The subunit interface of the Escherichia coli ribosome: Identification of proteins at the interface between the 30 S and 50 S subunits by crosslinking with 2-iminothiolane

The 70 S ribosomes of Escherichia coli were treated with 2-iminothiolane with the resultant addition of 110 sulfhydryl groups per ribosome. The modified ribosomes were oxidized to promote disulfide bond formation, some of which formed intermolecular crosslinks. About 50% of the crosslinked 70 S ribosomes did not dissociate when exposed to low concentrations of magnesium in the absence of reducting agent. Dissociation took place in the presence of reducing agents, which indicated that the subunits had become covalently linked by disulfide linkages. Proteins extracted from purified crosslinked 70 S ribosomes were first fractionated by polyacrylamide/urea gel electrophoresis. The proteins from sequential slices of these gels were analyzed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Monomeric proteins derived from crosslinked dimers appeared below the diagonal containing non-crosslinked proteins, since the second electrophoresis, but not the first, is run under reducing conditions to cleave the crosslinked species. Final identification of the proteins in each dimer was made by radioiodination of the crosslinked proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis in the presence of non-radioactive total 70 S proteins as markers. This paper describes the identification of 23 protein dimers that contained one protein from each of the two different ribosomal subunits. The proteins implicated must have some part of their structure in proximity to the other ribosomal subunit and are therefore defined as “interface proteins”. The group of interface proteins thus defined includes 50 S proteins that are part of the 5 S RNA: protein complex and 30 S proteins at the initiation site. Correlations between the crosslinked interface proteins and other functional data are discussed.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins

Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates.     

Cross-linking study on localization of the binding site for elongation factor 1 alpha on rat liver ribosomes

Complexes containing rat liver 80 S ribosomes, poly(uridylic acid), phenylalanyl-tRNA, elongation factor 1 alpha, and guanylyl(beta, gamma-methylene)-diphosphonate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 26 fractions by chromatography on carboxymethylcellulose. Each protein fraction was subjected to diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Four cross-linked pairs containing elongation factor 1 alpha were on the vertical line below the diagonal. The ribosomal protein spot of each pair was cut out from the gel plate and labeled with 125I. The labeled proteins were extracted from the gel and identified by two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both 60 S and 40 S subunits were identified: L12, L23, L39, S23/S24, and S26, three proteins of which had been found to be cross-linked also to elongation factor 2 (Uchiumi, T., Kikuchi, M., Terao, K., Iwasaki, K., and Ogata, K. (1986) Eur. J. Biochem. 156, 37-44). These results afford direct evidence that both elongation factors interact with partially overlapping sites on rat liver ribosomes.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Analysis of ribosomal proteins and ribosomal subunits of pea seeds by electrophoresis in polyacrylamide gel]

The amino acid composition of overall protein of ribosomes and ribosomal subunits of pea seeds has been found typical of ribosomal protein. Electrophoresis in polyacrylamide gel demonstrates that proteins extracted by the solution of 3 M LiCl-4 M urea from purified ribosomes of pea seeds move towards the cathode at pH 2.2 and separate into 41 components. Electrophoresis in a tris-glycine buffer at pH 9.2 does not reveal any substance corresponding to acid proteins. Similar distribution patterns are observed when ribosomal particles are isolated with or without triton (0,5%). The treatment of ribosomes by deoxycholate results in some changes, depending on the detergent concentration. All the protein components detected in ribosomes, except one, are present in the subunits. Proteins of large and small ribosome subunits produced 26 and 21 components respectively in polyacrylamide gel electrophoresis. The distribution patterns of proteins of the two subunits appear to be different. The majority of the components of the large and small subunits differ in mobility. The data obtained suggest considerable specificity of the protein composition of 60S and 40S subunits of 80S ribosomes in higher plants.     

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.     

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.     

