Acidic ribosomal proteins from eukaryotic cells. Effect on ribosomal functions.

Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be substituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.     

The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity

The acidic proteins, A-proteins, from the large ribosomal subunit of Saccharomyces cerevisiae grown under different conditions have been quantitatively estimated by ELISA tests using rabbit sera specific for these polypeptides. It has been found that the amount of A-protein present in the ribosome is not constant and depends on the metabolic state of the cell. Ribosomes from exponentially growing cultures have about 40% more of these proteins than those from stationary phase. Similarly, the particles forming part of the polysomes are enriched in A-proteins as compared with the free 80 S ribosomes. The cytoplasmic pool of A-protein is considerably high, containing as a whole as much protein as the total ribosome population. These results are compatible with an exchanging process of the acidic proteins during protein synthesis that can regulate the activity of the ribosome. On the other hand, cells inhibited with different metabolic inhibitors produce a very low yield of ribosomes that contain, however, a surprisingly high amount of acidic proteins while the cytoplasmic pool is considerably reduced, suggesting that under stress conditions the ribosome and the A-protein may aggregate, forming complex structures that are not recovered by the standard preparation methods.     

Protein kinases phosphorylating acidic ribosomal proteins from yeast cells

Phosphorylation of ribosomal acidic proteins ofSaccharomyces cerevisiae is an important mechanism regulating a number of active ribosomes. The key role in the regulatory mechanism is played by specific phosphoprotein kinases and phosphoprotein phosphatases. Three different cAMP-independent protein kinases phosphorylating acidic ribosomal proteins have been identified and characterized. The protein kinase 60S (PK60S), RAP kinase, and casein kinase type 2 (CK2). All three protein kinases phosphorylate serine residues which are localized in the C-terminal end of phosphoproteins. Synthetic peptides were used to determinate the amino acid sequence of phosphoacceptor site for PK60S. Peptide AAEESDDD derived from phosphoproteins YP1β/β′ and YP2α turned out to be the best substrate for PK60S. A number of halogenated benzimidazoles and 2-azabenzimidazoles were tested as inhibitors of the three protein kinases. 4,5,6,7-Tetrabromo-2-azabenzimidazole inhibits phosphorylation only of these polypeptides phosphorylated by protein kinase 60S, namely YP1β/β′ and YP2α, but not the other, YP1α and YP2β phosphorylated by protein kinases RAP and CK2. RAP kinase has been found in an active form in the soluble fraction ofS. cerevisiae. The enzyme uses ATP as a phosphate donor and is less sensitive to heparin than casein kinase 2. RAP kinase monophosphorylates the four acidic proteins. The ribosome-bound proteins are a better substrate for the enzyme. Multifunctional CK2 kinase phosphorylate all four acidic proteins. The kinase phosphorylates preferentially serine or threonine residues surrounded by cluster of acidic residues. The enzyme activity is stimulatedin vitro by the presence of polylysine and inhibited by heparin. Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21 st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

Characterisation of eukaryotic ribosomal proteins.

A simple method of two-dimensional polyacrylamide gel electrophoresis is described which affords: (1) high resolution of eukaryotic ribosomal proteins; (2) good recovery of protein in the transfer from first to second dimension; and (3) characterisation of the separated proteins in terms of molecular weights and other electrophoretic properties. Using this method, we have characterised 70 proteins in rabbit reticulocyte ribosomes, 30 from the small subunit and 40 from the large subunit. The molecular weight distribution is compared with those obtained by other authors after fractionation of the proteins in two dimensions.     

Conformation and biological activity of acidic ribosomal proteins from different organisms.

Acidic ribosomal proteins L7L12 from Escherichia coli, A2 from Bacillus stearothermophilus, and HL20 from Halobacterium cutirubrum are considered analogous based on amino acid composition and are presumed to be involved in similar functions on the ribosome. The conformation of these proteins has been studied by circular dichroic spectroscopy and correlated with their ability to support polyphenylalanine synthesis in vitro. Only proteins L7L12 and A2 showed comparable biological activity and conformational responses to changes in various conditions (salts, denaturing agents, helix-promoting solvents, temperature). L7L12 and A2 have highly ordered secondary structures, 48 ± 5 and 40–65%, respectively. Data for A2 show a wide range because of the established spontaneous partial denaturation of this protein at room temperature. Protein HL20, on the other hand, could not restore protein synthesis activity to L12 depleted E. coli ribosomes and its conformation is characterized by a very low content of α-helices (18.5%).     

