Mitochondrial ribosome assembly in Neurospora crassa. Purification of the mitochondrially synthesized ribosomal protein, S-5

In Neurospora, one mitochondrial ribosomal protein (S-5, Mr = 52,000) is synthesized intramitochondrially and is presumably encoded by mitochondrial DNA. We have developed a rapid method for the purification of S-5 which takes advantage of its high affinity for carboxymethyl-Sepharose in the presence of 6 M urea. Using this method, S-5, at purity greater than 95%, can be prepared by column chromatography in a single batch elution step. The amino acid composition of S-5 was determined. Judged by the contents of hydrophilic and basic amino acids, S-5 is more similar to Escherichia coli and yeast ribosomal proteins than to other mitochondrial translation products which are hydrophobic membrane proteins.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

Identification of 12 new yeast mitochondrial ribosomal proteins including 6 that have no prokaryotic homologues

Mitochondrial ribosomal proteins were studied best in yeast, where the small subunit was shown to contain about 35 proteins. Yet, genetic and biochemical studies identified only 14 proteins, half of which were predictable by sequence homology with prokaryotic ribosomal components of the small subunit. Using a recently described affinity purification technique and tagged versions of yeast Ykl155c and Mrp1, we isolated this mitochondrial ribosomal subunit and identified a total of 20 proteins, of which 12 are new. For a subset of the newly described ribosomal proteins, we showed that they are localized in mitochondria and are required for the respiratory competency of the yeast cells. This brings to 26 the total number of proteins described as components of the mitochondrial small ribosomal subunit. Remarkably, almost half of the previously and newly identified mitochondrial ribosomal components showed no similarity to any known ribosomal protein. Homologues could be found, however, in predicted protein sequences from Schizosaccharomyces pombe. In more distant species, putative homologues were detected for Ykl155c, which shares conserved motifs with uncharacterized proteins of higher eukaryotes including humans. Another newly identified ribosomal protein, Ygl129c, was previously shown to be a member of the DAP-3 family of mitochondrial apoptosis mediators.     

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.     

The Modular Adaptive Ribosome

The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5’UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments.     

Ribosomal proteins

Summary Ribosomal proteins of E. coli and yeast were separated by gel filtration on dextran (Sephadex) and polyacrylamide (Bio-Gel) columns. Both gels revealed a valuable separation of the proteins. Finally only Bio-Gel columns were used, since their polyacrylamide matrix is more resistant to the applied organic acids.The wide distribution of the molecular weights for both the E. coli and yeast ribosomal proteins was confirmed. E. coli ribosomal proteins were separated into three main groups by a single chromatography on Bio-Gel P-10. The same was true for yeast ribosomal proteins. Rechromatography of these protein groups resulted in a further valuable resolution. The fractionated proteins are recovered without any loss and they are very useful for further purification by other procedures, especially on a large scale basis.     

Identification and comparative analysis of the large subunit mitochondrial ribosomal proteins of Neurospora crassa

The mitochondrial ribosome (mitoribosome) has highly evolved from its putative prokaryotic ancestor and varies considerably from one organism to another. To gain further insights into its structural and evolutionary characteristics, we have purified and identified individual mitochondrial ribosomal proteins of Neurospora crassa by mass spectrometry and compared them with those of the budding yeast Saccharomyces cerevisiae. Most of the mitochondrial ribosomal proteins of the two fungi are well conserved with each other, although the degree of conservation varies to a large extent. One of the N. crassa mitochondrial ribosomal proteins was found to be homologous to yeast Mhr1p that is involved in homologous DNA recombination and genome maintenance in yeast mitochondria.     

Structural characterization of yeast acidic ribosomal P proteins forming the P1A-P2B heterocomplex

Acidic ribosomal P proteins form a distinct lateral protuberance on the 60S ribosomal subunit. In yeast, this structure is composed of two heterocomplexes (P1A-P2B and P1B-P2A) attached to the ribosome with the aid of the P0 protein. In solution, the isolated P proteins P1A and P2B have a flexible structure with some characteristics of a molten globule [Zurdo, J., et al. (2000) Biochemistry 39, 8935-8943]. In this report, the structure of P1A-P2B heterocomplex from Saccharomyces cerevisiae is investigated by means of size-exclusion chromatography, chemical cross-linking, circular dichroism, light scattering, and fluorescence spectroscopy. The circular dichroism experiment shows that the complex could be ranked in the tertiary class of all-alpha proteins, with an average alpha-helical content of approximately 65%. Heat and urea denaturation experiments reveal that the P1A-P2B complex, unlike the isolated proteins, has a full cooperative transition which can be fitted into a two-state folding-unfolding model. The behavior of the complex in the presence of 2,2,2-trifluoroethanol also resembles a two-state folding-unfolding transition, further supporting the idea that the heterocomplex contains well-packed side chains. In conclusion, the P1A-P2B heterocomplex, unlike the isolated proteins, has a well-defined hydrophobic core. Consequently, the complex can put up its structure without additional ribosomal components, so the heterodimeric complex reflects the intrinsic properties of the two analyzed proteins, indicating thus that this is the only possible configuration of the P1A and P2B proteins on the ribosomal stalk structure.     

Mammalian mitochondrial ribosomal proteins (2). Amino acid sequencing, characterization, and identification of corresponding gene sequences

Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.     

