Modification of the accessibility of ribosomal proteins after elongation factor 2 binding to rat liver ribosomes and during translocation

Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.     

Regulation of the activity of eukaryotic peptide elongation factor 1 by autocatalytic phosphorylation

Highly purified peptide elongation factor 1 from rabbit reticulocytes liberates the terminal phosphate from [gamma-32P]GTP and incorporates it into its own protein. Approximately one phosphate residue becomes bound by one molecule of the factor. Only the eEF-1 alpha subunit of the factor (Mr 53 000) becomes phosphorylated as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by autoradiography and by the incubation of [gamma-32P]GTP with individual subunits of the elongation factor separated by chromatofocusing in the presence of 5 M urea. The phosphorylation also takes place, though to a lesser extent, if the factor is incubated with Na2H32PO4, probably due to the presence of endogenous GTP bound in the molecule of the factor. The content of endogenous GTP in various factor preparations was 0.21-0.43 mol/mol factor. Phosphorylation of the peptide elongation factor is ribosome-independent, acid-labile and apparently autocatalytic since no other proteins are required for this reaction. Preincubation of the factor with GTP or with inorganic phosphate results in the phosphorylation of the factor and is followed by an enhanced binding of phenylalanyl-tRNA to 80S ribosomes in the presence of poly(U). This is accompanied by a dephosphorylation of the factor protein and thus the reversible autophosphorylation of the factor apparently activates its binding site for aminoacyl-tRNA. This is supported by the observation that sodium fluoride, which inhibits the dephosphorylation of the factor, blocks the factor-catalyzed binding of aminoacyl-tRNA to ribosomes. The incorporation of phosphate into factor protein also inhibits the formation of an eEF-1 X GDP complex, which is inactive in protein synthesis. Thus GDP liberated by the GTPase activity of the factor cannot affect its binding site for aminoacyl-tRNA. This may be the other reason for the enhanced activity of the phosphorylated factor. The autocatalytic GTP-dependent phosphorylation of the peptide elongation factor 1 apparently modifies its function and may thus play a regulatory role in protein synthesis.     

Ribosomal proteins in growing and starved Tetrahymena pyriformis. Starvation-induced phosphorylation of ribosomal proteins.

The complements of ribosomal proteins in growing and starved cells of Tetrahymena pyriformis strain GL were examined by two-dimensional gel electrophoresis. In growing cells, the 40-S ribosomal subunit contained 30 proteins, 4 of which migrated toward the anode at pH 8.6, while the 60-S ribosomal subunit contained 46 proteins, 9 of which migrated toward the anode at pH 8.6. When exponentially growing cells were transferred into a non-nutrient medium pronounced phosphorylation of a single 40-S ribosomal subunit protein, S6, was induced. The phosphorylation was very specific; more than 99.5% of the [32P]phosphate incorporated into ribosomal proteins was associated with S6. Phosphate was incorporated into S6 as O-phosphoserine and O-phosphothreonine. Two-dimensional gel electrophoresis indicated that the complement of proteins associated with the ribosomes isolated from starved cells differed from that of growing cells. Careful examination, however, suggested that except for the phosphorylation of certain ribosomal proteins in starved cells, the observed differences did not reflect starvation-induced changes in vivo, but most probably different levels of artifactual modifications (limited proteolysis) during the preparation of the ribosomes.     

Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.     

Presence of guanine nucleotide-binding proteins in a plant hypocotyl microsomal fraction

The presence of specific guanine nucleotide-binding proteins in a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction was investigated. Polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of nitrocellulose blots with [alpha-32P]GTP and [gamma-32P]GTP indicated the presence of four specific and distinct GTP-binding proteins with molecular masses of approx. 23.4 kDa, 24.8 kDa, 26.6 kDa and 28.5 kDa. Binding of [alpha-32P]GTP could be completely prevented by 30 microM GDP or 10 microM guanosine 5'[gamma-thio]triphosphate. This report presents evidence for the presence in a microsomal fraction from zucchini hypocotyls of Gn-proteins as defined by Bhullar and Haslam (1987) Biochem.J. 245, 617-620. The four plant proteins resemble animal Gn-proteins when molecular weights and GTP-binding specificities are considered.     

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.     

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).     

