that occur at the E site. The specificity of 13-deoxytedanolide for eukaryotic ribosomes is explained by its extensive interactions with protein L44e, which is an E site component of archaeal and eukaryotic ribosomes, but not of eubacterial ribosomes. In addition, protein L28, which is unique to the eubacterial E site, overlaps the site occupied by 13-deoxytedanolide, precluding its binding to eubacterial ribosomes. Girodazole is specific for eukarytes and archaea because it makes interactions with L15 that are not possible in eubacteria.     

Codon-anticodon interaction at the P site is a prerequisite for tRNA interaction with the small ribosomal subunit

The arrival of high resolution crystal structures for the ribosomal subunits opens a new phase of molecular analysis and asks for corresponding analyses of ribosomal function. Here we apply the phosphorothioate technique to dissect tRNA interactions with the ribosome. We demonstrate that a tRNA bound to the P site of non-programmed 70 S ribosomes contacts predominantly the 50 S, as opposed to the 30 S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30 S binding. Protection patterns of tRNAs bound to isolated subunits and programmed 70 S ribosomes were compared. The results suggest the presence of a movable domain in the large ribosomal subunit that carries tRNA and reveal that only approximately 15% of a tRNA, namely residues 30 +/- 1 to 43 +/- 1, contact the 30 S subunit of programmed 70 S ribosomes, whereas the remaining 85% make contact with the 50 S subunit. Identical protection patterns of two distinct elongator tRNAs at the P site were identified as tRNA species-independent phosphate backbone contacts. The sites of protection correlate nicely with the predicted ribosomal-tRNA contacts deduced from a 5.5-A crystal structure of a programmed 70 S ribosome, thus refining which ribosomal components are critical for tRNA fixation at the P site.