The extraction of proteins from eukaryotic ribosomes and ribosomal subunits

Proteins were extracted from rat liver ribosomes and ribosomal subunits: with 67% acetic acid (in the presence of 3.3 mM, 33 mM, or 67 mM Mg) with 2 M LiCL in 4 M urea; with 0.25 N HCI; with 1% SDS; and after RNase digestion. The most efficient extraction and the best recovery were either with acetic acid in the presence of 33 mM or 67 mM Mg, or with LiCI-urea. Protein extracted with acetic acid, LiCi-urea, or with HCI had little or no contamination with RNA. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis: the proteins extracted with acetic acid were the most soluble in the sample gel solution; their electrophoretograms displayed the maximum number of spots and the smallest number of derivatives or altered proteins. Preparations of protein extracted with SDS or RNase were relatively insoluble in the sample gel solution, and proteins extracted with HCI showed a large number of derivatives. All things considered, the most satisfactory method for the extraction of protein from eukaryotic ribosomes is with 67% acetic acid in the presence of 33 mM MgCl2.     

Functional aspects of ribosomal proteins.

A short personal recollection of HG Wittmann is given with emphasis on his basic contribution to the structure of the ribosome, in particular the ribosomal proteins. With these considerations in mind, two interrelated problems are reviewed here. The first relates to the internal symmetry both in tRNA and in the tetrameric L12-protein complex. The second problem to be addressed relates to the dynamics of transfer RNA in the ribosome and the role of L12 proteins in this process. The importance of electrostatic repulsion in the maintenance of the mutual spatial orientation of tRNAs and L12 in the ribosome is emphasized in relation to a pendulum model for how L12 may steer translocation.     

Structural homologies in alanine-rich acidic ribosomal proteins from procaryotes and eucaryotes.

The amino acid composition and amino-terminal sequence have been determined for the alanine-rich, acidic ribosomal 'A' protein (equivalent to Escherichia coli L7/L12) from three procaryotic cell types that live under extreme environmental conditions (Arthrobacter glacialis, Clostridium pasteurianum, and Bacillus stearothermophilus) as well as from wheat germ, a eucaryote source. These data are compared with previously published 'A' protein sequences from other procaryotes and eucaryotes. All the procaryotic 'A' proteins, with the exception of the very acidic 'A' protein from Halobacterium cutirubrum, show similar charge, size, and amino acid composition, as well as an extensive sequence homology in the N-terminal region. Some differences are observed between gram-negative and gram-positive bacteria. The 'A' proteins from eucaryotes contain two tyrosine molecules, an amino acid absent in procaryotic 'A' proteins, as well as a reduced number of valine residues and an increased amount of aspartic acid. The N-terminal sequence of wheat germ 'A' protein shows considerable homology with other eucaryotic 'A' proteins and also with H. cutirubrum. It also shows some sequence homology with E. coli 'A' proteins.     

Purification and characterization of two ribosomal proteins of Saccharomyces cerevisiae. Homologies with proteins from eukaryotic species and with bacterial protein EC L11

The nature of the protein phosphatases involved in the regulation of glycolysis/gluconeogenesis, fatty acid synthesis and cholesterol synthesis in rat liver has been investigated using L-pyruvate kinase, ATP-citrate lyase, acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase as substrates. The results show that protein phosphatases-1, 2A and 2C are the only significant protein phosphatases in rat liver acting on these four substrates. The relationship of these three enzymes to other protein phosphatases described in the literature is discussed.     

The acidic proteins of eukaryotic ribosomes. A comparative study

The acidic proteins extracted by 0.4 M NH4Cl and 50% ethanol from ribosomes from Saccharomyces cerevisiae, wheat germ, Artemia salina, Drosophila melanogaster, rat liver and rabbit reticulocytes have been studied comparatively in several structural and functional aspects. All the species studied have in the ribosome two strongly acidic proteins with pI values not greater than pH 4.5., which appear to be monophosphorylated in the case of S. cerevisiae, A.Salina, D. melanogaster and wheat germ. Rat liver proteins are multiphosphorylated, as possibly are those from reticulocytes. The molecular weight of these acidic proteins as determined by SDS electrophoresis ranges from around 13,500 to 17,000 and, except in the case of yeast, of which both proteins have the same molecular weight, the size of the two proteins in the other species differs by approx. 1,000-2,000. In general, the size of the proteins increases with the evolutionary position of the organism, as seems to be the case with the degree of phosphorylation. From an immunological point of view the ribosomal acid proteins of eukaryotic cells are partically related, since antisera against yeast protein cross-react with proteins from wheat germ, rat liver and reticulocytes. Bacterial proteins L7 and L12 are very weakly recognized by the anti-yeast sera. Anti-bacterial acidic proteins do not cross-react with any of the protein from the species studied. The proteins from all the species studied are functional equivalents and can reconstitute the activity of particles of S. cerevisiae deprived of their acidic proteins.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seems to have an “inside-out” orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins lableled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic ³²P. We conclude that the phagolysosome is probably oriented in an “inside-out” configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.     