Affinity labeling of eukaryotic initiation factor 2 and elongation factor 1 alpha beta gamma with GTP analogs

As part of an attempt to understand the specific function and role of each subunit in multisubunit protein synthesis factors, we have attempted to identify the nucleotide binding peptides of eukaryotic initiation factor 2 (eIF-2). To ensure that the interactions were of a specific nature, two general controls were used: first, other protein factors with characterized GTP binding activity were tested; second, all affinity labeling was checked for nucleotide specificity by protection with the authentic nucleotide at a 10-fold molar excess over the affinity reagent. Results with a number of GTP modifying reagents ([alpha-32P]GTP, [alpha-32P]GDP, oxidized [alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GDP, and 5'-p-[8-3H]fluorosulfonylbenzoyl guanosine) indicate that appropriate conditions for both nucleotide and subunit specific labeling have been achieved. Under these conditions all reagents modified the beta subunit of eIF-2. Complementary studies with subunit-deficient forms of eIF-2 also suggest that the beta subunit of eIF-2 is involved with GTP binding. Coupled with other data suggesting that the gamma subunit of eIF-2 might be involved in GTP binding and amino acid sequence data of eIF-2 gamma from which a part of a GTP binding consensus sequence can be localized, support is provided for the concept of alternate GTP binding domains or a GTP binding domain shared between different subunits of eIF-2.     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

Protein synthesis in brine shrimp embryos. Dormant and developing embryos of Artemia salina contain equivalent amounts of chain initiation factors 2.

Dormant and developing embryos of Artemia salina contain equivalent amounts of eIF-2, the eukaryotic initiation factor which forms a ternary complex with GTP and Met-tRNAf. The factor was purified from 0.5 M NH4Cl ribosomal washes by (NH4)2SO4 fractionation, followed by chromatography on heparin-Sepharose, DEAE-cellulose, hydroxyapatite and phosphocellulose. Purified preparations from dormant and developing embryos have similar specific activities and nucleotide requirements. The mobility of both proteins in dodecylsulfate gel electrophoresis is indistinguishable, and each contains three major polypeptide chains of molecular weight 52 000, 45 000 and 42 000. Both proteins are also immunologically identical, and each stimulates amino acid incorporation in a cell-free system of protein synthesis. The binding of [35S]Met-tRNAf to 40-S ribosomal subunits is catalyzed by eIF-2 isolated from dormant or developing embryos and is dependent upon GPT and AUG. Binding of [35S]Met-tRNAf to 40-S ribosomal subunits, and ternary complex formation with eIF-2, GTP, and [35S]Met-tRNAf is stimulated 2--3-fold by a factor present in the 0.5 M NH4Cl ribosomal wash and which elutes from DEAE-cellulose at 50 mM KCl. This protein does not exhibit GTP-dependent binding of [35S]Met-tRNAf. Binding of GDP and GTP was investigated with purified eIF-2 from developing embryos. The factor forms a binary complex with GDP or GTP, and eIF-2-bound [3H]GDP exchanges very slowly with free nucleotides. Our results suggest that eIF-2 does not limit resumption of embryo development following encystment, nor does it limit mRNA translation in extracts from dormant embryos.     

Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme-deficient or double-stranded RNA-inhibited reticulocyte lysates

The recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2.GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(alpha-32P). RF and eIF-2(alpha P) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K+. The association of eIF-2(alpha-32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F-, indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(alpha P) in the absence of F-. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2.GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF.     

Adaptive Potential of Wheat Ribosomes toward Heat Depends on the Large Ribosomal Subunit and Ribosomal Protein Phosphorylation

In a study of the translational efficiency of ribosomal subunits as a function of an in vivo temperature pretreatment, ribosomes were isolated from heat-pretreated (36°C) and reference (20°C) wheat seedlings (Triticum aestivum L.). The efficiency of recombined subunits in translating polyuridylic acid was assessed. A threefold increase in the rate of incorporation of phenylalanine by ribosomes from heat-pretreated plants was due to the large ribosomal subunit. This adaptive temperature effect was not correlated with a higher thermal stability of ribosomes or subunits from heat-pretreated seedlings, and two-dimensional gel electrophoresis failed to detect structural alterations of ribosomal proteins. Phosphorylation of ribosomal proteins in vitro showed no differences between ribosomes or subunits from heat-pretreated and reference plants. Incubation with [³²P]orthophosphate in vivo led to twice the amount of phosphate in ribosomal proteins from heat-pretreated wheat seedlings. This result is important with respect to the evaluation of the molecular basis of enhanced translational efficiency of ribosomes isolated from heat-pretreated wheat seedlings.     

Affinity labeling of the P site of Drosophila ribosomes: a comparison of results from (bromoacetyl)phenylalanyl-tRNA and mercurated fragment affinity reactions

The binding site of the peptidyl group of peptidyl-tRNA in the P site of Drosophila ribosomes was probed with (bromoacetyl)phenylalanyl-tRNA (BrAcPhe-tRNA). This affinity label binds specifically to the P site by virtue of its ability to participate in peptide bond formation with puromycin following its attachment to ribosomes. As many as nine ribosomal proteins may be labeled under these conditions; however, the majority of the labeling is associated with three large-subunit proteins and two small-subunit proteins. Two of the large-subunit proteins, L4 and L27, are electrophoretically very similar to the proteins labeled by the same reagent in Escherichia coli ribosomes L2 and L27. Reexamination by a different two-dimensional gel system of the ribosomal components labeled by a second P site reagent, the 3' pentanucleotide fragment of N-acetylleucyl-tRNA which is derivatized to contain mercury atoms at the C-5 position of all three cytosine residues, shows two major and three minor labeled proteins. These proteins, L10/L11, L26, S1/S4, S13, and S20, are likely present in the binding site of the 3' end of peptidyl-tRNA, a site that appears to span both subunits. These results have allowed us to construct a model for the protein positions in and near the peptidyl-tRNA binding site of Drosophila ribosomes.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

