Phosphorylation in vitro and in vivo of ribosomal proteins from Saccharomyces cerevisia.

Crude ribosomes from Saccharomyces cerevisiae cultures were phosphorylated in vitro when incubated in the presence of [gamma-32P]ATP. Analysis of the ribosomal proteins with two-dimensional electrophoresis revealed that of the 29 proteins identified in the small subunit, only protein S6 was phosphorylated. Of the 37 proteins identified in the large subunit, one was highly phosphorylated (L3) and two only slightly phosphorylated (L11 and L14). The protein kinase activity associated with the ribosomes was extracted with 1 M KCl and was not dependent on adenosine 3':5'-monophosphate; it preferentially phosphorylated casein and phosvitin, but was less active on histones. Structural ribosomal proteins were also phosphorylated in vivo when the yeast cultures were incubated with [32P]orthophosphate; the radioactivity resistant to hydrolysis by hot perchloric acid was incorporated into the proteins of the two subunits. Radioactive phosphoserine was found by subjecting hydrolysates of ribosomal proteins to high-voltage electrophoresis. After two-dimensional electrophoresis, one poorly phosphorylated protein (S10) was identified in the small subunit. In the large subunit, one protein (L3) was highly labelled, and two proteins (L11 and L24) only slightly labelled.     

Sulfhydryl groups on yeast ribosomal proteins L7 and L26 are significantly more reactive in the 80 S particles than in the 60 S subunits.

The sulfhydryl-directed fluorescent reagent, 5-iodoacetamidofluorescein (IAF), reacts differently with proteins from the 60 S ribosomal subunit of Saccharomyces cerevisiae when this subunit is free as opposed to being contained within the 80 S ribosome. When the 80 S ribosomes and the free 60 S subunits were labeled with IAF, the specific fluorescence intensity (fluorescence intensity unit/A260 60 S subunit) of the subsequently derived 60 S was 16.3 and 5.4, respectively. Gel analysis showed that proteins L7 and L26 were selectively labeled and contained greater than 90% of the total fluorescent label, when 80 S ribosomes were labeled. When free 60 S subunits were labeled, six additional proteins were labeled. Both types of modified 60 S subunits were equally capable to support protein synthesis in vitro. Reassociation of the IAF-labeled derived and free 60 S subunits with unmodified 40 S subunits resulted in a maximum of 5-7% decrease and a 3-fold increase, respectively, in the fluorescence intensity without a shift in the emission maxima. The data suggest that ribosomal proteins L7 and L26 contain SH groups that respond to ribosomal subunit association and become more reactive in the intact ribosome than in the subunit. The environments of some or all of the additionally labeled proteins are also sensitive to subunit reassociation.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo

Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

Identification of the ribosomal proteins phosphorylated by the ribosome-associated casein kinase type II from cryptobiotic gastrulae of the brine shrimp Artemia sp

Phosphorylation of the ribosomal proteins by the extra-ribosomal protein kinase was investigated "in situ" and with purified 40 S or 60 S ribosomal proteins from cryptobiotic embryos of Artemia sp. Ribosomal proteins that were most readily phosphorylated in 80 S ribosomes included S6 and S8 of the 40 S subunit and proteins L9, L13 and L18 of the 60 S subunit. Several additional polypeptides were phosphorylated when purified 40 S or 60 S ribosomal proteins were separately incubated in the reconstituted system. The possible functions of ribosomal phosphorylation in protein synthesis will be discussed.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

Phosphorylation of wheat germ initiation factors and ribosomal proteins

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M_r = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

Group fractionation and determination of the number of ribosomal subunit proteins from Drosophila melanogaster embryos

Proteins were extracted from ribosomes and (for the first time) from ribosomal subunits of Drosophila melanogaster embryos. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. The electrophoretograms displayed 78 spots for the 80S monomers, 35 spots for the 60S subunits, and 31 spots for the 40S subunits. On the basis of present information, we propose what we believe to be a reliable and convenient nomenclature for the proteins of the ribosomes and each of the subunits. A pair of acidic proteins from D. melanogaster appears to be very similar in electrophoretic mobility to the acidic proteins L7/L12 from Escherichia coli and L40/L41 from rat liver. The electrophoretogram of proteins from embryonic ribosomes shows both qualitative and quantitative differences from those of larvae, pupae, and adults previously reported by others. The proteins of the 40S subunit range in molecular weight from approximately 10,000 to 50,000, and those from the 60S subunit range from approximately 11,000 to 50,000.     

Biochemical changes in hereditary progressive muscular dystrophies. Defect of protein synthesis in fibroblasts, muscle tissues and blood cells

80S ribosomes and ribosomal subunits were isolated from fibroblasts, muscle tissues and blood cells of patients with different muscular dystrophies (MD) as well as of controls and were used for in vitro measurement of ribosomal protein synthesis (RPS) in a poly(U)-directed polyphenylalanine synthesis system. The activity of ribosomes from the patients showed a disease-dependent decrease compared to normal controls. Examination of hybrid 80S ribosomes consisting of 40S and 60S subunits of patients and the corresponding control cells revealed that the loss of RPS activity was related to one or both of the ribosomal subunits depending on the type of MD.     

Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3'':5''-cyclic monophosphate.

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2' dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2'-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

The binding of ribosomal subunits to endoplasmic reticulum membranes

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60-80% of added 60S subunits and 10-15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane-60S subunit-40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.     

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.     

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.     

The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were lethal. We propose that ARB1 functions as a mechanochemical ATPase to stimulate multiple steps in the 40S and 60S ribosomal biogenesis pathways.