Comparative immunological and electrophoretic studies on ribosomal proteins of Bacillaceae

Summary The ribosomal proteins from several Bacillus species were compared by two-dimensional gel electrophoresis and immunological methods. The results revealed great heterogeneity among most Bacillus species. Comparison of ribosomal proteins from Bacilli with those of E. coli by two-dimensional gel electrophoresis showed little similarities, while structural homologies could be found by immunological methods. SDS two-dimensional gel electrophoresis revealed that the molecular weight of ribosomal proteins is conserved in all tested bacteria.Paper No. 68 on Ribosomal Proteins. Preceding paper is by Geisser et al., Molec. gen. Genet. 127, 111–128 (1973).     

Isolation and characterization of fourteen ribosomal proteins from small subunits of yeast.

A method for preparation of a large amount of ribosomal subunits from Saccharomyces cerevisiae by a Ti-15 zonal rotor is described. The proteins of the small subunits (ca. 50 000 A260 units) were separated into 22 fractions by chromatography on carboxymethylcellulose columns. Fourteen proteins were then purified from the ten chromatographic fractions by filtration through Sephadex G-100 or Sephacryl S--200. The isolated proteins are YP 6, YP 7, YP 9, YP 12, YP 14', YP 14', YP 28, YP 38, YP 45, YP 50, YP 52, YP 58, YP 63, and YP 70. The molecular weight and amino acid compositions of these proteins are presented.     

Isolation and characterization of twenty-three ribosomal proteins from large subunits of yeast.

The proteins of large ribosomal subunits from Saccharomyces cerevisiae were separated into 25 fractions by chromatography on columns of carboxymethylcellulose (CMC). Twenty-three proteins were then purified from the 12 CMC fractions by filtration through Sephadex G-75, Sephadex G-100, and Sephacryl S-200, and/or by phosphocellulose column chromatography. The isolated proteins are YP 1, YP 2, YP 9, YP 11, YP 13', YP 16, YP 18, YP 26, YP 39, YP 41, YP 42, YP 42', YP 44, YP 45, YP 47', YP 52a, YP 53, YP 55, YP 59, YP 62, YP 68, YP A1, and YP A2. The molecular weight and amino acid composition of these proteins are presented.     

Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

Summary Revertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.Paper No. 60 on Ribosomal Proteins. Preceding paper is by B. Wittmann-Liebold, Hoppe-Seyler's Z. physiol. Chemie, in press.     

Structure/function analysis of yeast ribosomal protein L2

Ribosomal protein L2 is a core element of the large subunit that is highly conserved among all three kingdoms. L2 contacts almost every domain of the large subunit rRNA and participates in an intersubunit bridge with the small subunit rRNA. It contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the peptidyltransferase center. Here, screening of randomly generated library of yeast RPL2A alleles identified three translationally defective mutants, which could be grouped into two classes. The V48D and L125Q mutants map to the globular domain. They strongly affect ribosomal A-site associated functions, peptidyltransferase activity and subunit joining. H215Y, located at the tip of the extended domain interacts with Helix 93. This mutant specifically affects peptidyl-tRNA binding and peptidyltransferase activity. Both classes affect rRNA structure far away from the protein in the A-site of the peptidyltransferase center. These findings suggest that defective interactions with Helix 55 and with the Helix 65-66 structure may indicate a certain degree of flexibility in L2 in the neck region between the two other domains, and that this might help to coordinate tRNA-ribosome interactions.     

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.     

Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly

Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.     

Isolation and properties of two protein kinases from yeast which phosphorylate casein and some ribosomal proteins.

Three fractions of protein kinase from postribosomal supernatant of Saccharomyces cerevisiae, active in phosphorylation of casein, were resolved on DEAE-cellulose. Two of these fractions: protein kinase 1 and protein kinase 3, were further purified about 1000 and 1800-fold respectively. The kinase 1 appeared to exist as a monomer with a molecular weight of 50 000 and utilized only ATP as phosphoryl donor. The protein kinase 3 was an aggregated form of enzyme with a molecular weight of above half a million and used both ATP and GTP for protein phosphorylation. Both isolated enzymes showed variations in respect to Michaelis constants, and inhibitory effects exerted by monovalent cations and nucleotide phosphates. The activity of the kinases was not affected by the presence of cAMP (adenosine 3':5'-monophosphate) or cGMP, however, only protein kinase 1 appeared to be a cAMP nucleotide-independent enzyme. Despite these differences both enzymes equally phosphorylated two strongly acidic proteins of the 60-S ribosome subunit, possibly related to L7, L12 of Escherichia coli.     

Phylogenic studies on ribosomal proteins

Two-dimensional electrophoresis of ribosomal proteins makes it possible to evaluate the phylogenic distance between any set of two species. The evaluation is based on the number of spots having the same electrophoretic mobility in the two species. The two-dimensional finger-prints of ribosomal proteins from mammals, reptiles and birds, which have diverged 300 million years ago, are identical. For more remote species with respect to mammals, the number of comigrating spots gradually decreases. For as remote species from mammals as plants, one third of the spots have still the same mobility. Only five proteins from E. coli comigrate with ribosomal protein from mammals. The low evolution of ribosomal proteins indicate the high degree of internal organization of the ribosome.     

Modification of yeast ribosomal proteins. Phosphorylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins labelled in vivo with 32PO43- revealed that the proteins S2 and S10 of the 40S ribosomal subunit, and the proteins L9, L30, L44 and L45 of the 60S ribosomal subunit, are phosphorylated in vivo. Most of the phosphate groups appeared to be linked to serine residues. Teh number of phosphate groups per molecule of phosphorylated protein species ranged from 0.01 to 0.79. Since most of the phosphorylated ribosomal proteins appear to associate with the pre-ribosomal particles at a very late stage of ribosome assembly, phosphorylation is more likely to play a role in the functioning of the ribosome than in its assembly.     

Modification of yeast ribosomal proteins. Methylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.     

YmL9, a nucleus-encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles.

The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP-L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP-L9 gene is essential for mitochondrial function and is located on chromosome XV or VII. YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C-terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.-R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787-788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.-H., Yen, T. S. B., Wang, Y.-F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919-8934] are postulated to be true nucleus-encoded mitochondrial ribosomal proteins.     

Isolation of eukaryotic ribosomal proteins: purification and characterization of S25 and L16.

Proteins were extracted from rat liver ribosomal subunits with ethanol and ammonium chloride. The extract from the 40S subunit contained mainly S25, but smaller amounts of a number of other proteins were found as well; the extract from the 60S subparticle had L16 in addition to P1, P2, S25, and several other proteins. S25 and L16 had not been purified before. The former was isolated from the ethanol-ammonium chloride extract by stepwise elution from carboxymethylcellulose with LiCl, chromatography on phosphocellulose, and filtration through Sephadex G-75; L16 was purified by elution from carboxymethylcellulose with LiCl (in steps). The molecular weight of the two proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; and amino acid composition was determined also.     

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.