Study on mammalian ribosomal protein reactivity in situ. III. Effect of trypsin on 40S and 60S subunits.

Rat liver 40S and 60S ribosomal subunits were treated with increasing concentrations of trypsin. The activity of both trypsin-treated subunits, when assayed for polyphenylalanine synthesis, progressively decreased, but the 60S subunits were inactivated at much lower trypsin concentrations than were the 40S ones. The sedimentation coefficients of trypsin-treated subunits were identical to those of control subunits when sucrose gradients containing 0.5 M KCl were used. When the sucrose gradients were prepared with a low salt buffer (80 mM KCl), dimer formation was observed with control subunits, but not with trypsin-treated ones. Two-dimensional gel electrophoresis analysis of the proteins extracted from trypsin-treated subunits revealed that all ribosomal proteins in the subunits were accessible to the enzyme. However, several proteins were more resistant to trypsin in compact subunits than when they were free or in unfolded subunits. Proteins of the 60S subunits were generally digested by lower trypsin concentrations than those of the 40S subunits. From the quantitative measurements of the undigested proteins, a classification of the proteins from both subunits according to their trypsin sensitivity was established. These results were compared with those previously obtained concerning ribosomal protein reactivity to chemical reagents.     

Phosphorylated and other modified forms of eukaryotic ribosomal protein S3 analysed by two-dimensional gel electrophoresis.

Proteins were isolated from the 40S ribosomal subunits of baby-hamster kidney fibroblasts and subjected to two-dimensional gel electrophoresis. When the cells were pretreated with cyclic AMP or 2-deoxyglucose a more basic derivative of ribosomal protein S3 or S3a was often observed, apparently similar to that previously reported to occur early in liver generation. This derivative was not a dephosphorylated form of protein S3, which protein does not appear to be phosphorylated in normal cells; nor did it correspond to the proteolytic fragment, S3b. It appears to be an oxidation product of protein S3 or S3a, as it can be eliminated by thorough reduction of the ribosomal protein before electrophoresis. In contrast with previous results with Krebs II ascites cells, starvation of baby-hamster kidney fibroblasts of glucose did not cause extensive phosphorylation of ribosomal protein S3.     

Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.

The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.     

Studies on the distribution of ribosomal proteins in mammalian ribosomal subunits.

Murine L5178Y cell ribosomes were dissociated into subunits either with potassium chloride in the presence of puromycin or with the chelating agent EDTA. The proteins of ribosomal subunits obtained by these different methods were compared by means of bidimensional polyacrylamide gel electrophoresis. KCl-derived 60S and 40S subunits were shown to contain 38 and 31 proteins respectively, 3 of them having identical electrophoretic mobilities. Preparations of EDTA-dissociated ribosomal subparticles contained different proportions of these proteins, and 11 major spots were shared between the EDTA-derived large and small ribosomal subunits. Furthermore, 10 proteins absent from subunits treated by high concentrations of KCl were reproducibly found in EDTA-treated ribosomal subparticles.     

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.     

Characterization of ribonucleoproteins and ribosomes isolated from lymphocytic choriomeningitis virus.

Disruption of purified lymphocytic choriomeningitis (LCM) virus with Nonidet P-40 in 0.5 M KCl followed by sucrose gradient centrifugation in 0.3 M KCl led to the isolation of two viral nucleoproteins (RNPs) as well as 40S and 60S ribosomal subunits. The largest viral RNP sedimented heterogenously at 123S to 148S and was associated with 23S and 31S viral RNA. The other viral RNP sedimented at 83S and was associated with 23S viral RNA. The buoyant density in CsCl was determined to be 1.32 g/cm3 for the viral RNP. Densities of 1.52 and 1.60 g/cm3 were determined for the 40S and 60S subunits, similar to those of the BHK-21 cells subunits dissociated by 0.5 M KCl. The viral RNPs were partly sensitive to RNase.     

Heterogeneity of native ribosomal 60-S subunits in Ehrlich ascites tumor cells cultured in vitro.

Native large ribosomal subunits in cultured Ehrlich ascites tumor cells analyzed by high-resolution CsCl isopycnic centrifugation consist of at least two classes of particles with densities of 1.57 g/cm3 (LI) and 1.59 g/cm3 (LII), respectively. A wash with 0.5 M KCl converts LI into particles with the density of LII particles. Incubation of derived large subunits (density 1.59 g/cm3) with 0.5 M KCl wash of reticulocyte ribosomes leads to the formation of particles with the density of LI particles. A protein with a molecular weight of 57000 present in the high-KCl wash of 60-S native subunits was virtually absent in the KCl wash of 40-S subunits or polyribosomes suggesting that specific protein factors may be present on some native 60-S subunits. Possible functions of these protein factors are discussed.     

Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells.

Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products from native 40 S subunits after phosphorylation in vitro by a cyclic nucleotide-independent protein kinase. Phosphoserine was the major phosphorylated amino acid of the proteins phosphorylated in vivo and in vitro.     

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.     