The course of the assembly of ribosomal subunits in yeast

The course of the assembly of the various ribosomal proteins of yeast into ribosomal particles has been studied by following the incorporation of radioactive individual protein species in cytoplasmic ribosomal particles after pulse-labelling of yeast protoplasts with tritiated amino acids. The pool of ribosomal proteins is small relative to the rate of ribosomal protein synthesis, and, therefore, does not affect essentially the appearance of labelled ribosomal proteins on the ribosomal particles. From the labelling kinetics of individual protein species it can be concluded that a number of ribosomal proteins of the 60 S subunit (L6, L7, L8, L9, L11, L15, L16, L23, L24, L30, L32, L36, L40, L41, L42, L44 and L45) associate with the ribonucleoprotein particles at a relatively late stage of the ribosomal maturation process. The same was found to be true for a number of proteins of the 40 S ribosomal subunit (S10, S27, S31, S32, S33 and S34). Several members (L7, L9, L24 and L30) of the late associating group of 60-S subunit proteins were found to be absent from a nuclear 66 S precursor ribosomal fraction. These results indicate that incorporation of these proteins into the ribosomal particles takes place in the cytoplasm at a late stage of the ribosomal maturation process.     

Molecular cloning of some components of the translation apparatus of fission yeast Schizosaccharomyces pombe and a list of its cytoplasm ic proteins genes]

Full-length cDNAs of four new genes encoding cytoplasmic ribosomal proteins L14 and L20 (large ribosomal subunit) and S1 and S27 (small ribosomal subunit) were isolated and sequenced during the analysis of the fission yeast Schizosaccharomyces pombe genome. One of the Sz. pombe genes encoding translation elongation factor EF-2 was also cloned and its precise position on chromosome I established. A unified nomenclature was proposed, and the list of all known genetic determinants encoding cytoplasmic ribosomal proteins of Sz. pombe was compiled. By now, 76 genes/cDNAs encoding different ribosomal proteins have been identified in the fission yeast genome. Among them, 35 genes are duplicated and three homologous genes are identified for each of the ribosomal proteins L2, L16, P1, and P2.     

A new nomenclature for the cytoplasmic ribosomal proteins of Saccharomyces cerevisiae.

The availability of the complete sequence of the Saccharomyces cerevisiae genome has allowed a comprehensive analysis of the genes encoding cytoplasmic ribosomal proteins in this organism. On the basis of this complete inventory a new nomenclature for the yeast ribosomal proteins is presented.     

Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins: improved resolution with Triton X-100

The nonionic detergent Triton X-100 binds in varying proportions to specific ribosomal proteins and decreases the relative mobility of these proteins during electrophoresis. When Triton X-100 binds to these ribosomal proteins in the first-dimension gel, the resolution of the ribosomal proteins in the second-dimension gel pattern is greatly improved. Maximum binding of Triton X-100 to the ribosomal proteins is dependent on pH, urea concentration, and the complete reduction of cysteine and methionine. After first-dimension electrophoresis the Triton X-100 in the gel does not interfere with the binding of sodium dodecyl sulfate to the ribosomal proteins and the molecular weight of these proteins can still be estimated directly from the second-dimension slab gel.     

Isolation of yeast ribosomal proteins L3 and L2 for immunological studies

We report on a rapid method for the isolation and purification of the yeast ribosomal proteins L3 and L2 using a simple instrumentation. Preparative dodecyl sulfate polyacrylamide gel electrophoresis was applied to the separation of cytoplasmatic ribosomal proteins of the large subunit from the yeast Saccharomyces cerevisiae. The polypeptides were removed from gel slices by electrophoretic elution. Subsequent analytical electrophoresis showed groups of proteins in all but two fractions. The latter were further analysed by a two-dimensional gel electrophoresis system which disclosed the purity of two polypeptides. They were identified as L3 and L2. Their molecular masses were 51.5 and 44 kDa as estimated from the gels. A possible application to the isolation of other yeast ribosomal proteins is discussed. An antiserum against the polypeptide L3 was raised in a rabbit. Applying an enzyme-linked immunosorbent assay (ELISA) we were able to determine the relative antibody concentration. Its specificity was demonstrated by immunoblotting.     

Cellular site of synthesis of ribosomal proteins in yeast.

Looking for messenger RNA coding for yeast ribosomal protein, we devised a method to identify polysomes involved in ribosomal protein synthesis. Analysis of nascent protein elongated in vitro demonstrated that ribosomal proteins are synthesized both on membrane-associated and free polysomes.     

Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast cells. It is concluded that the heterodimeric complex of the P1/P2 proteins is formed preferentially. Formation of homodimers (P1/P1 and P2/P2) can also be observed, though with much less efficiency. Regarding that, we propose to describe the double heterodimeric complex as a protein configuration which forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.     

Immunologically related proteins in cytoplasmic and mitochondrial ribosomes of yeast Saccharomyces cerevisiae

Summary Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques. Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits. The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.Abbreviations cyto cytoplasmic - mito mitochondrial     

Extended N-terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria

We have determined the N-termini of 26 proteins of the large ribosomal subunit from yeast mitochondria by direct amino acid micro-sequencing. The N-terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r-) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N-termini of the mature proteins, the putative leader peptides responsible for mitochondrial matrix-targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found.