Biosynthesis of ribosomal proteins by poly(A)-containing mRNAs from rat liver in a wheat germ cell-free system and sizes of mRNAs coding ribosomal proteins

(1) Poly(A)-containing mRNAs from total polysomal RNA of regenerating rat liver were incubated with [3H]leucine in a wheat germ cell-free system. Ribosomal proteins were purified as described previously [1], and with two-dimensional gel electrophoresis. The proteins on the gel except for less basic protein had appreciable radioactivity, whereas the surrounding areas had very low radioactivity. Acetic acid-soluble proteins labeled in this system were subjected to three-dimensional gel electrophoresis [2]. Except for L1 and L2 proteins, each of the ribosomal proteins, including less basic ones, showed a major radioactive peak coinciding with the protein band on SDS gel. Thus, the wheat germ cell-free system completely translates almost all mRNAs for individual ribosomal proteins. Equimolar amounts of almost all ribosomal proteins were synthesized in the presence of the saturating concentration of mRNAs. (2) Free polysomes from regenerating rat liver were fractionated into three sizes. Each class of polysomes was incubated with [3H]leucine. Ribosomal proteins with molecular weights of 40 000 to 21 000 were mainly synthesized by Fraction B (5-14 monomeric ribosomes), L1 and L2 [2] with 60 000 and 54 000, by Fraction C (greater than 15 monomeric ribosomes) and B, and ribosomal proteins smaller than 20 000 by Fractions A (less than pentamer) and B. (3) mRNAs from rat liver total polysomes were fractionated into seven classes by size and each was translated in the wheat germ extract. Ribosomal proteins with molecular weights of 54 000 to 30 000 were mainly synthesized by mRNAs of 12 to 14.5 S, ribosomal proteins of 35 000 to 22 000 by those of 9.5 to 12 S, ribosomal proteins of 22 000 to 13 000 by those of 7 to 9.5 S, and smaller ribosomal proteins by those smaller than 7 S. These results indicate that individual ribosomal proteins are synthesized by monocistronic mRNAs, the lengths of which are proportional to the molecular weights of the corresponding ribosomal proteins.     

Two-step purification of the major phosphorylated protein in reticulocyte 40S ribosomal subunits

In reticulocytes, a single ribosomal protein, S13, has been shown to be phosphorylated by the cAMP-regulated protein kinases. The 40S ribosomal subunits were phosphorylated in vitro with [gamma-32P]ATP to facilitate the identification of S13 during the two-step purification procedure. Total ribosomal protein from the 40S subunit was fractionated by phosphocellulose chromatography in urea, and S13 was purified to homogeneity by gel filtration on Sephadex G-100. The protein was identified by the radioactive phosphate, by molecular weight, and by the migration characteristics in a two-dimensional polyacrylamide gel electrophoresis system. Thin-layer electrophoresis of partial acid hydrolysates of S13 showed that more than one phosphorylated residue was present in the same oligopeptide, indicating at least some of the phosphoryl groups were clustered in the protein molecule.     

Studies on interaction of 5 S RNA with ribosomal proteins

Proteins of the large ribosomal subunit of rat liver (TP 60) were immobilized by diffusion transfer onto nitrocellulose after two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Incubation of the TP 60 blots with 32P-labeled 5 S RNA under defined ionic conditions (300 mM KCl, 20 mM MgCl2) resulted in specific binding to a limited set of ribosomal proteins consisting of proteins L3, L4, L6, L13/15 and--to a lesser extent--L7 and L19. Under identical conditions, blots with proteins of the small ribosomal subunit (TP 40) did not bind 5 S RNA.     

Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.     

Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLa S3 cells

Nucleoside-diphosphate (NDP) kinase-associated [alpha-32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [alpha-32P]GTP-incorporating proteins (approx. Mr 20,000) could be separated from NDP-kinases (approx. Mr 80,000) by 5-25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [alpha-32P]GTP incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [3H]GDP, but no other nucleoside 5'-diphosphates, was also bound to these two proteins in the presence of Mg2+ (5 mM). Moreover, incubation of [alpha-32P]GTP with either G1 or G2 in the presence of Mg2+ (5 mM) resulted in the formation of [32P]GDP and Pi. The data presented here indicated that the guanine nucleotide-binding activity, the GTPase activity, and the molecular weight (approx. Mr 20,000) of NDP-kinase-associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins).     

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.