Disproportionate accumulation of 18S and 28S ribosomal RNA in cultured normal rat kidney cells treated with picolinic acid or 5-methylnicotinamide

Normal rat kidney cells treated with the pyridine derivative picolinic acid, or 5-methylnicotinamide, an inhibitor of ADP-ribosylation, are unable to process 28S rRNA and accumulate 60S ribosomal subunits in the cytoplasm. Synthesis of polyA(+) RNA, rRNA precursors, and the processing of 18S rRNA into 40S ribosomal subunits are almost unaffected. Serum starvation and treatment of cells with histidinol, cycloleucine, nicotinic acid, or 1,10-phenanthroline do not elicit this alteration in rRNA metabolism. Ribosomal subunits synthesized before picolinic acid addition have different stabilities after picolinic acid treatment. The 40S subunits are degraded while the 60S subunits are more stable, demonstrating that a compensatory mechanism exists to maintain preferentially existing subunits when they are no longer being synthesized. The results suggest that ADP-ribosylation is necessary for proper processing of 28S rRNA and therefore for formation of mature 60S ribosomal subunits.     

Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.     

Interaction of the intermediate filament protein vimentin with ribosomal subunits and ribosomal RNA in vitro

If in a low ionic strength extract of Triton X-100-resistant residual cell structures derived from Ehrlich ascites tumour (EAT) cells Mg²⁺ was chelated by EDTA, vimentin became associated with unfolded ribosomal subunits. The first molecular characterization of this association has shown that (1) vimentin binds to the RNA moiety of the ribosomes, (2) vimentin has a higher affinity for unfolded small ribosomal subunits or 18S rRNA than for unfolded large ribosomal subunits or 28S rRNA, (3) the limited degradation of vimentin by the vimentin-specific, Ca²⁺-activated proteinase, with the formation of a 48 Kd breakdown product, abolishes its affinity for rRNA, (4) the association products are rather sensitive to moderate concentrations of KCl and Mg²⁺, and (5) reductive alkylation of vimentin with pyridoxal-5-phosphate and NaBH₄ has no effect on the affinity of vimentin for rRNA. Actin and tubulin do not interact with EAT cell rRNA under the above ionic conditions.     

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.     

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.     

Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination

The SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p-beta-galactosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post-ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the 'run-off' polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.     

Synthesis of native 60S and 40S ribosomal subunits in Yoshida rat ascites hepatoma AH-130 cells: correlation with the rate of cell growth

The 60Sn and 40Sn subunit ribosome synthesis declined significantly in Yoshida rat ascites hepatoma AH-130 cells from the log phase to the plateau phase of the in vivo growth. Two main classes of 40Sn particles with protein/RNA ratios of 1.82 (p2) and 1.20 (p3) and a minor "heavy" one with protein/RNA ratio of 0.96 (p1) could be distinguished reproducibly by their ultraviolet absorption after sucrose zone sedimentation. The p2 particles appeared the dominating class in log phase cells. In plateau phase cells a decrease of p2 and an increase of p3 particles was observed. Under these conditions the p1 particles and the peaks corresponding to 60Sn subunits and to 80S ribosomes were also increased. Newly synthetized 40Sn particles banded in the p3 region of the gradient and p2 particles originated from them. These particles entered into the ribosomal cycle and contained poly(A) RNA. Formation of radioactive 80S couples by subunits entering into the ribosomal cycle was markedly stimulated in log phase cells and almost completely blocked in cells at the plateau phase of growth.     

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.     

Protein topography of the 40 S ribosomal subunit from Saccharomyces cerevisiae as shown by chemical cross-linking

Protein-protein cross-linking was used to examine the spatial arrangement of proteins within the 40 S ribosomal subunits of Saccharomyces cerevisiae. Purified ribosomal subunits were treated with either 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate under conditions such that the ribosomal particle was intact and that formation of 40 S subunit dimers was minimized. Proteins were extracted from the treated subunits and fractionated on Sephadex G-150 or by acid-urea-polyacrylamide gel electrophoresis. Cross-linked proteins in these fractions were analyzed by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Constituent members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Forty-two pairs involving 25 of the 32 40 S subunit proteins were identified. Many proteins were detected in several cross-linked dimers. These proteins with multiple cross-links form foci for the construction of a schematic model of the spatial arrangement of proteins within the 40 S subunit.     

Linear ubiquitin fusion to Rps31 and its subsequent cleavage are required for the efficient production and functional integrity of 40S ribosomal subunits

The post-translational modifier ubiquitin is generated exclusively by proteolytic cleavage of precursor proteins. In Saccharomyces cerevisiae , cleavage of the linear precursor proteins releases ubiquitin and the C-terminally fused ribosomal proteins Rpl40 (Ubi1/2 precursor) and Rps31 (Ubi3 precursor), which are part of mature 60S and 40S ribosomal subunits respectively. In this study, we analysed the effects of ubi3 mutations that interfere with cleavage of the ubiquitin–Rps31 fusion protein. Strikingly, the lethal ubi3 + P77 mutation, which abolished cleavage almost completely, led to a rapid G1 cell cycle arrest upon genetic depletion of wild-type UBI3 . Under these conditions, the otherwise unstable Ubi3+P77 protein was efficiently assembled into translation-competent 40S ribosomal subunits. In contrast to the cleavage-affecting mutations, deletion of the ubiquitin moiety from UBI3 led to a decrease in 40S ribosomal subunits and to the incorporation of the 20S pre-rRNA into polyribosomes. Altogether, our findings provide additional evidence that the initial presence of the ubiquitin moiety of Ubi3 contributes to the efficient production of 40S ribosomal subunits and they suggest that ubiquitin release is a prerequisite for their functional integrity.