Molecular weight hetergeneity of proteins of the ribosomes and ribosomal subunits from dry pea seeds]

The molecular weight distribution of the total protein of ribosomes and ribosomal subunits isolated from dry pea seeds was studied by electrophoresis in polyacrylamide gel, containing sodium dodecyl sulfate. It was demonstrated that overall protein of 80 S ribosomes is separated into a number of fractions with molecular weights of 10000-64000. Treatment of ribosomes with 0.5 per cent tritone, 0.5 per cent and 1 per cent deoxycholate does not change the general pattern of the molecular weight distribution of ribosomal proteins. The large subunit reveals 19 protein zones (14 major and 5 minor zones), their molecular weights are varying from 10000 to 54000. The majority of proteins of the large subunit have molecular weights of 14000--32000. The molecular weights of 17 protein zones of the small subunit (7 major and 10 minor zones) vary from 10000 to 64000. The majority of proteins of both large and small subunits have molecular weights of 14000--32000. Electrophoretic separation of proteins in the split gel confirmed the fact that the proteins of large subunit differ in molecular weights from those of the small subunit. Thus, ribosomal proteins of pea seeds are shown to produce a typical (for 80S ribosomes) pattern of molecular weight distribution under polyacrylamide gel electrophoresis in the presence of sodium dodecul sulphate.     

Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli.

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

Analysis of the protein composition of yeast ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis

The number of proteins in yeast ribosomal subunits was determined by two-dimensional polyacrylamide gel electrophoresis. The 40S subunit obtained after dissociation of ribosomes at high ionic strength contains 30 different protein species (including six acidic proteins). The 60S subunit, obtained in the same way contains 39 different species (including 1 acidic protein). While the total number of protein species found in yeast ribosomes, thus, is in close agreement with those reported for other eukaryotic organisms, the distribution between acidic and basic proteins is quite different. When the ribosomes were dissociated at low ionic strength, four extra protein spots appeared in the electropherograms of both 40S and 60S subunits. We consider these proteins to be nonribosomal.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

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.     

Immunological properties of isolated protein fractions from Artemia ribosomes

Acid-soluble ribosomal proteins from cysts of Artemia salina were separated by high-resolution polyacrylamide gel electrophoresis at pH 4.3. Three distinct protein bands, occurring in different parts of the electrophoretic pattern, were used for immunization in rabbits, and the γ-globulin fractions of the antisera were prepared. These preparations produced precipitation lines in agarose gel with protein extracted from whole 80S ribosomes and from 60S and 40S ribosomal subunits. With γ-globulin preparations from non-immune or anti-ovalbumin sera no reactions were obtained.     

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.     

Cross-linking study on protein neighborhoods at the subunit interface of rat liver ribosomes with 2-iminothiolane

Rat liver 80 S ribosomes were cross-linked with 2-iminothiolane. Proteins extracted from the cross-linked 80 S ribosomes were separated into 25 fractions by chromatography on carboxy methylcellulose. Each protein fraction was analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Eight pairs characteristic of 80 S ribosomes were detected which did not appear when isolated 40 S and 60 S subunits were cross-linked, and the cross-linked proteins were analyzed in similar manners. The cross-linked components were radioiodinated and then analyzed by two-dimensional gel electrophoresis, followed by autoradiography. Eight kinds of cross-links between 60 S subunit proteins and 40 S subunit proteins were identified as follows: SA30 (acidic protein with Mr 30,000)-LA33 (acidic protein with Mr 33,000), S2-LA33, S2-L11, S3a-L11, S4-L5, S25-L5, S4-L24 and S6-L24.     

High heterogeneity within the ribosomal proteins of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.Patrick Giavalisco, Daniel Wilson are contributed equally to this work.     

Electrophoretic analysis of soluble proteins and esterase, superoxide dismutase and acid phosphatase isoenzymes of members of the protozoan family trichomonadidae

Polyacrylamide gel electrophoresis was used to compare the proteins and isoenzymes of esterase, superoxide dismutase, and acid phosphatase in soluble, whole-cell extracts of four strains of Trichomonas vaginalis, two strains of Trichomonas gallinae, and one strain each of Tritrichomonas foetus, Tritrichomonas augusta, Tetratrichomonas gallinarum, and Pentatrichomonas hominis. Intraspecific, interspecific, and intergeneric differences were found in protein and isoenzyme profiles. At least four to seven isoenzymes were detected among the ten strains for each of the three enzymes studied. Each strain usually contained one or two isoenzymes of both esterase and acid phosphatase, and two or three isoenzymes of superoxide dismutase.