Functional roles of 50-S ribosomal proteins.

Ribosomal proteins previously inactivated by treatment with fluorescein isothiocyanate have been incorporated into 50-S ribosomal subunits during reconstitution from particles disassembled by 2 M LiCl in the presence of an excess of the modified proteins. The reconstituted particles show alterations in some functional activities resulting from the incorporation of the inactive ribosomal proteins added exogenously. Of the fluorescein-isothiocyanate-treated proteins incorporated, L24 and L25 drastically affect all the activities tested and these proteins possibly play a fundamental role in determining the overall structure of the particle. Proteins L16 and L10 are apparently involved both in the GTP hydrolysis dependent on elongation factor G and in peptidyl transferase activity but the modified protein L11 only affects GTPase activity indirectly and interferes with the ribosome assembly process involving proteins L7 and L12. Protein L1 may be involved with peptidyl transferase activity while proteins L7 and L12, in agreement with many reports in the literature, affect the factor-dependent hydrolysis of GTP.     

Primary structures of five ribosomal proteins from the archaebacterium Halobacterium marismortui and their structural relationships to eubacterial and eukaryotic ribosomal proteins

The complete amino acid sequences of ribosomal proteins L9, L20, L21/22, L24 and L32 from the archaebacterium Halobacterium marismortui were determined. The comparison of the sequences of these proteins with those from other organisms revealed that proteins L21/22 and L24 are homologous to ribosomal protein Yrp29 from yeast and L19 from rat, respectively, and that H. marismortui L20 is homologous to L30 from eubacteria. H. marismortui ribosomal protein L9 showed sequence homology to both L29 from yeast and L15 from eubacteria. No homologous protein was found for H. marismortui L32. These results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes.     

Extraribosomal functions of bacterial ribosomal proteins

Ribosomal proteins (r-proteins) constitute a considerable part of the cell proteome. Although their primary role in the cell is to serve as integral components of protein synthesis machinery, ribosomes, many of them have functions beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with other cell components. Extraribosomal activities of some ribosomal proteins were observed as early as the 1970s–1980s. In recent years, both the list of moonlighting r-proteins and the repertoire of their additional functions beyond the ribosome was greatly expanded, mainly owing to new techniques developed for dissecting RNA/DNA-protein or protein-protein interactions within functional complexes involved in various cell processes. The review surveys information on the extraribosomal functions demonstrated experimentally or presumed for bacterial r-proteins.     

Variation of ribosomal proteins with bacterial growth rate.

The composition of ribosomal proteins has been examined as a function of the growth rate of Escherichia coli cells. Seven sets of cultural conditions, utilizing different combinations of carbon and nitrogen sources, were employed to provide a 36-fold spread in growth rate. The cellular content of most of the ribosomal proteins in ribosomes decreased to a similar extent in the very slow-growing cultures. Major exceptions were proteins S6 and L12, which exhibited a much more pronounced decrease , and S21, which exhibited an increase. None of the proteins remained invariant with growth rate.     

Stoichiometry and structure of a complex of acidic ribosomal proteins

The acidic proteins B-L13 (homologous to Escherichia coli protein L7/L12) and B-L8, from the 50 S subunit of Bacillus stearothermophilus ribosomes, form a stable complex. Trypsin digestion of ribosomes generates an N-terminal fragment of B-L13 (approximately residues 1 to 47) which can associate with B-L8, displacing intact B-L13, and bind to B-L13-deficient ribosomes. Displacement of B-L13 from the B-L8 · B-L13 complex by the B-L13 N-terminal fragment causes a change in gel electrophoretic mobility of the complex, and titration of the complex with fragment indicates unambiguously that it contains four molecules of B-L13. Evidence is presented that B-L13 forms a dimer in solution, and that the dimer associates intact with B-L8. Reconstituted 50 S subunits in which B-L13 is replaced by its N-terminal fragment have the same functional properties as 50 S subunits missing B-L13 altogether: polypeptide synthesis is reduced but not abolished; ability to bind elongation factor EF-G and GTP is severely reduced; and peptidyl transferase activity and ability to associate with a 30 S subunit · Phe-tRNA · poly(U) complex are unaffected (relative to intact 50 S subunits).